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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

Succinate dehydrogenase flavocoenzyme (‘SD-flavin’), previously shown to be an 8α-substituted riboflavin derivative containing a tertiary nitrogen homoconjugated to the flavin nucleus, was subjected to further hydrolysis and to reduction under acid conditions. Both conditions resulted in the liberation of 1 mole of histidine per mole of flavin. This proves histidine to be the covalent link between flavin and peptide backbone in succinate dehydrogenase and imidazole to be the tertiary nitrogen function homoconjugated to the flavin. 8α-Histidyl-riboflavin has been synthesized starting from riboflavin chemically and shown to be completely identical with the natural product in optical, ESR and NMR spectra, pH-fluorescence curve and behavior on thin-layer and paper chromatography, as well as paper electrophoresis. [This equation cannot be represented into ASCII Text] 3. Both the natural compound isolated by acid hydrolysis of flavin peptide and the synthetic one contain two isomers, which may be separated by high voltage electrophoresis. The isomers appear to be the N(1)- and N(3)-imidazole substituted compounds. Digestion of the flavin peptide with aminopeptidase M yields only one isomer but on treatment with 6-N HCl this is gradually converted to a mixture of the two isomers. The absolute assignment of the natural isomer is suggested as 8α-[N(3)-histidyl]-riboflavin on the basis of imidazole quaternization with CH3I, reductive cleavage of the flavin-imidazole bond and identification of the methyl-histidine liberated as 1-methyl-histidine. [ABSTRACT FROM AUTHOR]

4 UDP-trisaccharides present in the mammary gland and colostrum of the ewe were isolated and identified. The nucleotides, extracted by means of trichloracetic acid, were purified in the following way: 2 ion-exchange chromatographies on Dowex 1 (formate), filtration on Sephadex G-25, paper electrophoresis, pH 5, followed by paper chromatography in 3 different solvents. Using these fractionation techniques, 4 uridine derivatives (X1', X2', Y1', Y2') with positive reaction to the Warren neuraminic acid test, were obtained. UDP-5', which constitutes the nucleotidic part of the 4 derivatives, was defined more accurately after various acid or enzymatic analyses. The isolation of UDP-5' was made by ionexchange or paper chromatography and the total and labile phosphate was estimated. The Warren positive substances released from by neuraminidase or mild acid hydrolysis were identified as N-glycolyl-neuraminic acid for the X1', Y1' nucleotides, and N-acetyl-neuraminic acid for the X2', Y2' nucleotides. The behaviour of asialylated nucleotides to ion-exchange and paper chromatography is described as well as that of asialylated oligosaccharides. The 4 asialylated oligosaccharides contain galactose and an N-acetyl-D-hexosamine, the latter being N-acetyl-D-glucosamine in the case of X1' and X2', and N-acetyl-D-galactosamine in the case of Y1' and Y2'. Total analysis of UDPoligosaccharides shows a 1:1:1:1 ratio between uridine, N-acetyl-D-hexosamine, galactose, and neuaminic acid. The 1-P-trisaccharides and the trisaccharides were identified after phosphodiesterase hydrolysis followed by phosphatase hydrolysis. The N-acetyl-D-hexosamine carried the reducing group (sodium borohydride). Some structural characteristics of the 4 nucleotides were determined using β-galactosidase, neuraminidase, periodate, and periodate borohydride. Two of the nucleotides contain N-acetyl-glucosamine and are identical to those previously isolated from the goat colostrum. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharide of Escherichia coli U41/14 (serotype 03 :K2ab(L) : H2) an unusual amino sugar was isolated. By sequential oxidation with periodate and hypoiodite and by oxidation with periodate, followed by detection of acetaldehyde formed, the ammo sugar was characterized as a 2-amino- dideoxyaldose. The hydrochloride of the amino sugar, in comparison to those of 2-amino-2, 6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2, 6-dideoxy-D-mannose, 2-amino-2, 6-di- deoxy-D-allose, and 2-amino-2, 6-dideoxy-D-gulose, was studied with aid of ion exchange column chromatography. The N-acetylated amino sugar was compared with the N-acetylated reference amino sugars with regard to their relative mobihties in paper chromatography. The ammo sugar alcohols and reference amino sugar alcohols were studied using paper electrophoresis in molybdate buffer. On the basis of the results obtained the unknown amino sugar was identified as 2-amino- 2,6-dideoxymannose (rhamnosamine). Its optical rotation showed it to be of the L-configuration. This is the first report on the natural occurrence of L-rhamnosamine. [ABSTRACT FROM AUTHOR]

A hitherto unknown amino sugar was discovered in lipopolysaccharides isolated from strains of chemotype III of the photosynthetic bacterium Rhodopseudomonas palustris. The sugar (2-3% of the lipopolysaccharides dry weight) is detected as a shoulder on the descending part of the glucosamine peak obtained when acid hydrolysates of the lipopolysaccharides are passed through an amino acid analyzer. It was separated from the other amino sugars of the lipopolysaccharide (i,e. glucosamine, galactosamine, quinovosamine) by high voltage paper electrophoresis and subsequent paper chromatography. The mass spectrometric analysis of the isolated sugar as deuterium-reduced alditol acetate revealed the presence of a 6-0-methyl-2-amino-2-deoxyhexose. Demethylation of the isolated sugar afforded glucosamine and the demethylation of its ninhydrin degradation product yielded arabinose, which showed that the parental amino sugar is glucosamine. Authentic 6-O-methyl-D-glucosamine exhibited identical properties in paper chromatography and high-voltage electrophoresis as did its alditol acetate derivative in gas-liquid chromatography and mass spectrometry. These data prove that the unknown amino sugar in lipopolysaccharides of Rhodopseudomonas palustris is 6-0-methyl-glucosamine. The strong positive optical rotation of the isolated sugar indicates its D-configuration. 6-0-Methyl-D-glucosamine is part of the O-specific chain and not of the lipid A moiety of the lipopolysaccharide. Periodate-oxidation studies revealed that the sugar, like the other amino sugars of this lipopolysaccharide, does not occupy non-reducing terminal positions to any appreciable extent. [ABSTRACT FROM AUTHOR]

The role of Β-glucose 1,6-bisphosphate as a cofactor in the reaction catalyzed by the phosphoglucomutase for Β-glucose 1-phosphate (Β-phosphoglucomutase) has been examined in purified extracts of Euglena gracilis var. bacillaris. 1. The incubation of Β-glucose 1-[32P]phosphate with Β-phosphoglucomutase in presence of high concentrations (0.1 mM) of a commercial preparation of α-D(+)-glucose 1,6-bisphosphate (prepared synthetically) yielded a labelled compound running electrophoretically and chromatographically as the sugar bisphosphate. Specificity studies with Β-phosphoglucomutase and muscle phosphoglucomutase (α-phosphoglucomutase) strongly suggest that the compound formed is Β-glucose 1,6-[32P]bisphosphate. The results would also indicate that the commercial preparation of α-D(+)-glucose 1,6-bisphosphate contains Β-glucose 1,6-bisphosphate as contaminant. 2. When synthetic Β-glucose 1-phosphate preparations were purified by paper chromatography and then incubated with Β-phosphoglucomutase, no glucose 6-phosphate could be detected. On the other hand the reaction readily took place when challenged with the chromatographic fraction that runs as sugar bisphosphate. Upon analysis with etectrophoresis, chromatography and weak acid hydrolysis, and experiments with Β-phosphoglucomutase and α-phosphoglucomutase it was concluded that this compound is Β-glucose 1,6-bisphosphate. 3. It was established that the natural α-D(+)-glucose 1,6-bisphosphate not only fails to sustain the reaction catalyzed by Β-phosphoglucomutase but rather inhibits the reaction when added to the whole Β-phosphoglucomutase system, it was also demonstrated that Β-glucose 1,6-bisphosphate acts as inhibitor of the α-phosphoglucomutase system. 4. These results show for the first time that Β-glucose 1,6-bisphosphate is an active and necessary participant in the reaction catalyzed by Β-phosphoglucomutase. The role played by Β-glucose 1,6-bisphosphate in this reaction would be essentially the same as that of the α-anomer in the reaction catalyzed by α-phosphoglucomutase. [ABSTRACT FROM AUTHOR]

Acidic capsular polysaccharide of Vibrio parahaemolyticus A55 (05:K15) was esterified with ethylene oxide and then reduced with sodium borohydride. Hydrolysis of the reduced material gave two hexosamines identical with mannosamine (2-amino-2-deoxy-mannose) and gulosamine (2-amino.2-deoxy-gulose) on paper and ion-exchange chromatograms. This fact suggested that the K15 antigen contained mannosaminuronic acid (2-amino-2-deoxy-mannuronic acid) and gulosaminuronic acid (2-amino-2-deoxy-guluronie acid). Direct acid hydrolysis of the antigen gave two 2-amino-2-deoxy-hexuronic acids. These two were identified as mannosaminuronic and gulosaminouronic acids by paper chromatography and paper electrophoresis, being compared with synthesized standard samples. Titration of acid form of the polysaccharide indicated that about 91.5% of the polysaccharide was composed of the N-acetyl-hexosaminuronic acids. [ABSTRACT FROM AUTHOR]

