• Journal of Life Science
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• v.8 no.4
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• pp.410-415
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• 1998

Procine pepsin hydrolysis of hexapeptide L-S-pNF-Nle-A-OMe in the presence of dipeptide L-L generates a new peak on HPLC analysis of reaction mixtures that is not seen when enzyme is incubated with either peptide alone. The peaks can be detected spectroscopically at either 214 or 254 nm, the latter consistent with a new peptide containing the p-nitro-F residue. The data suggest acyl transpeptidation between E(L-S-pNF) and L-L to form L-S-pNF-L-L. Consistent with this inference are (1) the ability of L-L-NH$_{2}$ and inability of Boc-L-L to undergo a similar transpeptidation reaction, and (2) the data from electrospray mass spectrum. This synthesis requires that Nle-A-L-OMe be released before L-S-pNF, an order opposite to that proposed on the basis of product inhibition kinetics. Consistent with this inference are reciprocal solvent isotope effects ; normal isotope effects of 1.736$\pm$0.121 on the formation of Nle-A-L-OMe and 2.281$\pm$0.184 in the formation of L-S-pNF, coupled to an inverse isotope effects of 0.576$\pm$0.045 on the formation of L-S-pNF-L-L. Because transpeptidation precedes faster in D$_{2}$O, the isotopically-sensitive step must occur after release of Nle-A-L-OMe. Isotopically-enhanced transpeptidation is consistent with the Uni-Bi iso memchanism postulated on the basis of an isotope effects on Vmax but not on Vmax/Km$^{1)}$ and confirmed by isotope effects on the onset of inhibition by pepstatin$^{2)}$.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.360-363
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• 1994

A colorimetric assay for $\gamma$-glutamyl transpeptidase ($\gamma$-CTP; E.C 2.3.2.2) employing 2, 4, 6-trinitrobenzene sulfonate (TNBS) to detect the amount of disappeared acceptor via transpeptidation, has been developed. Under the experimental conditions using L-$\gamma$-glutamyl ethyl ester and L-phenylalanine as $\gamma$-glutamyl donor and acceptor, respectively, it was found that the decreased absorbance of yellow color at 420 nm was strictly related to the amount of L-$\gamma$-glutamyl-L-phenylalanine (L-$\gamma$-Glu-L-Phe) formed, which was determined by DEAE-cellu-lose column chromatography. Concentrations of the enzyme and $\gamma$-glutamyl products were able to be determinedin the nanogram and nanomoles per milliliter range, respectively, with high precision and reliability. This novel assay system may therefore be a useful means for understanding of catalytic function of the $\gamma$-CTP spectrophotometrically without any usage of sophisticated instruments.

• BMB Reports
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• v.38 no.3
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• pp.343-349
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• 2005

Gamma-glutamyltransferase (GGT, EC 2.3.2.2) which hydrolyzes glutathione (GSH), is required for the maintenance of normal intracellular GSH concentration. GGT is a membrane enzyme present in leukocytes and platelets. Its activity has also been observed in human neutrophils. In this study, GGT was purified from Triton X-100 solubilized neutrophils and its kinetic parameters were determined. For kinetic analyses of transpeptidation reaction, $\gamma$-glutamyl p-nitroanilide was used as the substrate and glycylglycine as the acceptor. Apparent $K_m$ values were determined as 1.8 mM for $\gamma$-glutamyl p-nitroanilide and 16.9 mM for glycylglycine. The optimum pH of GGT activity was 8.2 and the optimum temperature was $37^{\circ}C$. It had thermal stability with 58% relative activity at $56^{\circ}C$ for 30 min incubation. L-serine, in the presence of borate, was detected as the competetive inhibitor. Bromcresol green inhibited neutrophil GGT activity as a noncompetetive inhibitor. The neutrophils seem to contain only the isoenzyme that is present in platelets. We characterized the kinetic properties and compared the type of the isoenzyme of neutrophil GGT with platelet GGT via polyacrylamide gel electrophoresis (PAGE) under a standart set of conditions.

