Search

Several electrophoretic and chromatographic systems are described for the separation of di- and tripeptides with N-terminal methionine. The most effective methods of separation are electrophoresis in barbital-triethylamine-acetic acid, pH 8.0 and chromatography in n -butanol-acetic acid-water, 18:2:5. Increased resolution may be obtained by using two or more systems in combination. These methods were intended to separate and identify the oligopeptides formed during the initiation of eukaryotic protein synthesis, but may also be applied to the separation of small peptides generally.

THE importance of the amino-acids and oligopeptides and the complexity of their mixtures often encountered stimulate a demand for more powerful analytical tools to complement those already available. Paper electrophoresis does not show satisfactory resolving power, particularly in the separation of the neutral amino-acids, at the low potential gradients (5–10 V./cm.) usually applied. Its value is greatly enhanced, however, by the application of high potential gradients, most likely because the great reduction in time required for the molecules to traverse a given distance minimizes the detrimental effects of diffusion and moisture changes in the paper strip. Technical difficulties arising from increased heat generation can be overcome by modification of the design of basic equipment and by efficient cooling.

Forty-six analogues of angiotensin II were obtained with the solid-phase method for peptide synthesis. The peptides were purified, using the conventional procedures; homogeneity and purity were established after paper, thin-layer chromatography, paper electrophoresis, amino acid analysis, elemental analysis, and enzymatic degradation by aminopeptidase. The biological activity of all compounds was compared with that of angiotensin II on the blood pressure of anesthetized rats. The same test was used to establish the antagonistic effect of several analogues against angiotensin II.

Summary Porcine pepsin and penicillopepsin catalyze transpeptidation reactions which involve the transfer in high yield of N-terminal leucine from Leu-Tyr-Leu, and Leu-Tyr amide to intact substrate with the subsequent release of Leu-Leu. With the latter substrate porcine pepsin also forms Leu-Leu-Leu. With penicillopepsin small amounts of Leu-Leu-Tyr-Leu and Leu-Leu-Tyr amide resp. were obtained as intermediates from Leu-Tyr-Leu and Leu-Tyr amide. No radioactivity was incorporated into Leu-Leu when Leu-Tyr-Leu and [14C]-leucine (1:6) were incubated with the enzymes. It was concluded that the transpeptidation proceeds via covalent acyl intermediate.

We have recently shown that the monomeric subunit of mesodiaminopimelic acid containing bacterial peptidoglycans, i. e. the disaccharide tetrapeptide N-acetyl glucosaminyl-N-acetyl muramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine can replace whole killed mycobacteria in Freund's adjuvant. We now report further structural simplifications of the active molecule; natural N-acetyl-muramyl-tripeptides have been found active; the smallest adjuvant molecule found is a synthetic N-acetyl muramyl-dipeptide prepared by Sinay et al. (1).

The major protein component, Band 15, of the chromatophores of Rhodopseudomonas spheroides is associated with most of the pigments and phospholipids. The primary structure of Band 15 has been further characterized. Cyanogen bromide cleavage produced 3 oligopeptides which were present in equimolar amounts. The sum of the molecular weights of the oligopeptides derived from cyanogen bromide cleavage of Band 15 was 8600. This value compares favorably with the value of 11000 calculated from the methionine content of the protein. A C-terminal sequence, NH2…Tyr-Ser-Glu-Glu-(Leu,Ala,Ala,Val,Val,Ala,Ala)-GlyCOOH, is proposed. A tryptic map of the protein has been obtained and the amino acid composition of each tryptic peptide determined.

Since most of the protein cross-linking reagents in use are strongly hydrophobic, their length cannot be increased beyond approximately 20 A between the protein-reactive groups, before denaturation of most proteins becomes noticeable at already a very few cross-links per molecule. The synthesis of longer reagents, coupling to lysine or cysteine side chains, and containing strongly hydrophilic oligoproline chains, is described. As they bear an azodye, linking the oligoproline parts, the cross-links effected are amenable to a mild cleavage by reduction with dithionite. A trifunctional reagent was constructed by reacting trimesinic acid chloride with β-alanine ethyl ester; the carboxyl groups of this amino acid could then be activated for protein cross-linking by reactions leading to the hydrazide, and azides. To compare the new reagents with the compounds in use at present, they were tested out on hemoglobin. The amount of reagent molecules coupled to the protein, and the fractions bifunctionally attached, as well as interchain linking were determined. The “long” reagents reached a distinctly higher efficiency in interchain cross-linking in this system, while showing smaller denaturing effects upon the protein. Thus, more than 11 reagent residues could be coupled to the hemoglobin tetramers without changes in its spectrum indicating denaturation of the heme environment, while shorter and more hydrophobic reagents had permitted the attachment of not more than four to six crosslinks.