9-S RNA was prepared from duck reticulocytes by dodecylsulfate treatment of the ribonucleoprotein particle derived from EDTA-treated reticulocyte polyribosomes. The messenger content of this RNA was assayed by microinjection into oocytes of the frog Xenopus laevis. The 9-S RNA was found to direct the synthesis of a molecular species that eluted from Sephadex G-100 with added duck reticulocyte haemoglobin. When analysed on a cation-exchange resin this material was found to contain the three globin-chain species characteristic of duck reticulocyte haemoglobin. Moreover, paper electrophoresis at pH 6.5 and pH 3.5, and paper chromatography showed that these globin-like species made in the oocyte were virtually indistinguishable, in terms of their histidine-containing tryptic peptides, from the corresponding globin chains derived from duck reticulocyte haemoglobin. A comparison between the products made following the injection into oocytes of reticulocyte 9-S RNA from rabbits, mice or ducks proved that it is the exogenous messenger that gives rise to the information content of the proteins whose synthesis is elicited by the injected RNA. The results also showed that the oocyte contains translational systems that are neither cell-type nor species specific, and that duck globin messenger RNAs require no reticulocytespecific factors for their translation in oocytes. The construction of a saturation curve showed that there is a linear relationship between the amount of messenger injected and the amount of haemoglobin synthesized, up to the point at which haemoglobin synthesis constitutes over 35% of the total protein being made. Consequently the frog oocyte provides a convenient assay system for small quantities of duck globin mRNAs, and the messenger activity of as little as 1 ng of 9-S RNA can be detected very simply. Thus a combination of the duck reticulocyte and frog oocyte may provide a useful system for studying post-transcriptional control, and the formation of mRNA, in a eukaryotic cell. [ABSTRACT FROM AUTHOR]

The polysaccharide moiety of the lipopolysaccharide (polysaccharide I) from Escherichia coli F492 (08:K27-:H-) and the polysaccharide (polysaccharide II) from the rfa mutant F612 (derived from F492) were isolated by extraction with 450% aqueous phenol at 65 °C. Polysaccharide I was obtained in 1–2% yield (based on dry bacteria). It contained mannose (83.5%), glucose (5.7%), galactose (3.4%), heptose (4.6%) and 2-keto-3-deoxy-mannosulonic acid (0.8%). Polysaccharide II was obtained in 0.8–1% yield (based on dry bacteria). It contained 99.2% mannose. With the method of Yphantis it was found that the molecular weights of polysaccharides I and II were 12400 and 10400, respectively. The difference accounted for the core oligosaccharide which was present in polysaccharide I and absent in polysaccharide II. The specific optical rotation was 43 °C for polysaccharide I and 42 °C for polysaccharide II. Both polysaccharides were permethylated. Subsequent hydrolysis, reduction and acetylation, followed by gas-liquid chromatography and mass spectrometry indicated that in both polysaccharides two-thirds of the mannose units were substituted at C-2 and one-third at C-3. These results were confirmed by periodate oxidation. From polysaccharide II nine oligosaccharides were obtained after partial acid hydrolysis by chromatography on Biogel P2, paper chromatography and paper electrophoresis of the borate complexes. The oligosaccharides were reduced with sodium borodeuterate (which labelled the reducing mannose units) and methylated. After hydrolysis, reduction and acetylation the products were analyzed by gas chromatography and mass spectrometry. It is concluded that the poIysaccharides I and II contain about 20 repeating units of α-mannosyl-1,2-α-mannosyl-1,2-α-mannose which are joined through &alph;-1,3 linkages. [ABSTRACT FROM AUTHOR]

Nicotinic acid in Rhodotorula glutinis is synthesized from tryptophan via N′-formyIkynurenine, kynurenine, 3-hydroxykynurenine and 3-hydroxyanthranilic acid. Auxotrophic mutants, with blocked synthesis at various steps between tryptophan and 3-hydroxyanthranilic acid, can grow with 100 μg/ml anthranilic acid (concentration in the agar-agar). The usually required concentration of substances such as nicotinic acid or other substances after the blocked step is about 2 μg/mI. Auxotrophs with a block between 3-hydroxyanthranilic acid and nicotinic acid are not able to grow with anthranilic acid. Therefore mutants requiring nicotinic acid with a blocked step between tryptophan and 3-hydroxyanthranilic acid can synthesize nicotinic acid from anthranilic acid, a substance formed in the first step of tryptophan synthesis from chorismic acid, by eluding the blocked pathways The same ability has been found in prototroph revertants with the original block of the auxotrophs but with a second mutation in the gene of anthranilate synthetase. As a result of this mutation the feedback inhibition by tryptophan is disturbed and anthranilic acid is produced in excess. When the culture medium of the prototrophs is analysed by paper chromatography after adding anthranilic acid to the growing culture, 3-hydroxyanthranilic acid as one of the formed products can be found. Summarizing these facts it is evident, that there exists a "short-cut"-pathway of nicotinic acid synthesis in Rh. glutinis, which is realized by direct hydroxylation of anthranilic acid to 3-hydroxyanthranilic acid. This "short-cut"pathway however is only possible, when anthranilic acid in the cell is present in excess. [ABSTRACT FROM AUTHOR]

This article is the sixth and last of a series devoted to the primary structure of bovine β-casein [1–5]. In the preceding paper, we presented a partial sequence of this protein in which 156 out of the 209 amino-acid residues were exactly positioned [5]. Only a few data were given on the tryptic peptides T2 and T3 [1] which corresponded to the unknown part of the molecule. In the present communication, we establish the amino-acid sequence of these 2 peptides, and discuss the salient features of the β-casein molecule. Bovine β-casein A2 is a phosphoprotein constituted of a single polypeptide chain containing 209 amino-acid residues. Among them are five phosphoseryl residues. From sequence data, it can be written: Asp4, Asn5, Thr9, Ser11, SerP5, Glu17, Gln22, Pro35, Gly5, Ala5, Val19, Met6, Ile10, Leu22, Tyr4, Phe9, Trp, Lys11, His5, Arg4. Its exact molecular weight is 23982, a little higher than that of αs1-casein, which is 23616 [6]. The total number of 27 amide groups (Asn + Gln) agrees well with previous data obtained by Pion et al. [7] and Peterson et al. [8]. The average hydrophobicity, calculated according to Bigelow [9], is 1330 and it places β-casein among the most hydrophobic proteins. The sequence obtained emphasizes the uneven charge distribution along the peptide chain. A high negative net charge is located in the first 50 residues. The high proline residue content (16.7%) and the rather uniform repartition of this residue exclude the possibility of a high α-helix content. Furthermore, the complete absence of segments containing more than 2 adjacent prolyl residues seems to preclude the possibility of poly-L-proline structure that was expected from optical-rotatory-dispersion studies [10]. An interesting extension of the present work is the discovery by Gordon et al [11] that γ-, TS-, R- and S-caseins are fragments of β-casein likely originating from tryptic-like cleavages of the peptide chain next to the carbonyl groups of some lysyl residues. The γ-, TS. A2- and S-, TS. B- and R-caseins respectively represent residues 29 to 209, 106 to 209 and 108 to 209. These results explain the occurrence of only one phosphorus atom in the γ-caseins, the absence of phosphorus in the TS-, R- and S-caseins [12]. Localization of the substitutions which differentiate the known variants of bovine β-casein also explains the relationship between β-, γ-, R-, S- and TS-caseins [12,13]. The NH2-terminal tryptic phosphopeptide, previously analyzed by Peterson et al. [14], has been sequenced independently by Manson and Annan [15]. Their results are identical to those reported in a preceding paper [4]. [ABSTRACT FROM AUTHOR]

