The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. Chemical mechanism

The apparent glutathione oxidase activity of gamma-glutamyl transpeptidase is due to nonenzymatic oxidation and transhydrogenation reactions of cysteinylglycine, an enzymatic product formed from glutathione by hydrolysis or autotranspeptidation. Since cysteinylglycine reacts with oxygen more rapidly than does glutathione, the rate of disulfide formation is increased and either cystinyl-bis-glycine or the mixed disulfide of cysteinylglycine and glutathione forms as an intermediate product. Nonenzymatic transhydrogenation reactions of these disulfides with glutathione yield glutathione disulfide and thus account for the apparent glutathione oxidase activity of gamma-glutamyl transpeptidase. A sensitive assay for glutathione oxidation is described, and it is shown that covalent inhibitors of gamma-glutamyl transpeptidase abolish the oxidase activity of the purified enzyme and of crude homogenates of mouse and rat kidney.     

Mechanism of the acidosis induced adaptation in renal gamma-glutamyltransferase

Acidosis induces an adaptation in renal γ-glutamyltransferase activity. The mechanism responsible for this adaptation was studied in isolated kidneys from control and chronically acidotic rats perfused with either γ-glutamyl-p-nitroanilide or D-glutamine. The results clearly establish that acidosis increased the utilization of both γ-glutamyl donors and that the adaptation occurs on both the luminal (urine) and antiluminal (blood) border of tubule cells. Acidotic rat kidneys exhibited an apparent Vmax for γ-glutamyl-p-nitroanilide similar to that of the control while the apparent K_m was significantly reduced consistent with an increased affinity of the enzyme for the substrate in acidosis.     

Influence of detergents and organic solvent extraction on human gamma-glutamyltransferase activity.

Human serum and urinary gamma-glutamyltransferase (gamma-GT) have been found to react differently towards the detergents Triton X-100 and sodium lauryl sulphate (SDS). Serum gamma-GT was virtually unaffected by Triton X-100 at a concentration of 5% whereas urinary gamma-GT was 10-15% activated under similar conditions. There was a 100-fold difference in the response of serum and urinary gamma-GT to SDS. The enzyme activity in urine was completely destroyed by 0.02% SDS, whereas it required 2.0% to destroy the serum enzyme. These latter differences, however, were found to result from the environments of the serum and urinary enzyme rather than to intrinsic factors within the molecule. Extraction of serum gamma-GT from normal individuals with n-butanol resulted in little or no loss of activity from the aqueous phase, whereas more than 30% activity was lost from normal urines. Extraction using various mixtures of butanol and di-isopropyl ether (DIPE) produced largely similar results, except that 25% DIPE: 75% butanol mixture produced a marked loss of activity from serum. It is suggested that gamma-GT activity in human body fluids may be dependent on the presence of a lipid fraction.     

Regulation of the supramolecular structure and the catalytic activity of gamma-glutamyltransferase in the reversed micelle system

Regulation mechanisms of the supramolecular structure and the catalytic activity of a heterodimeric enzyme, gamma-glutamyltransferase, in the system of Aerosol OT (AOT) reversed micelles in octane have been studied. gamma-(3-carboxy-4-nitro)-glutamic acid anilide (L- and D-isomers) and glycylglycine were used as substrates to explore the enzyme-catalyzed hydrolase, autotransferase, and transferase reactions. For all types of reactions, the catalytic activity of gamma-glutamyltransferase as a function of the hydration degree has a shape of curves with three optima. The optima of the catalytic activity were detected at hydration degrees [( H2O]/[AOT] = 11, 17, and 26) when radii of the micelle's inner cavity are commensurate with the light and heavy subunits (Mr 21,000 and 54,000, respectively) of gamma-glutamyltransferase as well as with the dimer (Mr 75,000). As ultracentrifugation the change in hydration degree caused a reversible dissociation of the enzyme to the light and heavy subunits. Both subunits catalyze the hydrolase and transferase reactions, whereas the autotransferase activity was detected only for the heavy subunit. Dependencies of catalytic activities of the subunits on the hydration degree have one optimum each (at [H2O]/[AOT] = 11 and 17 for the light and heavy subunits, respectively). When mixing micellar solutions containing both subunits, a third optimum was detected corresponding to the dimer [( H2O]/[AOT] = 26).     