• Journal of the Korean Chemical Society
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• v.13 no.3
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• pp.233-240
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• 1969

The synthesis is described of new pepsin substrates of benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine ethyl ester and benzyloxycarbonyl-glycyl-L-tyrosyl-L-phenylalanyl-glycine for studies on the specificity of pepsin, and thin layer chromatographic examination of the peptides prepared showed the new substrates are homogeneous and also, same examination of the incubation mixtures showed that two synthetic substrates are cleaved by pepsin at the L-tyrosyl-L-phenylalanyl bond and hydrolysis of these substrates by pepsin is achieved without transpeptidation. It is found that synthetic peptides are moderately soluble with the amount of the substrate up to a concentration of 0.7 mM in aqueous sodium citrate buffers (0.04 M) in the pH range 1.8-4.0, thus obviating the necessity for the adding of an organic solvent in the assay mixture. The kinetic parameters for synthetic substrates are tabulated in the following table. The data in the table indicate that the susceptibility of synthetic peptides to peptic hydrolysis are relatively large and the change of the carboxyl-terminal group of synthetic substrate from glycine ethyl ester to glycine causes a small decrease in the susceptibility of the L-tyrosyl-L-phenylalanyl bond.

• YAKHAK HOEJI
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• v.3 no.1
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• pp.4-9
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• 1957

Effects of antibioties on micro-organism have been reported by many scientists, such as Krampitz and Werkman, Fisher, Gale and Rodwell, Klimick Cavalito and Bailey, Umbreit, etc. On the mechanism by which penicillin act, Fisher(1947), Platt(1947), and Cavallito, considered that penicillin might act on bacteria by inhibiting with the normal function of SH-group of glutathione in the metabolism of the cell. Resenbrance of penicillin to gultathione in structure and the inactivation of penicillin by cysteine make us approve of the above inhibiting theory of SH-group. Galland (1947) and Schmidt (1947) reported that penicillin inhibited the activity of ribonuclease, Phosphatase, and mononucleotidase. Gale (1948) discovered that the gram positive bacteria had lost the power to uptake glutamic acid by ribonucleic acid in the medium contained penicillin: growth of gram positive organism was inhibited by the results that penicillin inhibited the uptake of amino acid byribonucleic acid, acting on ribonucleic acid of gram positive bacteria. Hotchkiss (1950) cultured S. aureus in the medium contained glucose and amino acids, and studied the effect of penicillin on protein synthesis. Peptide formation in living cells was inhibited by penicillin, while amono acid was utilized as before the addition of penicillin. On the otherhand, Binkley (1951) found penicillin interfered hydrolase of glutath one, and Hans (1950) reported penicillin inhibited the transpeptidation. On the machanism by which streptomycin acts. Cohen (1947) reported steptomycin made a irreversible complex with desoxyribonucleic acid, by the fact that desoxyribonucleic acid formed the precipitates with diguanide group of steptomycin. Zeller (1951) reported, on the other hand, streptomycin inhibited diamine oxidease. Geiger (1947) and Umbreit (1949) reported that steptomycin inhibited condensation of oxaloacetate and pyruvate in E. Coli and Oginsky et al (1949) reported steptomycin inhibited oxaloacetate-pyruvate reaction in Kreb's cycle. On the mechanism by which terramycin acts, Hahn & Wisseman (1951) reported that the formation of adaptive enzyme was inhibited by terramycin in E. Coli cultivated in the medium contained loctose, and that the protein synthesis was inhibited by terramycin. However, effects of antibiotics on amino acid metabolism have not been discussed much in spite of its important role in living cells. Especislly, effects of anitibiotics on higher plants have scarcely been reported. Here, to prove the effect of antibiotics on higher plants, and the mechanism by which, through amino acid metabolism, they promote or inhibit growth of plants, amino acids in bean plants treated with penicillin, streptomycin, and terramycin were analyzed by paper chromatography. And to clarify the antagonis of cysteine (as SH-group) against penicillin, through amino acid metabolism, amino acids in bean plants treated with cystene and penicillin, at the same time, were also analyzed.