Previous reports [1-5], we have described some of the features of the primary structure of bovine αs1-casein. In the present work, the complete amino acid sequence has been established and the salient features of this phosphoprotein have been discussed. In the polyeptide chain, the region containing the phosphopeptides Tm1 (T1-T2), Tm1T2 and Tm1T1 [1,4,5], had only been slightly studied as yet. Because of the difficulties encountered in the breakdown of these phosphopeptides, hydrolysis with endopeptidases and exopeptidases were performed on both native and dephosphorylated peptides. It was confirmed that peptide Tm1T2 contains three hydroxy-amino acids (2 Ser, 1 Thr), but, instead of three [1], two phosphorus atoms were found with purified preparations. Partial acid hydrolysis and dephosphorylation using an orthophosphoric-monoester phosphohydrolase indicate that the two seryl residues m this peptide are O-phosphorylated. The remaining gap in the sequence of the central part of peptide Tm1T2 was bridged by studying two fragments obtained by papain digestion of the dephosphorylated preparation. Instead of four serines, postulated earlier from the results of the amino-acid composition of peptide Tm1T1 [1], five were shown to be present after detailed analysis of the fragments. Since the five phosphate groups could be ready removed by an orthophosphoric-monoester phosphohydrolase, the five seryl residues could be O-phosphorylated. The fragments obtained after hydrolysis with thermolysin of both native and dephosphorylated peptide Tra1T1 were further degraded using classical methods for the determination of the amino acid sequence. In the light of all the results obtained on this phosphoprotein (αs1-casein B), the total number of amino acid residues has been corrected to 199, instead of 198, as reported previously [1-5], and the molecular weight has been calculated to be 23616. The following amino-acid composition; Asp7, Asn8, Thr5, Ser8, SerP8, Glu25, Gln14, Pro17, Gly9, Ala9, Val11, Met5, Ile11, Leu17, Tyr10, Phe8, Lys14, His5, Trp2, Arg6, indicates that there is a higher number of acidic than basic residues in this protein. On the basis of the intrinsic dissociation constants of titratable groups in proteins [6], the negative net charge of the molecule was estimated to be 22 at pH 6.5 and 28 at pH 8.6. Since Bigelow's parameter for thc average hydrophobicity [7] of this protein is 1170, it could be considered to be a hydrophobic protein The high amount (8.5%) and uniform distribution of prolyl residues indicate that this protein has limited structuralcoiling possibilities. The polypeptide chain contains three hydrophobic regions, viz. 1-44, 90-113 and 132-199. The first two are characterized by the fact that basic residues predominate over acidic residues. The third region, where most of the aromatic residues are located, contains very few basic residues and is therefore of more acidic character. Two regions, 45-89 and 114-131, are hydrophilic. The former contains more than half of the total acidic residues, in particular seven of the total eight phosphoseryl residues. Another remarkable feature is the concentration of amino acids with carboxylic side chains, particularly glutamic acid, in the vicinity of two clusters of phosphoseryl residues. It may be noticed that the phosphoseryl residues m αs1-casein are arranged m a manner similar to those in β-casein [8]. In particular, in αs1-casein, the 62-70 region containing four phosphoseryl residues, is similar to the 13-21 region in β-casein [8]. It is interesting to point out that ail but one of the phosphoseryl residues always occur m position n with relation to a glutamyl or a phosphoseryl residue, which is in position n + 2. From this, it would appear that there is an enzymatic phosphorylation of the polypeptide chum (carried out by a phosphoryl kinase which may require a negative charge in the phosphorylation site) rather than a direct incorporation of phosphoserine into the polypeptide chain during protein synthesis. The location of the amino-acid substitutions that characterize the αs1-Casein D and the β-casein C variants has provided evidence for an enzymatic phosphorylation of casein [9]. The problem of the phosphorylated sites in αs1- and β-caseins will be discussed in a forthcoming paper. The repeating amino-acid sequences located in the two regions of αs1-casein, viz. 70-84 and 110-125, are also of interest. The primary structure of αs1-casein given here is that of the genetic variant B. The three other variants A, C and D have also been studied. The C and D variants differ from the B variant by the substitutions of Gly/Glu in position 192 [10] and ThrP/Ala in position 53 [9], respectively. The A variant is characterized by a deletion of thirteen amino-acid residues from position 14 to position 26 in the hydrophobic NH2-terminal part of the polypeptide chain [11]. Because of this deletion, there m an important change in the physico-chemical properties of αs1-casein [12, 13]. Some important features of the amino-acid-sequence determination should be noted. Firstly, the great value of maleic anhydride as a reversible-blocking reagent for amino groups of proteins [14], and the usefulness of thermolysin as a specific enzyme for degrading polypeptides [15]. Secondly, the difficulties encountered during the study of the phosphopeptides: these peptides were difficult to purify since they have similar anionic characters and only react slightly with ninhydrin during paper revelation; other difficulties have been the high destruction of phosphoserine during hydrolysis with HCl 5.7 N, the poor yield in the substractive Edman degradation when the NH2-terminal residue is a phosphoserine, and the failure of endopeptidases and exopeptidases to split off peptide bonds that are close to phosphate groups. Fortunately, alkaline phosphatase has proved to be a useful tool for removing phosphate groups, thus avoiding most of these difficulties. Since the amino-acid sequence of αs1-casein is now known, the degradation of this protein by coagulating and other proteolytic enzymes during cheese ripening may be studied. In particular, bitter peptides originating from αs1-casein may be located. As we have already briefly noted [4], it is obvious that one of the three bitter peptides isolated by Matoba et al. [16] from a tryptic hydrolysate of whole casein, was the segment 23-34 of the polypeptide chain of αs1-casein. In addition, the knowledge of the primary structure will be of help in understanding many aspects of the relations between structure and physical properties in this phosphoprotein. [ABSTRACT FROM AUTHOR]

This paper is the fifth of a series devoted to the primary structure of the bovine β-casein A². The first two communications dealt with the isolation, analysis and positioning in the peptide chain of the tryptic and “CNBr” peptides [1,2]. The last two described the sequence of 65 residues from the carboxyl-terminus and of 32 residues from the amino-terminus [3,4]. We report here a partial sequence which includes 156 residues (out of the 209 residues of the β-casein A²) exactly positioned in the peptide chain. Two segments including 53 residues (from position 36 to 68 and from 73 to 92) remain to be analyze. In addition to the tryptic and “CNBr” peptides originating from β-casein A², two peptides issued from a tryptic digest of β-casein B were used as starting material. The position of the substitution His→Gln which differentiates the β-casein A² from the β-casein A³, already given in a preliminary communication [5], is indicated, as it is for the substitution Ser→Arg which represents one of the two differences occurring between the β-casein A² and the β-casein B. Four of the five phosphorus atoms of the β-casein A² have been previously located in the NH2-terminal tryptic peptide [4]. The fifth has been located at position 35. All the phosphorus atoms of the β-casein A² occur as phosphoserine. The complete structure of the β-casein A² will be given and discussed in the next and last paper. [ABSTRACT FROM AUTHOR]

1. The purification of a halidohydrolase from cell-free extracts of Pseudomonas dehalogenans NCIB 9061 grown on glucose and monochloroacetate as carbon source, is described. 2. This enzyme catalyses the hydrolysis of some 2-halogen substituents on aliphatic carboxylic acids, releasing a halide ion and a hydrogen ion and causing inversion of the configuration. 3. The molecular weight as determined by chromatography on Sephadex G-100 was about 15000. 4. The optimum activity was at pH 9.4. Activity was rapidly and irreversibly lost below PH6.0. 5. The amino acid composition of the purified enzyme in its native state and after photooxidation in the presence of methylene blue is presented. 6. The variation of the kinetic parameters, V and Km between pH 7.0 and pH 10.0 suggest that a group with a pKa of about 7.8 is involved in its base form in the catalytic reaction. 7. The enzyme is rapidly inactivated by reaction with iodine and upon photooxidation in the presence of methylene blue, but the effect of thiol reagents suggests that a thiol group is not directly responsible for catalysis. 8. It is suggested that a histidine residue is implicated in the catalytic reaction and a mechanism is proposed which is consistent with the experimental results. [ABSTRACT FROM AUTHOR]

Two isogenic derivatives of Escherichia coli K12: strains EA 1 (Met-, Biot-, RCstr and EA 2 (Mei-, Biot-, RCrel) when grown in the presence of ethionine in place of methionine, synthesize ribosomes whose RNA is submethylated and whose proteins contain ethionine. Net ribosome synthesis in the presence of ethionine is greater in the RCrel strain than in the RCstr strain. The precedinig paper describes results obtained with the latter strain. Here we present further studies carried out with the isogenic RCrel strain EA 2 As in the case of the RCstr strain (previous paper) ribosomes synthesized by the RCrel strain grown in the presence of ethionine display defective association properties. A large fraction of these "ethionine" ribosomes is found as free subunits in crude extracts containing 10 mM Mg+ prepared from strain EA 2 grown in an ethionine-containing medium. These free "ethionine" subunits have sedimentation coefficients of 28 S and 45 S approximately while the "ethionine" subunits found associated as 70 S particles in crude bacterial extracts sediment at 30 S and 50 S respectively. Both classes of "ethionine" subunits (free, and associated) can be methylated in vitro in the presence of S-adenosyl methionine with or without the addition of an external source of methylases (100000 x g supernatant of a homologous crude bacterial extract) The latter observation shows that the "ethionine" subunits contain bound methylases. These enzymes do not seem to be normal ribosomal proteins nor to be bound specifically to "ethionine" ribosomes since (a) they are removed by washing the "ethionine" ribosomes with 1 M NH4Cl, and (b) they are found bound to normal methionine ribosomes from which they are also removed by washing with 1 M NH4Cl. [ABSTRACT FROM AUTHOR]

Commercial lima bean inhibitor was fractionated into four apparently homogeneous components as previously described by Jones, Moore and Stein in 1963. Reduced and alkylated component IV was hydrolyzed with trypsin and the resulting peptides were separated by ion exchange chromatography on Dowex 50 X2 or gel filtration on Bio-Gel P-6. Where necessary, further purification of the peptides was carried out by paper chromatography, paper electrophoresis or a combination of both. Seven peptides were obtained in pure form and their compositions are reported here. The amino acid sequence of six of these peptides was determined, using classical methods. When allowance is made for the occurrence of two homologous peptides, presumably resulting from heterogeneity m the original protein preparation, the tryptic peptides isolated, account for the complete composition of the protein. In an attempt to obtain overlapping sequences the tryptic digest was also performed on the reduced, alkylated and guanidinated protein. Five tryptic peptides, including one containing homoarginine as the carboxy-terminal residue, were isolated in pure form from the digest; their amino acid compositions are reported. [ABSTRACT FROM AUTHOR]