1. A low molecular weight inhibitor of maize DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) has been purified to chromatographic homogeneity from a high-speed supernatant fraction which also contains the soluble enzyme. 2. Chemical characterization of the inhibitor has revealed the presence of an aromatic nucleus which apparently contains an attached —COOH group and one or more sulfur-containing side chains. 3. Studies using known inhibitors of RNA polymerase for comparative purposes demonstrated that the maize inhibitor acts much the same as α-amanitin in its ability to interrupt polymerization. Initiation by maize RNA polymerase was greatly enhanced in the presence of the maize inhibitor. 4. On the basis of resistance to rifamycins and inhibition by α-aminitin, the soluble maize RNA polymerase was shown to be equivalent to the nucleoplasmic RNA polymerase II of animals.

The trichloroacetic acid-soluble fragments that are released during initial stages of attack of plasmin upon fibrinogen consist in large part of N-terminal segments from the βB chain. The β B fragments could be distinguished from others because they release fibrinopeptide B when treated with thrombin. Release of the β B fragments was partially preceded by faster release of only one other trichloroacetic acid-soluble fragment and followed by slower release of at least three and subsequently many others. No loss of coagulability of the fibrinogen occurred until the overall reaction with plasmin had progressed well beyond the stage of β B fragment release. Fibrinopeptide B itself was not cleaved from the fragments by plasmin until a late stage in the overall reaction. As could be predicted from studies of Iwanaga, S., Wallen, P., Grondahl, N., Henschen, A., and Blombach, B. [(1967) Biochim. Biophys. Acta 147, 606–609] fibrinopeptide A was fully retained as part of the trichloroacetic acid-precipitable hulk of the degraded fibrinogen up to the terminal stage. Gel electrophoresis showed that the large C-terminal fragment of the αA chain which others had described as initial product was released over the same period taken for release of the β B fragments. The rapid loss of B due to release of the β B fragments, and the retention of A contrasted sharply with the converse order in which fibrinopeptides are released from fibrinogen by thrombin. Full content of fibrinopeptide B in some preparations of cryoprofibrin indicated formation of this fibrinogen-fibrin complex is not critically dependent upon degradation by plasmin.

— The formation of N-acetyl-aspartyl-peptides in mouse brain homogenates is described. The formation is completely dependent on histamine and on an ATP-regenerating system, and partially dependent on the addition of N-acetyl-aspartate and certain other amino acids. The N-acetyl-aspartyl-peptide formation is probably non-ribosomal and is apparently not due to hydrolysis of preformed proteins.

A goitrogenic substance in soya beans has been further purified and characterized. It is a compound of low molecular weight, probably an oligopeptide composed of two or three amino acids or a glycopeptide consisting of one or two amino acids and a sugar. It inhibits iodine uptake by the thyroid in vivo and in vitro and decreases its organisation by the gland. Thyroid glands exposed in vitro to the agent incorporate relatively less radioiodine into diiodotyrosine than controls. The ratio of radioiodinated triiodothyronine to thyroxine is increased in these glands. J. Nutr. 103: 378-383, 1973.

The substrate specificity of hog plasma converting enzyme which was free of a second dipeptidylcarboxypeptidase activity was studied by employing a number of peptide analogues related to the C-terminus of the decapeptide substrate, angiotensin I. Tripeptides, even when related to angiotensin I such as Phe-His-Leu, are not substrates. N-terminal-blocked tripeptides and a tetrapeptide, Pro-Phe-His-Leu, are fair to good substrates at high substrate concentrations. Five substrates, the tripeptide to the heptapeptide, derived from the C-terminus of angiotensin I were studied in detail. The Michaelis-Menten constants and maximum velocities were measured. The second-order rate constants which are proportional to the percentage rate of hydrolysis of the substrate when [S] ⪡ Km were tabulated for the enzyme reaction. There is little specificity in V. However, there is specificity in Km but it develops slowly as the length of the amino acid sequence of the polypeptide increases from the C-terminus and approaches angiotensin I.

Es wird uber die Strukturklarung eines neuen pyroglutaminsaurehaltigen Peptids aus dem Gift vonAgkistrodon halys blomhoffii berichtet.

