Studies on serum gamma-glutamyl transpeptidase in primary idiopathic hypothyroidism patients.

Variations in the levels of serum gamma-glutamyl transpeptidase (GGT) were measured in control subjects and in 39 adult primary idiopathic hypothyroidism (PIH) patients. The serum GGT activity was low in PIH patients compared to that of control subjects. A more significant correlation was found between serum GGT and T3 (r = 0.766) but not with T4 (r = 0.476). The comparison of serum GGT with TSH has revealed that those two parameters are not parallel with each other (r = -0.454). No significant correlation between serum GGT activity, age, and sex in PIH patients and control subjects was observed. The present available data indicate that measurement of serum GGT might be useful as a marker index in PIH patients.     

Studies on the properties of the variants of gamma-glutamyl transpeptidase in human urine

Both the high molecular weight and the low molecular weight variants of urinary Y-glutamyl transpeptidase, displayed transpeptidase (pH optimum 8.6) and autotrans-peptidase (pH optimum 9.4) activities. Iodoacetamide inhibited the transpeptidase activity more efficiently than the autotranspeptidase activity with respect to both variants of Y-glutamyl transpeptidase. The high molecular weight form utilized L-glutamine as a better acceptor than L-cystine during the transpeptidation reaction whereas the reverse was the case with the low molecular weight variant. While phenylmethylsulphonyl fluoride-treated enzymes retained full activitiesper se, addition of maleic acid to the modified enzyme was found to inhibit the catalytic activities indicating a maleic acid-induced conformational change of the modified enzyme.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

gamma-glutamyl transpeptidase activity in human colostrum

gamma-Glutamyl transpeptidase (GGT) was determined in the colostrum and milk of 38 patients, 14 days postpartum. The results obtained were compared with the enzymatic activity in colostra of some animals. The human colostrum has been found to contain the highest enzymatic activity which decreases during the first 8 days and then remains stationary. The high GGT activity in the colostrum and milk and histochemical localization of the enzyme in the secretory epithelium of the milk gland indicate its participation in resorption processes of amino acids and peptides.     

Glutamine as a gamma-glutamyl donor for gamma-glutamyl transpeptidase: gamma-glutamyl peptide formation

This study demonstrates the formation of gamma-glutamyl peptides from glutamine and plasma amino acids, as catalyzed by gamma-glutamyl transpeptidase. It also establishes the effect of various amino acids in modulating the rate of glutamine utilization as well as the hydrolytic or transfer product formed. The mechanism of the utilization of glutamine as catalyzed by gamma-glutamyl transpeptidase, involves the formation of a gamma-glutamyl enzyme bound intermediate as the initial step, with release of the amide nitrogen as ammonium, NH+4, Figure 1. The gamma-glutamyl enzyme bound intermediate either reacts with the acceptor amino acids or water; reaction with amino acids yields gamma-glutamylpeptides via the transfer pathway and reaction with water yields glutamate via the hydrolytic pathway.     

Single-chain precursor of renal gamma-glutamyl transpeptidase

The two subunits of gamma-glutamyl transpeptidase (EC 2.3.2.2) are derived from a single-chain glycosylated precursor. A small fraction of the propeptide survives proteolytic processing in the rat kidney and has been purified by an immunoaffinity technique. The propeptide contains determinants for both the subunits and its amino acid composition resembles that of the dimeric enzyme. However, the propeptide exhibits less than 2% of the transpeptidase activity shown by the dimeric enzyme.     

Induction of gamma-glutamyl transpeptidase by hexachlorocyclohexane

Hexachlorocyclohexane (BHC) induced gamma-Glutamyl transpeptidase in rat liver. The enzyme was partially purified from normal BHC fed and fetal liver and also from hepatoma. The gel filtration and electrophoretic properties of the BHC-induced enzyme was compared against that of the other three. Chemical induced hepatoma showed an additional peak of activity in Sephadex G-200 filtration. The other enzymes could be cleaved by papain to give a fraction which cochromatographed with the additional peak of hepatoma enzyme. BHC-induced enzyme and normal enzyme had similar electrophoretic mobility but differed from that of hepatoma and fetal liver enzyme which showed a slightly slower movement.     

Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.

The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.     

Cell surface gamma-glutamyl transpeptidase in live cultures.

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.     

Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu

gamma-Glutamyl transpeptidase (gammaGTase) catalyzes the transfer of the gamma-glutamyl moiety of gamma-glutamyl-derived peptides, such as glutathione (gammaGlu-Cys-Gly), and anilides, such as gamma-glutamyl-7-amido-4-methylcoumarin (gammaGlu-AMC), to acceptor molecules, including water and various dipeptides. These acyl-transfer reactions all occur through a common acyl-enzyme intermediate formed from attack of an active site hydroxyl on the gamma-carbonyl carbon of gammaGlu-X with displacement of X. In this paper, we report that gammaGTase is potently inhibited by the gamma-boronic acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (gamma-boroGlu). We propose that gamma-boroGlu adds to the active site hydroxyl of gammaGTase to form a covalent, tetrahedral adduct that resembles tetrahedral transition states and intermediates that occur along the reaction pathway for gammaGTase-catalyzed reactions. Our studies demonstrate that gamma-boroGlu is a competitive inhibitor of the gammaGTase-catalyzed hydrolysis of gammaGlu-AMC with a K(i) value of 35 nM. Kinetics of inhibition studies allow us to estimate the following values: k(on) = 400 mM(-1) s(-1) and k(off) = 0.02 s(-1). We also found that gamma-boroGlu is an uncompetitive inhibitor of Gly-Gly-promoted transamidation of gammaGlu-AMC. This observation is consistent with the kinetic mechanism we determined for gammaGTase-catalyzed transamidation of gammaGlu-AMC by Gly-Gly to form gammaGlu-Gly-Gly. To probe rate-limiting transition states for gammaGTase catalysis and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K(m) for hydrolysis of gammaGlu-AMC and k(on) for inhibition by gamma-boroGlu are identical and equal unity, suggesting that the processes governed by these rate constants are both rate-limited by a step that is insensitive to solvent deuterium such as a conformational fluctuation of the initially formed E-S or E-I complex. In contrast, the solvent isotope effect on k(c) is 2.4. k(c) is rate-limited by hydrolysis of the acyl-enzyme intermediate that is formed during reaction of gammaGTase with gammaGlu-AMC. Thus, the magnitude of this isotope effect suggests the formation of a catalytically important protonic bridge in the rate-limiting transition state for deacylation.     

The primary structure of human gamma-glutamyl transpeptidase

A cDNA hybridizable to that of rat gamma-glutamyl transpeptidase (GGT) was cloned from a cDNA library of human fetal liver. The insert of the cDNA clone contained 1866 bp consisting of an open reading frame (ORF) of 1709 bp (569 amino acids (aa), N-terminal portion truncated) and a 135-bp 3'-untranslated region followed by a polyadenylated tail. In parallel, amino acid sequences of N-terminal portions of heavy and light chains of a purified human GGT were determined. Two stretches of amino acid sequences identical to the N-terminal sequences of heavy and light chains were found in the ORF. We therefore concluded that the clone is a cDNA for human GGT. From the amino acid sequence deduced from cDNA, the heavy and the light chains of the purified enzyme are estimated to be composed of 351 aa (Mr 38,336) and of 189 aa (Mr 20,000), respectively. The heavy chain is preceded by a signal peptide of at least 29 aa presumed to be cleaved by bromelain treatment. Six putative N-glycosylation sites are present in the heavy subunit region and one in the light subunit region. Primary structure and hydrophobicity profile are closely similar to those of rat GGT.     

Decrease in gamma-glutamyl transpeptidase activity in early amniotic fluid in fetal trisomy 18 syndrome.

A study was carried out in which activity of gamma-glutamyl transpeptidase was measured in 259 samples of amniotic fluid obtained at various weeks of pregnancy. Two hundred and twenty-eight of the babies subsequently delivered had no chromosome abnormality and served as controls, while in 31 various chromosome abnormalities were detected. Mean activity of gamma-glutamyl transpeptidase in the control samples at 15 weeks was 602 U/l. Activity in the samples obtained in cases of fetal chromosome abnormality was generally below this: it was below the 10th percentile in 74% of the samples and below the 2.5th percentile in 52% of the cases. It is concluded that assay of gamma-glutamyl transpeptidase activity is a rapid preliminary test for prenatal diagnosis of chromosomal abnormalities.