Using ion exchange chromatography, gel filtration and paper electrophoresis eight peptides were isolated in pure form from a chymotryptic digest of lima bean inhibitor component IV. They account for 76 out of the 84 residues of the protein. Complete or partial sequences of these peptides were determined using classical methods. This information, together with information obtained on the tryptic peptides and reported in the preceding paper allowed the deduction of the complete amino acid sequence of lima bean inhibitor IV. The resets show that the original protein preparation was heterogenous as reflected by the occurrence of conservative substitutions at positions 26, 37 and 39. A detailed examination of the sequence revealed the presence of a repetitive sequence with residues 50 through 61 clearly homologous to residues 23 through 34. Of added interest is the fact that the anti-chymotrypsin site of lima bean inhibitor, as recently determined by Krahn and Stevens, was found to be located in one of the homologous regions. This has led us to speculate that the anti-trypsin site could be located m the other homologous region and that this double-headed inhibitor, with independent sites for trypsin and chymotrypsin, may have evolved by gene duplication. [ABSTRACT FROM AUTHOR]

In paper [5] a simplified model for the hydrophobic binding sites of trypsin was proposed, showing where 1-amino-n-alkanes and α,ω-diamino-n-alkanes are bound while they competitively inhibit the catalyzed hydrolysis of a substrate ester. In the model, the enzyme bas two hydrophobic binding sites separated by an aqueous region which presumably contains the catalytic important residues. The first site binds up to 4 methylene groups of the aliphatic side chain adjacent to the ω-amino group: additional—CH2 groups in t, he chain extend into the aqueous region. If the chain is larger than 7 carbon atoms, the additional methylene or methyl groups are bound to the second hydrophobic site. This site is found to bind less strongly than the first site. This paper presents a thermodynamic investigation of the binding properties of trypsin in the formation of complexes with cationic esters and amids which bear an ammonium group on either the end of the acid side chain or the end of the alcohol or amine component. Free energy and in addition enthalpy and entropy of binding were measured as a function of the length of the inhibitor molecule and as a function of the position of the ester or amide carbonyl group. It was found that thc stability of a complex increases linearly with length of the molecule, and that the magnitude of increase depends on the position of the carbonyl group. Amides always exhibit a smaller increase than esters. Inhibitors with the ammonium group on the alcohol component, were bound with about, the same stability than the corresponding ω-aminoacidesters. The results are consistent, with the model. Positioning of the carbonyl group within the first or second binding site reduces stability due to restricted interaction of the hydrophobic residues on enzyme and inhibitor. Only when the carbonyl group falls into the aqueous region, the free energy for an inhibitor containing the ester group is similar to that for a 1-amino-n-alkane with identical chain length. Enthalpy and entropy as functions of the position of the carbonyl group show minima for the position within the aqueous region. This observation is correlated with the jump of enthalpy and entropy of complex formation of trypsin in the series of 1-amino-n-alkanes reported earlier. It is shown that the magnitude of free energy of complex formation with specific substrates is well estimated on basis of the model. As a consequence, the alternative binding of either the 2-N-acyl group or the alcohol or amine component, to the second hydrophobic site may imply a mode of less productive binding. It is emphasized that unproductive binding should be reflected in the Michaelis Menten constants for substrate amides whereas thc Michaelis Menten constant, for a substrate ester is normally not perturbed. [ABSTRACT FROM AUTHOR]

Malonic acid as well as its model substrate methylmalonic acid are covalently bound to two chemically different sites of the fatty acid synthetase complex of yeast. When radioactively labeled malonyl CoA or methylmalonyl CoA are incubated with the multienzyme complex, radio- active malonyl and methyhnalonyl enzymes are formed and may be precipitated with trichloro- acetic acid. Treatment with performic acid splits only a minor proportion of the (methyl) malonyl enzyme bonds. Most of them are resistant to the oxidant and, hence, prove to be non-thioester linkages. Methyl- [3-14C]malonyl enzyme was digested with pepsin. By DEAE-Sephadex chromatography of the peptic hydrolysate the radioactive (methyl)malonyl peptides. were separated into three different fractions A, B and C. In peptides B and C (methyl)malonic acid is bound by performic acid stable linkages, whereas (methyl)malonyl peptide A apparently is a thioester and can be split by oxidation. Peptide C is always a minor sateffite of peptide B, the quantitative relation of both varying somewhat in different preparations. it is concluded that both are derived from a common original peptide sequence in the enzyme. Methyl- [3-14C]malonyl peptide B was further purified by the following procedures: Dowex-50X2 chromatography, subtilisin S digestion, DEAB-Sephadex chromatography, Dowex-50X2 chromatography, high voltage paper electrophoresis and paper chromatography. Two methylmalonyl peptides, B1and B2, resulted from subtilisin S treatment. They finally were obtained in analytically pure form and proved to be a pentapeptide and a heptapeptide, respectively, with the following amino acid composition: B1 = Leu,AIa,Gly,Ser,His and B2 = Leu, Ala,Gly(2 × ),Glu,Ser,His, Leucine was the C-terminal amino acid in peptide B1. No cysteine was found in either peptide. A preparation of [3-14C]malonyl peptide B was found to be analytically pure after DEAE- Sephadex, Dowex-50X2 and paper chromatography. Its amino acid composition was identical to that of methylmalonyl B1peptide B2. The acyl peptides B and C were highly sensitive against alkaline hydrolysis. It is concluded that the (methyl)malonic acid in peptides B and C is bound to serine by an O-ester linkage which is unusually activated by some surrounding amino acid, possibly histidine, [3-14C]Malonal peptide A was shown to contain stoichiometric amounts of cysteamine, β-alanine, and organic phosphate, corresponding to the quantity of malonate bound. These data suggest a thioester linkage between malonate and the SH-group of enzyme-bound 4'-phospho- pantetheine. 4'- Phospho-pantetheine, therefore, represents the so called "central" SR-group in yeast fatty acid synthetase, The other malonyl binding site is thought to be the active center of the malonyl transferase in the complex. After alkali treatment of the purified fatty acid synthetase under defined conditions 4' -phospho - pantetheine was isolated from the bydrolysate and characterized as S-benzoyl-4'-phospho- pantetheine. One molecule of the multienzyme complex contained between 3.5 and 6 molecules of 4'-phospho-pantetheine. According to the amount of malonyl enzyme formed, not all of the enzyme-bound phospho-pantetheine residues accept malonic acid. Some may be involved in other functions. [ABSTRACT FROM AUTHOR]

This paper is the first one of a series devoted to the primary structure of the major protein of cow's milk, αs1-casein, a single-chain phosphoprotein, formerly reposed to have arginine as an NH2-termimal, and Leu-TrpOH as COOH-terminal residues. ryptic digestion of maleyl αs1-casein (genetic variant B), in theory bruited to arginyl bonds, yields eight peptidic fragments, Tm1 to Tm8, which were fractionated by column chromatography and, in some cases, more thoroughly purified by preparative paper electrophoresis and/or chromatography. The ammo acid composition and the phosphate content of these fragments were determined. The fragments Tm3 and Tm8 arise from a partial non specific cleavage of Tm5. Thus, six fragments only—2, 4, 5, 6 and 7—arise from the specific cleavage of the αs1-casein chain at arginyl bonds, a result in accordance with the reported number of six arginyl residues in the chain, one being NH2-terminal. The fragment Tm7, with two arginyl residues, and Tm2 devoid of arginine appear to rcpresent respectively the NH2- and COOH-terminal parts of the αs1-casein chain. These peptidic fragments—except Tm4 which is devoid of lysine—were further digested by trypsin at lysyl bonds, after eliminating the blocking maleyl groups. The resulting peptides were purified by the same methods as mentioned above, and the composition of these tryptic peptides was established. As a control, the tryptic peptides of αs1-casein were simultaneously purified by the same methods from a hydrolysate of the protein which had not been treated with maleic anhydride. In summary, this first step of our work gives: (a) the composition of ail the bovine αs1-casein tryptic peptides; (b) partial information on the location of these peptides in the protein chain: (c) confirmation that the molecular weight of the bovine αs1-casein monomer is approximately 23600. [ABSTRACT FROM AUTHOR]

A linear lattice of equivalent binding sites with nearest neighbor cooperativity is used as a basic model for a linear biopolymer displaying cooperative binding of small ligands. Complicating effects due to the dimerization of free ligands are taken into account. The theory implies two types of intrinsic binding processes: (1) the binding of an isolated ligand (nucleation), and (2) the binding of a ligand in the immediate neighborhood of an already bound one (aggregation). The ratio of the respective association constants determines the degree of cooperativity. It is generally shown how the equilibrium properties of the system can be derived by means of the matrix method. Sonic properties of special practical significance, such as the equilibrium concentrations of free and bound monomer ligands, are discussed in more detail. On the basis of a pertinent kinetic model chemical relaxation of strong cooperative bindinng is also examined. The paper gives particular emphasis to the formulation of quantitative relations which are most useful for an experimental application of the the oretical results. Appropriate procedures to plot measurable data and to evaluate basic parameters are proposed. They are applied to specific experimental systems in a subsequent set of papers. [ABSTRACT FROM AUTHOR]