Tetradecapeptide renin substrate (H∙Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser∙OH) (1–14), tridecapeptide (1–13), dodecapeptide (1–12), undecapeptide (1–11), two nonapeptides (1–9) and (1–9-Leu), heptapeptide (1–7), tetrapeptide (1–4), tetrapeptide (5–8), pentapeptide (4–8), hexapeptide (3–8), and heptapeptide (2–8) were synthesized by the solid phase method and by using an improved reaction vessel. The yield averaged 50–70%. Homogeneity and purity of peptides were established with elemental anlysis for C, H, and N, paper chromatography in three solvent systems, electrophoresis, amino acid analysis, and enzymatic degradation by aminopeptidase M.

Two D -alanine carboxypeptidases have been found to be present in protoplast membranes from Bacillus stearothermophilus . D -Alanine carboxypeptidase I removes the terminal D -alanine residue from the substrate UDP-MurNAc- L -Ala- D -Glu- meso -Dap- D -[ 14 C]Ala- D -[ 14 C]Ala; (MurNAc, N -acetylmuramyl; Dap, α, e-diaminopimelic acid); D -alanine carboxypeptidase II removes the penultimate D -alanine residue. Both enzymes have been obtained in a soluble form by treatment of protoplast membranes with butan-1-ol at 20 °C. The properties of D -alanine carboxypeptidase I in the membrane-bound state have been studied. The enzyme is inactivated by penicillin G but the inactivation can be reversed by treatment with β-lactamase. Inhibition of enzyme by penicillin G shows hyperbolic competitive kinetics. Penicillin G does not appear to act as a substrate analogue and compete for the entire active site of the enzyme. Under certain conditions the penicillin sensitivity of D -alanine carboxypeptidase I is lost without concomitant loss of catalytic activity. It is suggested that the enzyme has a penicillin-binding site that is separate from the substrate-binding site, a suggestion which is strongly supported by kinetic evidence.

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a whole range of amino acids, peptides and structurally related amino compounds were tested for acceptor function. No compound tested was an acceptor for the enzyme from strain albus G whereas the enzymes from strains R61 and K11 could utilize with varying efficiency a wide range of substances including peptides with N-terminal glycine or d-alanine, ω-amino acids, aminohexuronic acids, 6-aminopenicillanic acid and d-cycloserine. Certain peptides, when present in higher concentration, inhibited the transpeptidation observed at lower concentration. The enzyme from strain R39 would not use any dipeptide as an acceptor, but a few compounds that were not glycine or α-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid.

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.

A careful examination of the low-molecular-weight solutes released during mild acid hydrolysis of the lipopolysaccharide of Pseudomonas aeruginosa (N.C.T.C. 1999) revealed the presence of ethanolamine triphosphate. During storage, the compound decomposed to give ethanolamine pyrophosphate, identified in a previous study (Drewry et al., 1971); PP(i) may be a further decomposition product. Evidence for the attachment of ethanolamine triphosphate to a polysaccharide fraction was obtained, but the possibility that some was attached to the lipid A moiety was not excluded. Basic compounds released during the hydrolysis of lipopolysaccharide included amino acids, polyamines and oligopeptides.

The inhibitory effects on gastric acid secretion of bovine trypsinogen activation peptide [Val-(Asp)4-Lys] have been confirmed with the pure synthetic peptide. However the effects are considered to be too weak to be of physiological significance. Four related peptides and a gastrin octapeptide sequence [(Glu)5-Ala-Tyr-Gly] have similar effects.

A number of peptides containing methionine and glycine, such as Met-Met-Gly, Met-Met-Gly-Met, and Met-Met-Gly-Gly, were synthesized by conventional techniques. The growth response of Saccharomyces cerevisiae G1333, a methionine-requiring auxotroph, toward various di-, tri-, and tetrapeptides containing methionine was measured. The yeast mutant utilized and contained peptidase activity toward most of the peptides investigated. Growth curves varied with the amino acid sequence and composition of the peptide substrate. Only three free peptides, Gly-Met-Gly, Met-Met-Gly-Gly, and Met-Met-Met-Gly failed to satisfy the nutritional requirement of strain G1333. A cell-free extract derived from the auxotroph contained peptidases which liberated methionine from these three peptides. Peptides containing COOH-terminal methionine residues were observed to be better growth substrates than those peptides having different amino acid residues at this position. The yeast auxotroph grew on pentamethionine as well as on methyl ester derivatives of (Met)3, (Met)4, and (Met)5. A number of N-acetylated and t-butyloxycarbonyl di- and tri-peptides containing methionine, such as Ac-Met-Met, Ac-Met-Met-Met, Ac-Met-Gly-Met, Boc-Met-Met-Met, and Boc-Gly-Met-Met served as growth substrates. Several peptide derivatives, all containing a COOH-terminal glycine residue, Ac-Met-Gly, Ac-Met-Gly-Gly, Ac-Gly-Met-Gly, and Boc-Met-Met-Gly, failed to support growth of strain G1333. Comparison of the results obtained for the yeast auxotroph with corresponding data for methionine-requiring auxotrophs of E. coli suggests that the ability of both organisms to utilize similar peptide substrates is different.

