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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]

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]

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]

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]

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 NADPH-cytochrome c reductase (called Fraction II) has been solubilized with Triton X-100 from porcine liver microsomes and purified. 1. Fraction II can reconstitute with a cytochrome P-450 fraction (called Fraction I) solubilized with Triton X-100 from porcine kidney cortex microsomes to exhibit laurate ω-hydroxylation activity. 2. When the purified Fraction II is subjected to disc electrophoresis on polyacrylamide gel in the presence of 0.1% Triton X-100, it shows a single hand. In contrast, on electrophoresis in the absence of Triton X-100, a polydisperse state is obtained. Fraction II is estimated to have a molecular weight of about 400000 by gel-filtration with detergent, and it does not contain iron. 3. Either gel filtration on a Sephadex G-100 column or treatment with trypsin at 4 °C results in a conversion of Fraction II to a form, which migrates at a much faster rate on disc gel electrophoresis than dose Fraction II. This fast-moving form (called Fraction IIf) is indistinguishable from either steapsin or trypsin-solubilized NADPH-cytochrome c reductase, judging from several criteria including electrophoretic and sedimentation behaviors, and molecular size. 4. Fraction IIf is reconstitutively inactive in the hydroxylation, unless supplemented with spinach ferredoxin-NADP reductase. Incubation of Fraction IIf with trypsin at 30 °C results in a remarkable enhancement in the reconstitution activity in the presence of ferredoxin-NADP reductase. The results in this paper along with an earlier report [11] suggest that Fraction II may contain two different electron carrier components, possibly participating in the electron transfer from NADPH to cytochrome P-450. [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]

The mycelium of Neurospora crassa was cultivated in a minimal liquid medium in surface culture. The moist mycelium was lyophilized, powdered, and eluted using 0,14 M sodium chloride solution for extracting the proteins. The enrichment of these proteins was carried out by means of ultrafiltration (vacuum and pressure filtration). The extracted proteins of N. crassa were characterized by paper-, agar gel- and immuno­electrophoresis. The antisera were obtained by immunising rabbits for 21 weeks. By means of agar gel electrophoresis 5 zones of proteins were separated in N. crassa 3a6A; by means of immunoelectrophoresis 17 bands resulted from the same protein mixture. The electrophoretic mobilities of the precipitation arcs were calculated. The immunoelectrophoretic patterns of N. crassa E 5256 obtained by N. crassa 3a6A antiserum nearly agreed with the bands of N. crassa 3a6A antigen. However there were no corresponding results in the patterns obtained from N. crassa 46001-1-1a and N. crassa 6994a with the same antiserum. It may be assumed that these differences depend on the different genetic enzyme patterns of the wild and mutant strains; they are also responsible for the differences in the rates of synthesis depending on the duration of cultivation. [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

Peptide B, the N-terminal fragment of thioredoxin obtained by cyanogens bromide treatment was aminoethylated and digested with pepsin. Eight peptides were separated by high voltage paper electrophoresis and chromatography and characterized with respect to amino acid sequence. The results account for all 37 residues peptide B. The N-terminal sequence of peptide B was determined as: Ser-Asp-Lys-Ile-Ile-His-Leu-Thr-Asp-Asp-Ser-Phe. [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]

α-Lactalbumin was isolated from human milk in a yield of 1.8 mg/ml of milk. The purification procedure involved ammonium sulphate fractionation (30% to 80% saturation) and pH-4.0 precipitation, followed by gel filtration with Sephadex G-100. A final purification stage using DEAE-cellulose was necessary in some preparations. Peptides derived from the reduced, S-aminoethylated protein by treatment with cyanogen bromide and digestion of these CNBr fragments with trypain, chymotrypsin or thermolysin, were purified by gel filtration, ion exchange chromatography and high-voltage paper electrophoresis. From the sequences of these peptides it has proved possible to deduce unambiguously the complete primary structure of the protein. Comparison with bovine α-laotaIbumin shows an identity in 72% of the residues with a further 6% being chemically similar amino acids. The corresponding figures for the human α-Iactalbumin/human lysozyme comparison, are 39% and 12%, respectively. The significance of some of the amino acid replacements is discussed. [ABSTRACT FROM AUTHOR]

Low molecular weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudontoytas aeruginosa were isolated from the fraction containing the partially degraded polysaceharide, by successive chromatography on columns of Sephadex G-75 and G-10, followed as necessary) by preparative high voltage paper electrophoresis The major components identified were 2-keto-3-deoxyoctonic acid, basic amino acids (free and bound), inorganic orthophosphate, ethanolamine phosphate and ethanolamine pyrophosphate. Ethanolamine pyrophosphate has not previously been found in acid hydrolysates of lipopolysaceharides. Ethanolamine phosphate and some of the orthophosphate were apparently produced by breakdown of ethanolamine pyrophosphate [ABSTRACT FROM AUTHOR]

An immunoglobulin M from Waldenström's disease (IgM Ga) was hydrolysed by papain and a glycopeptide fraction was isolated by gel filtration on Sephadex G-100. This fraction was then successively resolved into fourteen glycopeptides by gel filtration on Sephadex G-50 and by preparative paper electrophoresis at pH 1.9. The amino acid and carbohydrate content of these glycopeptides was determined by ionexchange chromatography in an autoanalyzer. All of them contain aspartic acid and serine, but they are quite different from one another, in amino acid composition and sequence about, the carbohydrate-peptide linkage, especially with respect to histidine, tyrosine, threonine, valine, glutamic acid, lysine and proline. Furthermore, a number of glycopeptides contain only mannose and glucosamine, while others contain in addition, galactose, fucose, and N-acetylneuraminic acid, in variable amounts. These results suggest the presence of several linkage sites of the oligosaccharide chains on the H-chain and the true heterogeneity of these oligosaccharide chains. The peptide sequence of a few glyeopeptides was reported, especially the following: r-Asn(carbohydrates)-Ser-Thr, also present, in the Fc fragment of Immunoglobulin G. [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]