The purification and the amino acid sequence of the peptide containing the essential sulphydryl group of arginine kinase from Homarus vulgaris muscle is described. The fractionation of the tryptic digest of arginine kinase labelled with N-[1-14C]ethylmaleimide has been caused out using gel filtration, ion-exchange chromatography and paper chromatography. The composition of the radioactive peptide, isolated from the digest, is reported together with its amino acid sequence. A structural comparison is made between the peptide of arginine kinase and the corresponding active-cysteine containing peptide isolated from rabbit muscle creatine kinase. [ABSTRACT FROM AUTHOR]

Various observations cited at the beginning of this paper and in the Discussion suggested that phalloidine, a poisonous constituent of the green mushroom Amanita phalloides, would not be toxic by itself, but would be rendered toxic previously in the liver. Using a tritiated toxic derivative of phalloidine ([³H]desmethylphalloin) we found that: (a) in rats, 60% of the administered substance are excreted unchanged in the urine within the first day. A second radioactive substance of low concentration, moving more slowly in paper chromatography turned out as a product of the self-degradation of [³desmethylphalloin; (b) respectively 98% and 99.9% of the radioactivity were extracted with methanol from homogenates of livers of rats or mice which had received [³H]desmethylphalloin 1 to 2 hours before. Aqueous homogenates of liver of poisoned rats also contained radioactivity firmly bound to a high molecular weight substance as shown by gel chromatography. The radioactive substance was totally removed from the carrier presumably ribosomal material, by adding methanol, and was identified as [³]desmethylphalloin; (e) [³H]desmethylphalloin was not metabolized on incubation with oxygen and rat liver ribosomes in a NADPH-regenerating system. A radioactive substance extracted from liver microsomes of a poisoned rat also proved to be original toxin. As a result of these experiments it can be concluded that [³H]desmethylphalloin and most probably also phalloidine are not metabolized in the livers of rats and mice. [ABSTRACT FROM AUTHOR]

This paper is a part of a project on the mechanism of activation of intestinal sucrase and of intestinal sugar transport by Na+. It reports a formal kinetic analysis of the following aspects: 1. Investigation on the apparent Km for sucrose hydrolysis in intestinal rings and homogenates does not indicate the existence of any gross diffusion barrier for sucrose between lumen and brush-border bound sucrose, Therefore, it is possible to compare kinetic parameters of solubilized sucrase with kinetics parameters of the intestinal sugar uptake system. 2. Intestinal sucrases from hamster, rat, Greek turtle, man and rabbit (the latter two reported in previous papers) are all activated by Na+. The modes of activation and the apparent Michaelis constants for Na+ are different for each species considered.3. K+ and Li+ have effects similar to those of Na+. 4. The Ksapp = f (Na+]) functions indicate a non-compulsory reaction sequence in Na+-activation of sucrase. 5. Hamster and rat sucrases show a cooperative interaction between homologous sites. Papain solubilization does not make it disappear. 6. The pH-activity curves are different in each species. In some species the percent activation by Na+ depends on the pH. 7. The Kochsaft of small intestine contains low molecular weight substance(s) which decrease the color yield in the glucose-oxidase/peroxidase assay for glucose. [ABSTRACT FROM AUTHOR]

The complexes of poly A and poly U are interesting models for studying the structure of nucleic acids. In the first part of this paper, the thermal stability of these complexes has been studied as a function of both pH and ionic strength in alkaline solution. The thermal stability is measured by the half transition temperatures, or Tm's. There are four different kinds of transitions: Tm2-1 refers to the dissociation of the two-stranded complex, poly (A + U); Tm2-1 to the dissociation of the three-stranded complex, poly (A + 2U) [4]. Tm8-2 is the temperature of half dissociation of poly (A + 2U) into poly (A + U) [1], and Tm2-3 is the temperature of rearrangement of 2 poly (A + U) into poly (A + 2U) [3] (Fig. 1). It is found that Tm2-1, and Tm8-1 decrease as pH increases above neutrality (Fig. 2 and 3), but that Tm2-8 does not vary (Fig. 3). This effect is readily explained if it is assumed that the ionisation of uracil brings a change in the state of the free poly U, but that the complexes themselves are not modified. In the second part of the paper a simple thermodynamic formulation is given which represents satisfactorily the relationship between the four equilibria, as observed at neutral pH (Fig. 1). A more complex treatment is then presented. It includes quantitation of electrostatic interactions, according to the theory of Kotin [18] and of the free enthalpy term corresponding to the organisation (stacking) of poly A. This leads to an excellent representation of the experimental data (Fig. 5). In alkaline solution, one can calculate the free enthalpy change associated with the ionisation of poly U at the Tm. According to our observations, it is likely that this is the only factor responsible for the decrease of Tm. Therefore, we can write that the sum of the free enthalpy of complex formation in the same conditions, but at pH 7, and of the free enthalpy of ionisation is zero. This leads to a determination of ΔG both as a function of temperature and ionic strength: the variation of ΔG with these factors is shown to be in good agreement with our detailed model. However, the absolute values are different, probably because of uncertainties concerning the data for poly A stacking enthalpy and entropy. Apart from providing evaluation of the thermodynamic parameters for double and triple helix formation, our results lead us to emphasize the importance of the thermodynamic state of the unassociated polynucleotides. This may be particularly relevant when one considers the partially complementary secondary structures of RNA's. [ABSTRACT FROM AUTHOR]

In 2000 ml of pooled human male plasma unconjugated 3β, 17β-dihydroxyandrost-5-ene as diacetate was identified. The identification was based on paper chromatography, acetylation followed by thin-layer chromatography and final mass spectrometry, Furthermore aliquot parts of the extract after paper-chromatography were subjected to a protein-binding assay to assure the 17β-hydroxyl configuration. [ABSTRACT FROM AUTHOR]

Debate continues over the physical characteristics and even the existence of arginase isoenzymes. This paper gives additional support for such multiplicity and reports differences in physical characteristics among the various forms. After 2500-5000-fold purification of rat liver arginase, three molecular forms were separated on carboxymethyl-cellulose columns and were purified 2500-5000-fold, 800-1000-fold and 600-1000-fold, respectively. The molecular forms have also been identified by chromatography in the supernatant of tissue extracts. The isolation of these molecular forms by affinity chromatography, using Sepharose-lysine as a competitive inhibitor of arginase, shows only one main form, however. Kinetic studies were done for two of the molecular forms isolated, specifically the activation energy (Ea, the energy of denaturatization (Ed), Km, pH and the effect of divalent cations were determined. Significant differences were found for the Ea between the two molecular forms. The isolated isoenzymes are cationic at pH 5.5 and pH 8.8. However, they show different mobilities in electrophoresis. The molecular weight determination by gel filtration yields a value of 110000 to 115000 for both forms. The use of thin-layer immunochromatography plates, a combination of molecular weight and immunodiffusion technique, gave only one peak with the same molecular weight as that determined by gel filtration. The immunological studies showed that the isoenzymes have similar antigenic determinants. [ABSTRACT FROM AUTHOR]

Semliki-Forest-virus 42-S virion RNA is translated in a mouse ascites cell-free system. One of the major polypeptides made in vitro has been identified as Semliki-Forest-virus capsid protein by co-electrophoresis on dodecylsulfate -- polyacrylamide gels and by tryptic peptide analysis on paper. Another major product of the cell-free system has been characterised as envelope protein by peptide analysis but it does not co-electrophorese on gels with envelope proteins from purified virions. It is possible that the nascent, non-glycosylated polypeptide is partially degraded by proleolytic enzymes present in the cell-free extract, or alternatively that fragments of envelope protein are produced by premature termination on the viral RNA. Several other polypeptides are synthesised in the cell-free system in variable amounts but these have not been identified. The genetic content and the mechanism of translation of Semliki-Forest-virus 42-S RNA are discussed. [ABSTRACT FROM AUTHOR]

Pure mucin isolated from canine submaxillary glands was treated with alkaline borohydride and products of degradation were fractionated by a procedure that included ion-exchange chromatography, gel filtration, high-voltage electrophoresis and paper chromatography. Seven acidic, reduced oligosaccharides were isolated being divided into two distinct types: one, containing sialic acid and no glucosamine or sulfate (type A) and the other containing glucosamine and sulfate but no sialic acid (type B). The most complete oligosaccharide of type A designated component (I) was a tetrasaccharide α-L-fucopyranosyl-(1 → 2)-D-galactopyranosyl-[N-acetylneuraminyl-(2 → 6)]-2-acetamido-2-deoxy-D-galactitol. The most complex oligosaccharide of type B designated component (V) has a hexasaccharide with the following structure: α-L-fucopyranosyl-(1 → 2)-β-D-galactopyranosyl-(1 → 3, 4, or 6)-β-2-acetamido-2-deoxy-D-glucopyranosyl 3 or 4 sulfate-(1 → 6)-[α-L-fucopyranosyl-(1 → 2)]-β-D-galactopyranosyl-(1 → 3) 2-acetamido-2-deoxy-D-galactitol. All the other acidic isolated oligosaccharides were derived from these two main components. No oligosaccharide containing both sialic acid and glucosamine sulfate could be demonstrated. Based on these structural features, a possible biosynthetic pathway leading to the two different oligosaccharide chains is discussed. Variations in ratios of sugars observed in canine submaxillary mucin and especially the reciprocal relationship between sialic acid and fucose noted earlier (1962) by Dische. Pallavicini, Kavasaki, Smirnow, Cizek and Chien may reffect variations in the proportions of these two types of oligosaccharides. [ABSTRACT FROM AUTHOR]

