Γ-glutamyl Derivatives Research Articles

γ-Glutamyl peptides of Vigna radiata seeds

It has been reported elsewhere that Vigna radiata seeds contain a high concentration of γ-glutamyl- S-methylcysteine and its sulphoxide and that these compounds serve as index compounds for the chemotaxonomy of V. radiata. Beside these two γ-glutamyl peptides, nine other γ-glutamyl derivatives, of which three are new compounds, have been isolated and identified from V. radiata seeds. The structures of the new peptides, γ-glutamyl- S-methylcysteinyl-β-alanine ( S-methylhomoglutathione), γ-glutamyl- N δ-acetylornithine and γ-glutamyl-γ-glutamyl- S-methylcysteine, were confirmed by direct comparison with synthetic specimens. The other six γ-glutamyl derivatives were homoglutathione, γ-glutamyl glutamic acid, -aspartic acid, -phenylalanine, -leucine and -isoleucine.

Presence of di- and polyamines covalently bound to protein in rat liver

Acid hydrolysis of trichloroacetic acid precipitate from rat tissue (liver, kidney and testis) homogenate released significant amounts of acid-insoluble putrescine, spermidine and spermine. Following incubation of liver homogenate with [1,4- 14C]putrescine, 1.4% of total radioactivity and 1.0% of labelled diamine were recovered in the acid-insoluble fraction. Exhaustive digestion of acid-precipitable material with proteinases (Pronase, aminopeptidase M, carboxipeptidase A, B and Y) revealed the presence of di- and polyamines and of N 1-( γ-glutamyl)spermidine, N 1-( γ-glutamyl)sperminine and N 1, N 12-bis( γ-glutamyl)spermine. These derivatives were identified both by chromatographic analysis and by enzymatic digestion with purified γ-glutamylamine cyclotransferase. The finding of di- and polyamine γ-glutamyl derivatives in the proteinase-digested acid-insoluble fraction of homogenate may be considered as a proof of the in vivo transglutaminase-catalyzed binding of polyamines to proteins. This evidence suggests that di- and polyamines might have an important role in mammalian tissues through covalent binding to proteins by either one or both the primary amino groups.

72] Separation of γ-glutamyl amino acids by ion-exchange chromatography

Publisher Summary The formation of γ-glutamylcysteine and the γ-glutamyl derivatives of certain amino acids are catalyzed by γ-glutamylcysteine synthetase, and many γ-glutamyl amino acids are formed by transpeptidation. γ-Glutamyl amino acids are found in brain, urine, and blood. More than 70 γ-glutamyl derivatives are isolated from plants. The separation of some γ-glutamyl amino acids by ion-exchange chromatography is described in the chapter. The separation is carried out on a Durrum D-500 amino acid analyzer using a single-column sodium citrate procedure designed for the analysis of physiological fluids. The elution times of 26 γ-glutamyl peptides is presented diagrammatically. For comparison, the elution times of a standard mixture of amino acids chromatographed under the same conditions are also presented. The γ-glutamyl derivatives of alanine, glutamic acid, asparagine, glutamine, serine, and glycine, as well as glutathione and several γ-glutamyl-γ-glutamyl amino acids, all elute together before aspartic acid.

Variation of properties of Micrococcus glutamicus glutamine synthetase brought about by divalent cations.

The optimum pH of glutamine synthetase varied with the type and concentration of divalent cation present: pH 7.0~8.0 with 30mM Mg2+, 6.0~7.0 with 30 mM Mn2+ and 7.0~8.0 with 4mM Mn2+. The activity with 4 mM Mn2+ at pH 7.0~8.0 was almost the same as that with 30 mM Mg2+. Changes in Km values for substrates and in susceptibility to inhibition by certain metabolites were observed when 30 mM Mg2+ was replaced by 4 mM Mn2+. Addition of a small amount of Co2+ greatly stimulated the reaction rate in the presence of a suboptimal level of Mg2+. Optimum pH, Km values for substrates and the inhibitory effect of metabolites were also changed in the mixture of Mg2+ and Co2+. Glutamine synthetase was reactive toward hydroxylamine, methylamine and ethylamine under the standard conditions as well as toward ammonia, and similarly the kinetics of these reactions were altered by divalent cations. The reaction products, γ-glutamyl derivatives, were identified.

Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Upon exposure of primary monolayer cultures of hepatocytes and H35 hepatoma cells, methptrexate (MTX) is taken up by carrier-mediated mechanisms and converted to γ-glutamyl derivatives with one to four residues being added. Under conditions that result in 90% or greater conversion, the primary metabolite in both cell types is MTX with three additional glutamates (4-NH 2-10-CH 3PteGlu 4). When the time-dependent synthesis of MTX polyglutamates (4-NH 2-10-CH 3PteGlu 2 and higher) at extracellular concentrations of 10 and 100 μ m methotrexate is measured, both cell types exhibit linear synthesis for 4 to 6 hr, at which time an apparent steady state intracellular concentration of approximately 40 μ m is reached. The concentration of MTX polyglutamate synthesized is not due a restriction in MTX since the hepatocytes and H35 cells accumulated 400 and 138 μ m intracellular methotrexate, respectively, after 24 h in the presence of 100 μ m extracellular MTX. Examination of MTX polyglutamate formation following a 24-h incubation showed concentration dependence with respect to intra- and extracellular MTX. Saturation was reached at a medium concentration of approximately 2 μ m with both cell types which corresponded to 10 to 12 μ m intracellular MTX. Placement of cells at steady state in medium lacking MTX results in the rapid equilibration of all free intracellular MTX with the medium. The MTX polyglutamates leave the cell by a slow loss of intact polyglutamates and also by intracellular cleavage to MTX followed by efflux. The longer-chain-length γ-glutamyl derivatives (Glu 4–5) are more avidly retained by the cells than the shorter ones (Glu 2–3).