Conversion of glutathione to glutathione disulfide, a catalytic function of gamma-glutamyl transpeptidase.

A purification procedure, based on that previously used for rat kidney gamma-glutamyl transpeptidase, was used for the purification of glutathione oxidase (which converts glutathione to gluthathione disulfide). The two activities co-purified, the ratio of the activities remaining constant through all steps of the isolation procedure. The purified enzyme was separable into 12 isozymic species by isoelectric focusing. All 12 isozymes exhibited a constant ratio of transpeptidase to glutathione oxidase activities, strongly supporting the conclusion that conversion of glutathione to glutathione disulfide is a catalytic function of gamma-glutamyl transpeptidase. Modulation of oxidase activity by inhibitors and acceptor substrates of transpeptidase is discussed in relation to the possible glutathione binding sites involved in gamma-glutamyl transfer and oxidase activities of the enzyme.     

Mechanism of activation of cAMP-dependent protein kinase: in Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.     

Glutathione S-transferases of the bovine retina. Evidence that glutathione peroxidase activity is the result of glutathione S-transferase.

We have purified two isoenzymes of glutathione S-transferase from bovine retina to apparent homogeneity through a combination of gel-filtration chromatography, affinity chromatography and isoelectric focusing. The more anionic (pI = 6.34) and less anionic (pI = 6.87) isoenzymes were comparable with respect to kinetic and structural parameters. The Km for both substrates, reduced glutathione and 1-chloro-2,4-dinitrobenzene, bilirubin inhibition of glutathione conjugation to 1-chloro-2,4-dinitrobenzene, 1-chloro-2,4-dinitrobenzene inactivation of enzyme activity and molecular weight were similar. However, pH optimum and energy of activation were found to differ considerably. Retina was found to have no selenium-dependent glutathione peroxidase activity. The total glutathione peroxidase activity fractionated with the transferases in the gel-filtration range of mol.wt. 49000 and expressed activity with only organic hydroperoxides as substrate. Only the more anionic isoenzyme expressed both transferase and peroxidase activity.     

De novo synthesis of glutathione is a prerequisite for curcumin to inhibit hepatic stellate cell (HSC) activation

On liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type for hepatic fibrogenesis, become active, characterized by enhanced cell growth and overproduction of extracellular matrix (ECM). Oxidative stress facilitates HSC activation and the pathogenesis of hepatic fibrosis. Glutathione (GSH) is the most important intracellular antioxidant. We previously showed that curcumin, the yellow pigment in curry from turmeric, significantly inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. It is hypothesized that curcumin might inhibit HSC activation mainly by its antioxidant capacity. Results from this study demonstrate that curcumin dose and time dependently attenuates oxidative stress in passaged HSC demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation. Curcumin elevates the level of cellular GSH and induces de novo synthesis of GSH in HSC by stimulating the activity and gene expression of glutamate-cysteine ligase (GCL), a key rate-limiting enzyme in GSH synthesis. Depletion of cellular GSH by the inhibition of GCL activity using L-buthionine sulfoximine evidently eliminates the inhibitory effects of curcumin on HSC activation. Taken together, our results demonstrate, for the first time, that the antioxidant property of curcumin mainly results from increasing the level of cellular GSH by inducing the activity and gene expression of GCL in activated HSC in vitro. De novo synthesis of GSH is a prerequisite for curcumin to inhibit HSC activation. These results provide novel insights into the mechanisms of curcumin as an antifibrogenic candidate in the prevention and treatment of hepatic fibrosis.     