The results presented in this paper establish relationships between structural morphological and functional properties of cytoplasmic membrane vesicles isolated from an Escherichia coli unsaturated fatty acid auxotroph. The membranes were isolated from cells grown in the presence of either oleic, linoleic, linolenic or elaidic acids. High-angle X-ray diffraction studies show that the order-disorder transitions induced by temperature variations and associated with the paraffin chains of the lipids are a function of the fatty acid composition of the membranes. In some cases "cocrystallization" of various lipid species takes place within a single type of ordered domains. In other cases there is segregation of various lipid species into more than one distinct type of ordered domain. The various order-disorder transitions observed induce morphological changes in the hydrophobic core of the membranes which can be detected by freeze-etch electron microscopy. A random distribution of particles on the fracture faces is observed when the paraffin chains of the lipids are disordered. Upon ordering of the paraffin chains, particles are excluded from the ordered domains and as a consequence, smooth areas and areas with densely packed particles are observed. The ratio of the smooth surface to particulated surface is proportional to the amount of ordered paraffin chains present. Moreover, the size of the smooth domains is a function of the fatty acid composition of the membranes. Discontinuities in the rate of D-lactate-dependent proline uptake as a function of temperature correlate with the order-disorder transitions observed. The high energies of activation at low temperatures are attributed to decreased mobility of the carrier proteins upon aggregation. In contrast, phosphoenolpyruvate-dependent vectorial phosphorylation does not respond to the ordering of the paraffin chains. [ABSTRACT FROM AUTHOR]

This paper describes the isolation and the properties of DNA-dependent RNA polymerase from Lactobacillus curvatus. The enzyme is highly labile and shows a tendency to dissociate into fragments. Therefore. a special purification procedure also suitable for the isolation of labile RNA polymerases from other pro-karyotes was developed. Three enzyme species, E (core enzyme). Eσ (full enzyme) and Ey were obtained. The subunit composition corresponds to that of other prokaryote RNA polymerases. In contrast to Echerichia coli enzyme. the Β subunit (Mr = 145000) has a lower molecular weight than the Β subunit (Mr = 1.51000). The Β subunit was identified by its capacity to bind [³H]rifamycin. σ is extremely small (Mr = 44000). y is a peptide chain (Mr = 84000) present once in the Ey monomer. By incomplete dissociation, Β α2, Β'σ and Β'y complexes were obtained. y and 5 have never been found together in the same enzyme particle. In contrast to RNA polymerase from E. coli. Eσ from L. curvatus exhibits optimal activity in the presence of Mn2+ as bivalent metal ion. Co2+ and Mg2+ also activate though with considerably lower efficiency. All subunits except y were isolated in pure state. σ was catalytically active. y could only be obtained as a complex with Β'. On single-stranded DNA. Eσ and E are equally active. For the transcription of double-stranded DNA. (σ is absolutely required. Even the transcription of poly[d(A-T)] · [d(A-T)] is strongly stimulated by σ. σ from L. curvatus is able to replace σ from E. coli on E. coli core enzyme even for the formation of the stable preinitiation complex. This effect requires Mg2+, that is conditions optimal for the E. coli system. Thus, the core and not g appears to determine the requirement for the bivalent metal ion. Different double-stranded templates are transcribed with highly different efficiency. The ionic strength optimum is different for different templates. Ey in contrast to E exhibits a low, though significant background activity on double-stranded DNA. It is stimulated by σ to twice the specific activity of Eσ. Thus, σ and y act synergistically though they appear to exclude each other on the core. [ABSTRACT FROM AUTHOR]

Reports in the literature indicate that the flavin prosthetic group of Chromatium cytochrome c552 is not acid-extractable but is dissociated from the protein by various treatments, including prolonged incubation with urea solution, which are not expected to break covalent bonds. In the present paper it is established by isolation of peptic and of tryptic-chymotryptic flavin peptides that the flavin component is covalently linked to the protein. The peptide chain is attached to the 8α group of the FAD, as in other enzymes containing covalently bound flavin, as evidenced by a hypochromic shift of the near ultraviolet absorption maximum, the characteristics of the hyperfine EPR spectrum, and the release of 8-carboxy-FMN from the cytochrome on oxidation with cold performic acid with concomitant hydrolysis of the pyrophosphate linkage. The fluorescence of the flavin peptides, at the FAD, FMN and riboflavin level, is extensively quenched, with less than 1% of the quantum yield of fluorescence, compared with riboflavin, in the peptic peptide. On oxidation of the tryptic-chymotryptic peptide with performic acid the fluorescence increases to 50% of that given by an equimolar concentration of riboflavin. This increase is accompanied by a further hypochromic shift of the near ultraviolet absorption maximum. This behavior, the tendency of the flavin peptide to undergo autooxidation, and the positive chloroplatinic acid test resemble the properties of cysteinylflavin thioether and its peptides and suggest that the flavin is bonded to a cysteine residue, as in monoamine oxidase. The presence of cysteine in both flavin peptides from cytochrome c552 has been verified by the liberation of cysteine on acid hydrolysis. Despite these similarities to peptides containing a cysteinyl flavin thioether. the peptic and tryptic-chymotryptic peptides from cytochrome c552 show several properties which preclude a thioether linkage. Evidence is summarized to indicate that the flavin is linked via a thiohemiacetal bond to a cysteinyl residue in the polypeptide chain. Thus, the flavin released from the peptides by acid hydrolysis is in every respect identical to 8-formylriboflavin. Further, two flavin components were detected on high voltage electrophoresis after aminopeptidase M digestion of the peptic peptide, which had mobilities expected for the aminoacylflavin and a thiazolidine derivative. A thiohemiacetal structure also ac-counts for the much greater lability of the flavin peptide linkage than that found for monoamine oxidase. Thus from the evidence presented, it is concluded that the structure of the covalently bound flavin in Chromatium cytochrome c552 is 8α-S-cysteinyl-8α-hydroxy-FAD. [ABSTRACT FROM AUTHOR]

The previous paper in this series described the isolation of tryptic-chymotryptic and peptic flavin peptides from Chromatium cytochrome c552 in homogeneous form and presented evidence that the FAD component is covalently linked to a cysteinyl residue via a hemiacetal linkage to the 8α position of the isoalloxazine ring. The amino acid sequence of the peptic and tryptic-chymotryptic flavin peptides were found to be Tyr-Thr-Cys(FAD)-Tyr and Thr-Cys(FAD)-Tyr. respectively. However, the presence of the NH2-tyrosyl residue in the former, imparts markedly greater stability to the cysteinyl flavin linkage, and even results in essentially quenched fluorescence of the flavin after performic acid oxidation of the sulfur moiety. Osidatlon at 40°C, which destroys tyrosine, results in the same level of fluorescence as found when the tryptic-chymotryptic peptide is oxidized at 0 C. Removal of this tyrosyl residue from the peptic peptide, oxidized at 0°C, by aminopeptidase M also leads to an increase in fluorescence. Evidence for a tyrosyl-flavin interaction has been further obtained from CD spectra. The peptic peptide has a broad, positive Cotton effect with a maximum at 484 to 490 nm, which is enhanced after treatment with pyrophosphatase and phosphatase and negative bands at 305 and 375 nm. The tryptic-chymotryptic peptide, on the other hand. has a positive band at 340 nm, as m FAD. but only a small negative band at 380 nm. Thus the tyrosyl-flavin interaction appears to be responsible for the stabilization of the thiohemiacetal bond in the peptide and perhaps also in the protein. [ABSTRACT FROM AUTHOR]

A technique which releases ribosomes from membranes without the use of detergent described in the preceding paper is shown to allow the separation of two distinct pools of poly(A)-containing RNA in microsomes. Thus, using preparations from mouse myeloma tissue-culture cells, essentially 100 % release of immunoglobulin mRNA occurs under conditions in which only 50-60 % of the total microsomal poly(A)-containing RNA is released. The RNA which is not released is principally mitochondrial ribosomal RNA and the method thus provides a good preparative procedure for this fraction. Messenger ribonucleoprotein particles involved in immunoglobulin biosynthesis have been prepared from the released material. The heavy-chain mRNA-containing particle sediments in a neutral sucrose gradient at about 22 S, and that containing the light-chain mRNA at about 18 S. The ribosome-release technique has been used for the rapid preparation of light-chain mRNA in good yield and at over 60 % purity after a single sucrose gradient centrifugation. Further purification has been achieved by polyacrylamide gel electrophoresis. Using [32P]RNA, the contaminants can be estimated directly at the chemical leveI using a fingerprint assay. [ABSTRACT FROM AUTHOR]