Transglutaminases in mammalian reproductive tissues and fluids: relation to polyamine metabolism and semen coagulation

Unique forms of transglutaminases were found in rat coagulating (anterior prostate) gland and its secretion, and also in dorsolateral prostate but not in the ventral lobe of the gland. These forms of transglutaminases were especially reactive with saline-soluble seminal vesicle secretion proteins and their N, N-dimethylated derivatives as compared with N, N-dimethylcasein as amine acceptor protein substrates, and are electrophoretically separable from both the transglutaminase of rat liver and thrombin-activated Factor XIII of the blood clotting system. Transglutaminases of the type found in coagulating gland and dorsolateral prostate appear to be absent from a large number of other organs in the male rat, and also from the uterus, ovary, vagina, of virgin females and from ovary and the maternal and fetal parts of the placenta of late pregnancy. Studies on the role of coagulating gland transglutaminase in the clotting of seminal vesicle secretion protein that is the basis of semen coagulation are discussed. The aliphatic amines putrescine, spermidine and spermine are among the most active of low molecular weight primary amine compounds in rat tissues in serving as amine acceptor substrates for coagulating gland transglutaminases. Putrescine and related polyamines are incorporated into vesicular secretion proteins in the form of peptide bound γ-glutamyl derivatives, the free primary amine groups of which can then further react with other polypeptide-bound glutamine residues to form bis-(γ-glutamyl)polyamine cross-bridges in transglutaminase-catalyzed reactions.

Determination of pyrrolidone carboxylate and γ-glutamyl amino acids by gas chromatography

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.

New isoxazolinone amino acids from Lathyrus odoratus seedlings

In addition to β-(isoxazolin-5-one-2-yl)-alanine (I) and β-(2-β-D-glucopyranosyl-isoxazolin-5-one-4-yl)-alanine (III), previously found in Pisum sativum seedlings, two new amino acids and an amine containing the same heterocyclic ring have been isolated from Lathyrus odoratus seedlings. Their structures were determined as α-amino-γ-(isoxazolin-5-one-2-yl)-butyric acid (VI), 2-aminoethyl-isoxazolin-5-one (VII) and the γ-glutamyl derivative of the latter (V).

Glutathione Turnover in the Kidney; Considerations Relating to the γ-Glutamyl Cycle and the Transport of Amino Acids

The overall turnover of glutathione in kidney and liver was determined in studies in which L-[(14)C]glutamate was administered to mice. Turnover was much more rapid (about 5 times greater) in kidney than in liver. Studies were also carried out in which 5-oxo-L-[(14)C]proline was administered; the first order rate constants for glutathione synthesis from 5-oxoproline in liver and kidney were not far from those found for synthesis of glutathione from glutamate in these tissues. The findings are in accord with the fact that the activities of the enzymes of the gamma-glutamyl cycle are much higher in kidney than in liver. The findings of high turnover of glutathione in kidney and the rapid utilization of 5-oxoproline by this organ for glutathione synthesis are consistent with the function of the gamma-glutamyl cycle in vivo and the proposed role of gamma-glutamyl derivatives in amino-acid transport.

ALPHA- AND GAMMA-GLUTAMYL DERIVATIVES OF AMINOBENZOIC ACIDS.

ALPHA- AND GAMMA-GLUTAMYL DERIVATIVES OF AMINOBENZOIC ACIDS.

Determination of γ-glutamyl transpeptidase activity in human serum and urine

A simple and improved method for the synthesis of γ- l-glutamyl-α- and β-naphthylamide is described. A procedure for the determination of γ-glutamyl transpeptidase activity based on the colorimetric determination of α-naphthylamine liberated from its γ-glutamyl derivative is given.

The use of α-(N-γ- dl-glutamyl)-aminonitriles for the colorimetric determination of a specific peptidase in blood serum

The occurrence of a specific peptidase in human serum is described. The enzyme catalyses the hydrolysis of α-(N-γ- dl-glutamyl)-aminopropionitrile as well as of γ-glutamyl derivatives of other α-aminonitriles. A method is described for assay of the enzyme activity, based on the colorimetric determination of α-aminopropionitrile liberated during the enzymatic incubation ; as little as 0.2 μg of α-aminopropionitrile can be estimated. Some properties of the enzyme were investigated and its specificity is discussed. The activity in sera of normal human subjects was 10.9 units (1 unit = 1 μmole α-aminopropionitrile liberated from the substrate per 100 ml serum after a 2-h incubation at pH 8.6 and 37°). Changes in the activity of the peptidase were found in some pathological conditions. Besides human serum, the enzyme activity was found in sera of many animals, as well as in human urine and pathological exudations. No activity was found in human red blood cells.

Rearrangement of the amino-acid residues in peptides by the action of proteolytic enzymes.

MANY studies of the structure of proteins are based on enzymatic hydrolysis. Since methods of determining the order of the amino-acids in a peptide chain have been developed1, it is important to know whether the enzymatic hydrolysis gives products in which the sequence of the amino-acid residues is the same as in the parent protein. The possibility that enzymes bring about a rearrangement has been raised by Bergmann and Fraenkel-Conrat2, who showed that transamidation occurred. Thus, by the action of papain, hippuric anilide was formed more rapidly from aniline and hippuric amide than from aniline and hippuric acid. Similarly, Fruton showed that, by transamidation, isotopically labelled ammonia was incorporated in certain amides in the presence of papain3,4. It has recently been found5 that glutathione reacts with amino-acids to give their γ-glutamyl derivatives; this transpeptidation was brought about by an extract of sheep kidney.