The catalytic activity of the Src family kinases is required to disrupt cadherin-dependent cell-cell contacts

Despite the importance of epithelial cell contacts in determining cell behavior, we still lack a detailed understanding of the assembly and disassembly of intercellular contacts. Here we examined the role of the catalytic activity of the Src family kinases at epithelial cell contacts in vitro. Like E- and P-cadherin, Ca(2+) treatment of normal and tumor-derived human keratinocytes resulted in c-Yes (and c-Src and Fyn), as well as their putative substrate p120(CTN), being recruited to cell-cell contacts. A tyrosine kinase inhibitor with selectivity against the Src family kinases, PD162531, and a dominant-inhibitory c-Src protein that interferes with the catalytic function of the endogenous Src kinases induced cell-cell contact and E-cadherin redistribution, even in low Ca(2+), which does not normally support stable cell-cell adhesion. Time-lapse microscopy demonstrated that Src kinase inhibition induced stabilization of transiently formed intercellular contacts in low Ca(2+). Furthermore, a combination of E- and P-cadherin-specific antibodies suppressed cell-cell contact, indicating cadherin involvement. As a consequence of contact stabilization, normal cells were unable to dissociate from an epithelial sheet formed at high density and repair a wound in vitro, although individual cells were still motile. Thus, cadherin-dependent contacts can be stabilized both by high Ca(2+) and by inhibiting Src activity in low (0.03 mM) Ca(2+) in vitro.     

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.     

Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

The localization of renal glutathione oxidase activity studied in the isolated, perfused rat kidney

The metabolism of extracellular glutathione was studied in the isolated, perfused rat kidney. Both recirculating and single-pass perfusions were associated with rapid conversion of reduced glutathione to glutathione disulfide in the perfusate. Only a minor fraction of perfusate glutathione was recovered in urine; however, this fraction was markedly increased in the presence of the inhibitor of γ-glutamyltransferase, serine·borate. In contrast, serine·borate had no effect on either oxidation or disappearance of perfusate glutathione. The results indicate that renal glutathione oxidase activity is restricted to glutathione present in plasma, while γ-glutamyltransferase acts on glutathione in the glomerular filtrate.     

The renal excretory activity of atrial natriuretic factor is independent of renal prostaglandins in humans.

Since renal prostaglandins may contribute to natriuresis induced by endogenous atrial natriuretic factor (ANF), acute volume expansion (AVL), a known stimulus of ANF and prostaglandins, was induced in 8 healthy women in order to test whether the consequent sodium and water diuresis is altered by prostaglandin inhibition. AVL (i.v. infusion of a 2 liter 5% glucose solution in 1 h) was infused after placebo and after inhibition of prostaglandins with diclofenac (200 mg/day orally for 4 days), in a double blind randomized cross-over fashion. Urinary eicosanoids (PGE2, PGF2 alpha, 6-ketoPGF1 alpha, TXB2--RIA), plasma ANF (RIA) and urinary electrolytes were determined before, during and after AVL under both placebo and diclofenac regimes. During placebo, AVL induced sustained increases in plasma ANF (174% at peak, p less than 0.001 ANOVA), excretion of the four eicosanoids (149%-1172%, p less than 0.005-0.001), urinary volume (UV, 815%, p less than 0.001), natriuresis (UNa, 98%, p less than 0.005) and in kaliuresis (UK, 90%, p less than 0.001). Cyclooxygenase inhibition resulted in a reduction of over 70% in both baseline values and AVL-induced increase of eicosanoids. It did not alter either baseline levels or AVL-stimulated ANF, UV, UNa and UK in relation to placebo. The present results suggest that the diuretic and natriuretic activity of ANF is not mediated by renal PGs in humans.     

Manganese is essential for catalytic activity of Escherichia coli agmatinase.

Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.     