The positions of the disulfide bridges in toxin Il of Androctonus australis Hector were investigated using proteolytic enzymes. The resulting peptides were separated by column and paper chromatography and high-voltage electrophoresis. Determination of the amino acid compositions of the cystinecontaining peptides or their oxidized derivatives and, when necessary, dansyl Edman degradation allowed to locate the disulfide bonds in the toxin. The four disulfide bridges were found to link the half-cystine residues number 12 and 63, 16 and 36, 22 and 46, 26 and 48. These positions are probably the same in all scorpion neurotoxins. [ABSTRACT FROM AUTHOR]

A purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

This paper presents several lines of evidence indicating that the synthesis of vitamin-B12-dependent 5-methyltetrahydrofolate-homocysteine transmethylase ("methionine synthetase") is closely correlated to the intracellular level of vitamin B12 in manners different between bacteria which do and do not produce vitamin B12. 1. The intracellular level of vitamin B12 in Rhodospirillum rubrum was reduced to one fifth that of the normal cells when the organism was cultivated in a medium lacking cobalt. In accord with the decrease in vitamin B12 production, the specific activity of methionine synthetase was lowered to about 50%. It was of much interest that the enzyme was always present as the holoenzyme and the apoenzyme could not be detected even in the vitamin-B12-deficient cells containing a markedly decreased level of the holoenzyme. When the vitamin-B12-deficient cells were incubated in a cyano- cobalamin-supplemented medium, the specific activity of methionine synthetase began to increase after 2 h lag time and reached the maximum in about 6 h. This enhancement of the specific activity was suppressed if an appropriate inhibitor of protein synthesis was added to the medium. These facts strongly suggest that synthesis of protein de novo is essential for the enhancement of the specific activity by incubation with cyanocobalamin. Similar results were obtained in other vitamin B12 producers such as Streptomyces olivaceus and Corynebacterium simplex. 2. Chromatium D which is known to form cobalt-free corrinoids but not produce complete vitamin B12, was found to possess methionine synthetase as apoenzyme. When the vitamin-B12-free cells were incubated in the presence of cyanocobalamin, all the apoenzyme was readily converted to the holoenzyme within 2 h. As the formation of the holoenzyme proceeded, the level of the total enzyme (holo + apo) increased to a certain degree. The enhancement, however, was not affected by the inhibitors of protein synthesis, indicating that the increment could not have resulted from de novo synthesis of protein. A similar phenomenon was observed in Escherichia coli which does not produce vitamin B12 but possesses apoprotein of methionine synthetase. It is suggested that vitamin B12 synthesis is closely correlated to that of methionine synthetase in bacteria producing the vitamin, such as Rhodospirllum rubrum, Streptomyces olivaceus and Corynebacterium simplex. On the contrary, the apoprotein of the enzyme is synthesized independently of vitamin B12 in bacteria not producing the vitamin, such as Chromatium D and Escherichia coli. [ABSTRACT FROM AUTHOR]

High-affinity penicillin binding sites from which the antibiotic could not be removed by washings at 4 °C in 0.017 M K2HPO4 or 0.05 M Tris-HCl pH 7.5, were shown to occur in the isolated membranes of Streptomyces R61. These sites caused the attachment of 25 picomoles of [14C]benzylpenicillin per milligram membrane protein. Penicillins and cephalosporins competed for the same binding sites. The antibiotic concentrations which excluded [14C]benzylpenicillin from 50% of the binding sites were those which inhibited by 50% the membrane-bound transpeptidase. The same rate constant (about I × 10-4s-1) for the dissociation of the benzylpenicillin membrane complex at 37 °C and in 0.017 M K2HPO4, was calculated either from the release of the radioactivity (using [14C]benzylpenicillin) or from the recovery of the transpeptidase activity. These observations supported the conclusion that the high-affinity binding sites in the isolated membranes were the transpeptidase molecules. All the complexes formed between the membranes and the various penicillins and cephalosporins examined were reversible at 37 °C and in 0.017 M K2HPO4 at least with regard to the transpeptidase. Depending upon the antibiotics, the rate constants for the dissociation of these complexes varied from 3.3 × 10-3 to 0.73 ×10-4 s-1. The radioactivity released through the dissociation of [14C]benzylpenicillin membrane complex occurred mainly in the form of a compound which behaved as [14C]-benzylpenicilloic acid both by paper electrophoresis and thin-layer chromatography. It was impossible to choose between several possible mechanisms for the release of the antibiotic molecule. [ABSTRACT FROM AUTHOR]

1. A β-glucosidase (cellobiase) has been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus Trichoderma viride. 2. The crude material was pre-fractionated by chromatography on Bio-Gel P-10 and by DEAE-Sephadex chromatography as previously described [1]. Further fractionation methods utilized in the present paper included ammonium sulfate precipitation. SE-Sephadex chromatography, isoelectric focusing, and chromatography on Bio-Gel P-60. 3. A yield of 53 mg enzyme was obtained per 100 g commercial cellulase. The specific activity of the purified enzyme represented an approximately 60-fold increase from that of the commercial product. 4. The isolated enzyme was homogeneous in polyacrylamide gel electrophoresis at pH 8.0 by isoelectric focusing in a polyacrylamide gel and also in free zone electrophoresis. 5. No enzyme activity towards Avicel (mieroerystalline cellulose) or towards carboxymethylcellulose could be detected in the purified material under the assay conditions used. 6. A molecular weight of 47 000 was determined for the enzyme by dodecylsulfate-polyacrylamide gel electrophoresis. The same molecular weight value was obtained by chromatography of the native enzyme on Bio-Gel P-100 and also by chromatography of the reduced and alkylated enzyme on Sepharose 6B in 6 M guanidine-HCl. 7. The amino-acid analysis showed that the enzyme was rich in aromatic (9.5%) and acidic (19.0%) amino acids. The half-cystine content was 6 residues per molecule indicating 3 disulfide bridges, since no free sulfhydryl group was detectable. The purified enzyme contained no carbohydrate (as hexose). 8. The enzyme was isoelectric at pH 5.74 (10 °C) and the electrophoretic mobility was determined to be 1.49 × 10-5 cm² s-1 V-1 at pH 8.0. 9. The Km-values for the enzyme towards the substrates p-nitrophenyl-β-D-glucoside, cellobiose, and cellotetraose were found to be 0.28, 1.5, and 0.35 mM, respectively. [ABSTRACT FROM AUTHOR]

I. This paper describes some features of the protein synthesis in isolated rat liver mitochondria and reports on the stability of the product of mitochondrial protein synthesis in vitro. 2. Chase experiments performed after a 30-min pulse of [14C]leucine indicated that a large part of the product is degraded to an acid-soluble form (labile product) almost as rapidly as it is terminated and released from the mitoribosome. Only a small proportion of the completed product (between 20 and 40 %)is conversed in acid-insoluble form (stable product). 2. Both labile and stable products have an exceptional degree of hydrophobicity, shown by their marked resistance to acid and alkaline hydrolysis and by their solubility in organic solvents. 4. A 14C-labelled peptide with an apparent molecular weight of 12000-13000, as assessed by gel electrophoresis in the presence of dodecylsulfate, is released after heat treatment of mitoribosomes isolated from mitochondria pulsed with [14C]leucine. [ABSTRACT FROM AUTHOR]

The primary structure of sheep para-ℵA-casein, the N-terminal portion of ℵ-casein, was established. The reduced alkylated protein was subjected to digestion with trypsin. The resulting soluble peptides were purified by a combination of Dowex I X 2 chromatography and electrophoresis and chromatography on paper; the core peptides were solubilized in 50 % formic acid and filtered on Sephadex G-50. The amino-acid sequence of these peptides was determined chiefly with a Sequencer. Alignment of thc tryptic peptides into a single chain containing 105 amino acids was determined, from basic overlap peptides (tryptic core peptides with several basic amino acids; chymotryptic peptides). Some comparisons were achieved with cow para-ℵA-casein. [ABSTRACT FROM AUTHOR]

The isolated α-chain of sea anemone collagen was cleaved at methionyl residues with cyanogen bromide. Eleven unique peptides were isolated by ion-exchange and molecular-sieve chromatography, eight of them in approximately equimolar amounts. Characterization of the eleven peptides with regard to amino-acid composition and molecular weight demonstrated that the isolated peptides account for all of the amino acids and for the molecular weight of the α-chain within experimental error. Since the molecule of sea anemone collagen contains ten methionyl residues, the data of the present paper confirm earlier analytical and preparative results that this invertebrate collagen is made up of three identical α-chains. In comparison with known collagens, on the basis of quantitative and qualitative features of amino-acid compositions, the cyanogen bromide peptides of sea anemone collagen indicate genetical relationship of sea anemone collagen to basement membrane collagen. [ABSTRACT FROM AUTHOR]

In this paper we demonstrate that two compounds which are known to enhance the permeability of membranes to potassium, valinomycin, a cyclic dodecapeptide and dicyclohexyl-18-crown-6, a macrocyclic polyether, inhibit protein synthesis in reticulocytes and that this inhibition is not primarily due to changes in the intracellular potassium concentration. No effect is obtained by adding the drugs to a cell free system and the inhibition observed is likely to be membrane mediated. Dicyclohexyl-18-crown-6 and low concentration of valinomycin (10 nM) act reversibly probably by interacting with the lipid phase of the membrane. This interaction in turn affects the protein synthesizing apparatus by affecting ribosomal functions. Ribosomes once isolated from treated cells are perfectly normal in structural and functional respects. High concentrations of valinomycin, which change the K+ gradient between the inside and the outside of the cell, affect the protein synthesizing apparatus of reticulocytes irreversibly by blocking incorporation of amino acids at the level of elongation or termination. Ribosomes isolated from cells treated with high valinomycin concentrations are mostly in the form of polysomes which are inactive in cell free protein synthesis. The high valinomycin block on the ribosomal fraction can be relieved either by washing ribosomes with high salt or with puromycin. It is believed that with high concentrations of valinomycin an inhibitor is released (probably from the membrane) and attached to the ribosomes. This high valinomycin effect is believed to by a supplementary phenomenon which when added to the effect of low valinomycin renders the inactivation of ribosomal functions irreversible.