Several proteases from human kidney tissue were partially purified by ammonium sulfate precipitation followed by Sephadex G-75 gel filtration chromatography. The incubations of column fractions with 14C-tetradecapeptide renin substrate indicated four major renin activity peaks between the molecular weights of 25,000 and 80,000. The highest specific activity was found in Fraction B (mol. wt 39,500). Significant renin-like activity was also found in Fraction A (mol. wt 58,000) and Fraction C (mol. wt 33,500). None of the fractions contained angiotensinase activity. Disc-gel electrophoresis indicated that Fractions B and C were heterogeneous. The conversion of 14C-tetradecapeptide renin substrate to angiotensin I by major fractions was differentially inhibited by low concentrations of pepstatin. Both the rate of substrate conversion and the sensitivity to pepstatin were influenced by pH. Generally, the proteases were more sensitive to pepstatin at physiological pH. Regardless of pH, all of the kidney proteases were completely inhibited by 10−3 M pepstatin. The conversion of human substrate by human plasma proteases was found to be even more sensitive to pepstatin. The concentration of 10−5 M pepstatin essentially eliminated plasma renin activity. These results indicate that pepstatin can serve as an efficient inhibitor of both human kidney and plasma renin proteases and as such will be useful in the study of the kinetics of these systems in both research and clinical situations.

A series of peptidyl-tRNA analogs with varying peptide chain length, BrAc(Gly) nPhe-tRNAphe, n = 0 to 16 has been prepared. When bound to Escherichia coli 70 S ribosomes these all react covalently with certain ribosomal proteins. The overwhelming majority of the reaction is with 50 S ribosomal proteins L2, L16, L24, L26–L27 and L32–L33. The extent of reaction with each protein is a function of peptide chain length, making it possible to estimate the relative proximity of these proteins to the 3′-terminus of tRNA bound in the ribosomal P site. This fact, coupled with the findings of others about the length dependence of the binding and peptide donor activity of peptidyl-tRNAs suggests that there is actually a binding site for the growing peptide chain. If this is true, the results presented here permit the ordering of the proteins in this site: L2 is closest to the 3′-end of tRNA followed by L26–L27, L32–L33 and last L24. Evidence is also given that the direction of the growing peptide chain must point away from the A site.

Particulate preparations from sporulating cells of Bacillus sphaericus 9602 contained an endopeptidase activity that hydrolyzed the γ- d -glutamyl-( l ) meso -diaminopimelic acid linkages found in the spore cortical peptidoglycan of this organism. Diaminopimelic acid did not occur in the vegetative cell wall peptidoglycan, and the γ- d -glutamyl- l -lysine linkages found in this polymer were not hydrolyzed by the endopeptidase. The endopeptidase hydrolyzed (X)- l -alanyl-γ- d -glutamyl-( l ) meso -diaminopimelyl( l )- d -alanyl- d -alanine only after removal of the terminal d -alanine residue. The preparations contained an acyl- d -alanyl- d -alanine carboxypeptidase I activity which converted such pentapeptides into substrates for the endopeptidase and which was inhibited 50% by 4 × 10 −7 M benzylpenicillin. This activity also hydrolyzed the analogous pentapeptide substrates containing l -lysine. The preparations also contained an acyl- l -lysyl- d -alanine carboxypeptidase II activity that was not active on the meso -diaminopimelic acid-containing analogue. Neither this activity nor the endopeptidase was inhibited by 10 −3 M benzylpenicillin. The specificities of the carboxypeptidases were consistent with the exclusive presence of l -lysine C-termini in the vegetative peptidoglycan and of meso -diaminopimelyl- d -alanine C-termini in the spore cortical peptidoglycan of B. sphaericus 9602.