Cytokinins: 7-glucosylation is not a prerequisite of the expression of their biological activity

¹⁴C-labelled benzyladenine-7-glucoside (0.57 M) supplied to cytokinin-requiring Nicotiana tabacum (cv. Wisconsin 38) cells cultivated in liquid medium was slowly absorbed and resulted in rather large intracellular quantities of benzyladenine-7-glucoside (30 pmol/10⁵ cells) but did not show any biological activity, while 0.01 M benzyladenosine induced cell division. In this case the resulting intracellular quantity of benzyladenine-7-glucoside formed was 3.3 pmol/10⁵ cells. Also, N⁶-(²-isopentenyl)adenosine induced cell division in these tobacco suspensions without being significantly 7-glucosylated. These data provide strong evidence that the 7-glucosylation of cytokinins is not a prerequisite of the expression of their biological activity.Abbreviations bz⁶adenine benzyladenine - i⁶adenosine N⁶(²-isopentenyl)adenosine     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of the catalytic activity of glutathione transferases

Steady state, pre-steady state kinetic experiments, and site-directed mutagenesis have been used to dissect the catalytic mechanism of human glutathione transferase T2-2 with 1-menaphthyl sulfate as co-substrate. This enzyme is close to the ancestral precursor of the more recently evolved glutathione transferases belonging to Alpha, Pi, and Mu classes. The enzyme displays a random kinetic mechanism with very low k(cat) and k(cat)/K(m)((GSH)) values and with a rate-limiting step identified as the product release. The chemical step, which is fast and causes product accumulation before the steady state catalysis, strictly depends on the deprotonation of the bound GSH. Replacement of Arg-107 with Ala dramatically affects the fast phase, indicating that this residue is crucial both in the activation and orientation of GSH in the ternary complex. All pre-steady state and steady state kinetic data were convincingly fit to a kinetic mechanism that reflects a quite primordial catalytic efficiency of this enzyme. It involves two slowly interconverting or not interconverting enzyme populations (or active sites of the dimeric enzyme) both able to bind and activate GSH and strongly inhibited by the product. Only one population or subunit is catalytically competent. The proposed mechanism accounts for the apparent half-site behavior of this enzyme and for the apparent negative cooperativity observed under steady state conditions. These findings also suggest some evolutionary strategies in the glutathione transferase family that have been adopted for the optimization of the catalytic activity, which are mainly based on an increased flexibility of critical protein segments and on an optimal orientation of the substrate.     

The quaternary structure of E. coli inorganic pyrophosphatase is not required for catalytic activity

Inorganic pyrophosphataseSubunitQuaternary structureKinetic analysis     

Steady-state kinetic investigation of specific anion effects on the catalytic activity of yeast glutathione reductase

The catalytic activity of yeast glutathione reduetase at pH 7.6 is sensitive to the sodium phosphate buffer concentration and the presence of monovalent sodium salts in the assay medium. Low concentrations of sodium phosphate activate and high concentrations inhibit enzymatic activity. The optimal concentration is at about 0.06 m sodium phosphate. In the presence of 0.06 m sodium phosphate, addition of a variety of monovalent sodium salts results in inhibition of enzymatic activity, the inhibition being competitive with respect to NADPH and noncompetitive with respect to oxidized glutathione. At suboptimal concentrations of sodium phosphate, addition of monovalent sodium salts activates enzymatic activity. In addition, at suboptimal sodium phosphate concentration Lineweaver-Burk plots of initial velocity at constant NADPH concentration with oxidized glutathione as the variable substrate are nonlinear, being concave down. The nonlinear behavior can be eliminated by addition of 0.1 m sodium chloride. It is concluded that there are at least two specific anion binding sites at or near the enzyme active site. The anion inhibition is explained in terms of an ordered sequential mechanism for glutathione reduetase. The anion activation is analyzed in terms of a change of reaction pathway, the reactive enzyme species being dependent upon the oxidized glutathione concentration.