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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]

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]

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]

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]

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]

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]

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]

The ethanolic extract of 8-week-old parsley plants was chromatographed on Dowex-1×2 with a concave gradient using the buffer system of Hurlbert. After further purification on carboncelite columns, paper chromatography and high-voltage electrophoresis, 120 μmoles of a homogeneous UDP­sugar fraction were obtained. The sugars present in this fraction were identified after acid or enzymatic hydrolysis (Crotalus adamanteus venom, acid phosphatase) by various chemical and enzymatic tests. A modified Dische test which is specific for apiose and a quantitative deterruination of D­xylose with D­glucose—oxidase were developed. According to the analytical investigations the UDP­sugar fraction had the following composition (μmol): UDP-D-glucose (84), UDP-D-galactose (27), UDP-D-xylose (4), UDP-L-arabinose (2), UDP­fructose (0.42), UDP-L-rhamnose (2.4), and UDP­apiose (0.18). The presence of UDP­apiose in parsley strongly corroborates the proposed biosynthetic pathway for the formation of D-apiose via DDP-D-glucuronic acid. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]

For the elucidation of amino acid sequence around the reactive site of soybean trypsin inhibitor (Kunitz), fragments A and B were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further purification of the peptides was carried out by gel filtration on Sephadex G-25 or by high-voltage paper electrophoresis at pH 3.6 and 6.5. Nine peptides were obtained in pure form from fragment A and three peptides from fragment B, and their amino acid sequences were determined by the direct Edman method and by carboxy-peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptie peptides from fragments A and B were obtained from a chymotryptic hydrolysate of fragment AB by gel filtration on Bio-Gel P-4, and their ammo acid compositions and sequence analyses of some peptides made it possible to establish the amino acid sequence of an amino-terminal region of the inhibitor consisting of AB amino acid residues. The reactive site (Arg63-Ile64) of the inhibitor to trypsin was involved in this region. [ABSTRACT FROM AUTHOR]

Presents an investigation of the sequencing of 65 amino acid residues at the carboxyl-terminus. Use of methods such as enzymic or chemical cleavages of the peptide chain; Fractionation of peptides by Sephadex or Dowex column chromatography, paper elctrophoresis or chromatography.

Our previous studies [1,2] on maleyl bovine αS1-B casein were concerned with the isolation and amino acid composition of the tryptic peptides designated as Tm peptides. The sequence of the COOH-terminal Tm peptide containing 48 residues was also reported. In the present study, we report the overlaps of all the cyanogen bromide peptides as well as the Tm peptides from αs1-B casein. The αs1-B casein which contains five methionyl residues was cleaved with cyanogen bromide. The six expected peptides obtained by this treatment and designated as CN peptides, were separated initially on Dowex-50X2 and further purified by Sephadex column chromatography. The amino acid composition of these six purified CN peptides was determined. The results were in perfect agreement with the amino acid composition of αs1-B casein deduced from the analysis of Tm peptides. The three Tm peptides from αs1-B casek@ which contained methionyl residues were also cleaved by cyanogen bromide. The fragments obtained were purified by either paper chromatography or Sephadex column chromatography and the amino acid composition of these purified peptides was determined. Of the eight peptides thus obtained, three were found identical with the CN peptides obtained directly by BrCN treatment of the αs1-B ease@. The remaining three CN peptides from αs1-B casein for which identical fragments could not be located fun the Tm peptides, were digested with trypsin (in two eases after maleylation) and the resulting fragments were purified by Sephadex column chromatography. These trifle peptides provided the remaining gaps. This permitted in to locate the relative position of all the CN peptides in the αs1-B casein molecule. These results also indicate the location of all the Tm peptides except 2 in the αs1-B casein molecule. Further, the chymotryptic peptides from one of the CN fragments of αs1-B casein provided the missing overlaps. [ABSTRACT FROM AUTHOR]

In order to determine the primary structure of the bovine β-caseins, the A2 variant was first cleaved with trypsin and cyanogen bromide (CNBr). The tryptic hydrolyzate was separated by column chromatography on Dowex-50. After additional purifications, 13 peptides numbered Ti to T13 were isolated. The 14th tryptic peptide, T14, not eluted from the column, was directly obtained from the hydrolyzate by preparative paper electrophoresis. Amino-acid analyses were carried out on all these peptides. Peptide T1 contain 2 Arginyl residues, one of which is NH2-terminal. This peptide represents the NH2-terminal end of the β-casein. Its composition is identical to that reposed by Peterson et al. in 1958 [1] for a phosphopeptide isolated from the β-casein. Peptide T11 contains 2 Lysyl residues, one of which is NH2terminal. The COOH-terminal peptide was identified with peptide T4, devoid of basic aminoacids. The 14 tryptic peptides account for all the residues of the β-casein A2 when compared with the already published amino-acid analyses of the whole protein [2, 3]. Several other peptides, originating from incomplete or non-specific cleavages, were also obtained. Similarly, 7 peptides accounting for the 6 methionyl residues of the protein were obtained from the same variant of β-casein after cleavage with cyanogen bromide. Six of these peptides were separated on Sephadex G-50. The seventh peptide was obtained by chromatography on Dowex-50 of the whole CNBr-digest. After additional purifications, the ammo-acid compositions of the 7 pure peptides were determined. Peptide CN1 contains peptide T1. It represents the NH2terminal end of β-casein. The COOH-terminal end of β-casein was identified with peptide CN7. As in the case of the tryptic peptides, the 7 CNBr-peptides account for all of the amino-acid residues of β-casein A2. β-casein A2 has 208 amino-acid residues. Its molecular weight is very close to 24 000. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharide of the gram-negative bacterium Rhodopseudomonas capsulata (Athiorhodaceae) a lipophilic sugar was isolated by chromatographic procedures and characterized by mass spectrometry as belonging to the group of 3-O-methyl-6-deoxy-hexoses. Demethylation showed that the parental sugar had the same properties in borate electrophoresis as rhamnose. Comparison with an authentic sample of 3-O-methyl-rhamnose in gas liquid chromatography, paper chromatography and borate electrophoresis showed that the sugar under examination is 3-O-methylrhamnose (acofriose). Investigation of its optical rotation indicates that the sugar has the L-configuration. L-Acofriose (3-O-methyl-L-rhamnose) has not hitherto been found in lipopolysaccharides of gram-negative bacteria. [ABSTRACT FROM AUTHOR]