1. New flavins have been isolated from purified prepartions of an electron-transferring flavo-protein (ETF) from Peptostreptococcus elsdenii and glycolate oxidase from pig liver. The structures of these new species have been established as FAD and FMN derivatives of 6-hydroxy-7,8-dimethyl-isoalloxazine, the chemical synthesis of which is described in tlhe accompanying paper by Schöllnhammer and Hemmerich. The chromophores are yellow at pH 5 and green at pH 9 due to an ionization at pK 7.1. 2. 6-OH-FAD is bound by apo-ETF and its pK is decreased. The complex is reduced by NADH and it couples the oxidation of NADH to the reduction of dichlorophenolindophenol. Unlike the complex of FAD and apo-ETF, 6-OH-FAD · ETF does not couple the oxidation of NADH to the reduction of butyryl-CoA dehydrogenase. 3. 6-OH-FMN is bound by the FMN-specific protein apoflavodoxin from P. elsdenii and the pK is increased to ≈9. This complex is reduced by sodium dithionite and an intermediate, presumed to be the semi-quinone, is formed at half rduction. [ABSTRACT FROM AUTHOR]

1. The method of "photohydroxylation" of azaheteroaromatics, known in the phenazine and a alloxazine series, has been applied to flavin (= isoalloxazine) derivatives FI[SUBox]H. The products habe been identified as 6- and 9-hydroxyflavins (Fl[SUBox]-6/9-OH). On this basis it was possible to assign a 6-hydroxyflavin structure to the new flavocoenzyme chromophores described by Mayhew et al. in the accompanying paper. 2. In agreement with the data obtained by Dekker et al. in a recent laser study on alloxazine, we find that, the reaction proceeds via, addition of water, alcohols or carboxylic acids by the lowest excited singlet state of flavin with synchronous intramolecular hydrogen shift from the site of RO-addition [= C(6) or C(9)] towards N(5) and subsequent oxidation of the 6/9-hydroxy1.5-dihydroflavin formed. 3. The photoaddition reaction occurs in competition with photodehydrogenation of available CH-bonds by the lowest excited flavin triplet, in which case the CH-substrate may be provided by the solvent ROH or, when R = H, by methyl groups of a second flavin molecule (= flavin autophotolysis). This photodehydrogenation may be suppressed by O[SUB2]-dependent triplet quenching and/or by applying a positive charge to the flavin (RFl[SUBox]H[SUP+], R = H or alkyl) in order to inhibit the reaction with itself. 4. Differentiation of 6- and 9-hydroxyflavin isomers was possible by double resonance evaluation of the proton coupling between the residual Ar-H and the adjacent methyl group, by evaluation of the electron paramagnetic resonance hyperfine coupling for the residual Ar-H in the radical cations 1,5-RHFl-6/9-OH, and by Formation of cupric chelates from 6-hydroxy-flavin as compared to the lack of metal affinity in the 9-isomer. [SUP1]H-Nuclear magnetic resonance, electron paramagnetic resonance and visible spectra and their dependence on the state of ionisation of hydroxyflavins are discussed. Hydroxyflavin anions as well as their N(1)-alkylated... [ABSTRACT FROM AUTHOR]

The isolation and amino-acid and carbohydrate composition of three tryptic glycopeptides, containing 16, 17 and 20 amino-acid residues, respectively, from the neuraminidase-treated major form of human ceruloplasmin have been described earlier. It has also been suggested that the peptides possibly represent all of the carbohydrate-binding sites in the protein. The present paper describes the amino-acid sequences of these peptides as obtained by stepwise Edman degradation and carboxypeptidase digestions of the whole peptides and their fragments after cleavage by chymotrypsin, pepsin and thermolysin. The results confirm that the carbohydrate in all cases is attached via an asparagine (or aspartic acid) residue, that is followed by an X-Thr/Ser sequence. No sequence homologies are found when the peptides are compared to each other or to other glycopeptides of known sequence. [ABSTRACT FROM AUTHOR]

Purification of a lysozyme-digest resulted in the isolation of two main regions of the molecule, namely sequence (57-107) denoted loop fragment (see the accompanying paper), and sequence (1-27) linked to sequence (122-129) through disulfide bridge I-VIII (Jolles et al., 1963), denoted NC-peptide. Inhibition of lysozyme-anti-lysozyme precipitation showed that NC-peptide reacted with about 10% of all goat anti-lysozyme antibodies. It was suggested that these antibodies react with a conformation-dependent determinant including tyrosine 20 and 30. In water or in aqueous salt solution, NC-fragment retained about 20% of its "native" alpha-helical amount (i.e. the amount of helicity this fragment possesses when integrated in the lysozyme-molecule). Reduction and alkylation of the I-VIII disulfide bridge abolished most of the spatial conformation, as was obvious from the circular dichroism curve isolated N-peptide. Long-range interactions between the N and C terminal parts of the molecule, therefore, seem to stabilize the native lysozyme molecule. [ABSTRACT FROM AUTHOR]

1. Adenylate kinase (ATP: AMP phosphotransferase) has been purified 490-fold from porcine muscle with a final yield of 60 mg/kg muscle. 2. The amino-acid composition is Asp11 Asn2, Thr14, Ser11, Glu19,, Gln6, Pro6, Gly19>, Ala6, Cyst2, Val17, Met6, Ile9, Leu18, Tyr7, Phe5, Lys21, His2, Arrg11. 3. The protein molecules a single polypeptide chain of 194 amino-acid residues with an acetylmethionine at the N-terminus and a lysine residue at the C-terminus. 4. Cyanogen bromide cleavage of carboxymethylated adenylate kinase yielded six fragments which were further degraded by using trypsin, chymotrypsin, thermolysin, subtilisin or α-protease. Sequence data on the resulting peptides are summarized in the present report, full details are given in a supplementary paper which has been deposited at CNRS from where copies can be obtained. 5. The primary structure of porcine adenylate kinase was given. [ABSTRACT FROM AUTHOR]

The neurohypophysial hormones of a chondrostean, the sturgeon (Acipenser sp.), have been purified by adsorption onto neurophysin, dissociation of the complex hormone-protein by trichloroacetic acid precipitation and isolation of active peptides, from the supernatant solution, by paper chromatoelectrophoresis. Arginine vasotocin has been characterized by amino acid composition, chromatographic and electrophoretic migrations and pharmacological properties as well. The amount of arginine vasotocin (about 50 nmol per g pituitary powder) is intermediary between those found for bony fishes (about 1000 nmol/g) and cartilaginous fishes (about 5 nmol/g). A second hormone, which can be classified in the oxytocin-like type by its electrophoretic migration and its pharmacological properties, has been disclosed. The very weak amount did not allow chemical identification. However the chromatographic behaviour and the pharmacological "profile" indicate that this hormone differs from the six known oxytocin-like peptides. [ABSTRACT FROM AUTHOR]

Exogenously supplied thymine is only poorly utilized by wild-type cells of Escherichia coli for the synthesis of their DNA. It appears that the lack of incorporation of exogenous thymine is due to a lack of endogenous deoxyribosyl groups, which are required for the synthesis of thymidine. Data to be presented in this paper indicate that the ability of different thymine auxotrophs to grow on progressively lower thymine concentrations is a function of their capacity to increase the internal pool of deoxyribose 1-phosphate and/or the level of thymidine phosphorylase. Thymine incorporation in wild-type cells could be observed if the cells possessed an increased deoxyribose 1-phosphate pool. The kinetics of thymine uptake in wild-type cells, in a mutant lacking thymidine phosphorylase and in thymine auxotrophs show that the initial rates of thymine uptake are almost identical and proportional to the external thymine concentration. The experiments in vivo led us to conclude that the incorporation of exogenous thymine occurs via thymidine, which is synthesized from thymine and deoxyribose 1-phosphate, catalyzed by thymidine phosphorylase. In accordance with this studies in vitro with purified thymidine phosphorylase showed that th3Tnidine is synthesized from deoxyribose 1-phosphate and thymine rather than through a deoxyribose transfer from a deoxynucleoside. [ABSTRACT FROM AUTHOR]