The structure of a crustacean hormone secreted by nerve cells, the red-pigment-concentrating hormone (blanching hormone) of the shrimp, Pandalus borealis, has been determined. About 100 μg of hormone, purified from shrimp eyestalks, was used. The structure, which is < Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, was revealed by thermolysin digestion and separation by Sephadex G-25 chromatography of the resulting two fragments. One fragment was the NH2-terminal peptide, < Glu-Leu-Asn, whose structure was determined by carboxypeptidase digestion and identification of the products in the automatic amino acid analyser and by thin-layer chromatography; that of the other, Phe-Ser-Pro-Gly-Trp-NH2, which was a COOH-terminal fragment, was determined by the dansyl-Edman technique. The COOH-terminal residue was identified by comparison of its 1-dimethylaminonaphthalene-5-sulfonyl (Dns) derivative with synthetic Dns-Trp-NH2 by polyamide thin-layer chromatography.

RNA was isolated from calf lens polysomes and crude nuclear preparations. Template activity, base composition, sedimentation behaviour, and electrophoretic properties were compared with corresponding RNA fractions from rat liver. 8–9-fold stimulation of amino acid incorporation was obtained in the cell-free test system derived from Escherichia coli. Template activity was widely distributed throughout sucrose density gradients. However, the major activity consistently was found at the bottom of the tubes. All fractions from the gradient revealed a heterogeneous pattern when subjected to polyacrylamide gel electrophoresis. The bottom fraction constistently showed a diffuse pattern instead of discrete bands. Only RNA fractions extracted at 65° and at pH 8.3 showed a G-C content which was significantly different from ribosomal RNA.

A variety of oligopeptides and polypeptides was subjected to the action of Aeromonas amino-peptidase and the sequential release of amino acid residues was determined quantitatively. The residues that proved susceptible to hydrolysis included all hydrophobic, aromatic and basic amino acids plus proline. Aspartyl, glutamyl and cysteic acid residues were not removed from the amino-terminus of any substrate tested, even at high enzyme concentrations but in contrast, asparagine, glutamine and aminoethylated cysteine were released. Glycine was generally resistant to hydrolysis, but was slowly released from some substrates dependent upon the adjacent residues. The specificity and ease of purification of this aminopeptidase should make it a useful tool for sequence determination.

Normal human gastric juice has been examined for the presence of peptides. Five pooled resting samples have been found to contain a decapeptide of low chromatographic mobility. The sequential structure of this peptide was shown by analysis, and by comparison with a synthetic sample, to be Leu-Ala-Ala-Gly-Lys-Val-Glu-Asp-Ser-Asp Two of the gastric juice samples also contained a peptide of slightly higher Rf value in smaller quantity. This second peptide, though not characterized completely, appeared to have the structure of a nonapeptide. The amino acid composition differed from that of the decapeptide in that leucine was absent; alanine was the N-terminal amino acid. These peptides have not previously been reported to be present in normal human gastric juice. The authors have not observed any of these peptides in a small number of samples from pernicious anaemia patients.

The interaction of melittin, a lytic peptide found in bee venom, with lecithins was studied. The single tryptophan residue present in melittin served as a fluorescence probe. Melittin and lecithins do form a complex, whereby the maximal emission wave-length shifts from 350 to 330 nm. This indicates a transfer of the indole ring from water to a hydrophobic environment. The melittin-lecithin complex can be isolated by chromatography on Sephadex G-200. This interaction is dependent on the concentration of lecithin, the ratio of peptide to lipid, and the fluidity of the micelles. Using again fluorescence measurements, no interaction between melittin and cholesterol could be detected. Lysolecithin, the product of the phospholipase A2 reaction, does form a complex with melittin, from which it can apparently be displaced by lecithin. A possible mechanism for the synergism of melittin and phospholipase A2 is discussed.

The [3H]phenylalanine-containing peptides synthesized from l -[3H]phenylalanyl RNA in a cell-free system from Escherchia coli, containing poly (uridylic acid), elongation factors and GTP at 10 m m Mg2+, have been separated after alkaline treatment into three fractions: phenylalanylphenylalanine (Phe)2, oligophenylalanine [Oligo-(Phe)] and polyphenylalanine [poly(Phe)]. Under conditions at which in the absence of inhibitors peptide chain elongation proceeds so that (Phe)2 and oligo(Phe) constitute a substantial proportion of the total Phe-containing peptides formed, amicetin, lincomycin, spiramycin and erythromycin affect the formation of (Phe)2 to a much smaller extent than they do the formation of oligo (Phe) and poly(Phe). On the other hand, thiostrepton and siomycin A cause a decrease in (Phe)2 while affecting poly(Phe) formation to a much lesser extent; this result parallels that obtained with tetracycline in the same system [Biochim. Biophys. Acta240, 137; 247, 632 (1971)], and is compatible with the proposal that thiostrepton and siomycin A inhibit primarily the enzymic binding of aminoacyl RNA to the ribosome.