Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.     

The role of glutathione and glutathione S-transferases in fatty acid ozonide detoxification

The ozonide derived from methyl linoleate was shown to cause a dose dependent inhibition of the phagocytosis of rat alveolar macrophages exposed in vitro to concentrations varying from 10(-5) to 10(-4) M. Vitamin C was demonstrated to detoxify the ozonide. In analogy to their behaviour on exposure to ozone, vitamin E supplemented cells demonstrated a decreased and glutathione depleted cells an increased sensitivity towards the compound. The characteristics of antioxidant protection of cells against the ozonide were thus comparable to those for protection against ozone. Preincubation with glutathione also detoxified the ozonide model compound. Survival of rat alveolar macrophages exposed to a toxic concentration of the ozonide (86 microM final concentration), measured by phagocytosis of the cells, increased significantly (P less than 0.01) from 23 to 54% after a 2.5-h preincubation of the ozonide with glutathione (5 mM final concentration). The detoxification of methyl linoleate ozonide by glutathione could be catalyzed by the rat liver glutathione S-transferases. After a 2.5-h preincubation of the ozonide (86 microM final concentration) with glutathione and glutathione S-transferases (final concentrations, respectively, 5 mM and 0.01 mg/ml), its toxicity was completely abolished, as demonstrated by the 98% survival (P less than 0.001) of subsequently exposed cells. A Km(app) (at 1 mM glutathione) for the ozonide of 0.80 mM and a Vmax(app) (at pH 6.5) of 94 nmol glutathione converted X min-1 X mg protein-1 or (at pH 7.4) of 34 nmol glutathione converted X min-1 X mg protein-1, were found. This glutathione S-transferase catalyzed detoxification of the potential intermediates in ozone induced cell damage, offers a new viewpoint on the role of glutathione in the protection of cells against ozone.     

Studies of the relationship between the catalytic activity and binding of non-substrate ligands by the glutathione S-transferases.

The dimeric enzyme glutathione S-transferase B is composed of two dissimilar subunits, referred to as Ya and Yc. Transferase B (YaYc) and two other transferases that are homodimers of the individual Ya and Yc subunits were purified from rat liver. Inhibition of these three enzymes by Indocyanine Green, biliverdin and several bile acids was investigated at different values of pH (range 6.0-8.0). Indocyanine Green, biliverdin and chenodeoxycholate were found to be effective inhibitors of transferases YaYc and YcYc at low (pH 6.0) but not high (pH 8.0) values of pH. Between these extremes of pH intermediate degrees of inhibition were observed. Cholate and taurochenodeoxycholate, however, were ineffective inhibitors of transferase YcYc at all values of pH. The observed differences in bile acids appeared to be due, in part, to differences in their state of ionization. In contrast with the above results, transferase YaYa was inhibited by at least 80% by the non-substrate ligands at all values of pH. These effects of pH on the three transferases could not be accounted for by pH-induced changes in the enzyme's affinity for the inhibitor. Thus those glutathione S-transferases that contain the Yc subunit are able to act simultaneously as both enzymes and binding proteins. In addition to enzyme structure, the state of ionization of the non-substrate ligands may also influence whether the transferases can perform both functions simultaneously.     

Binding of nonsubstrate ligands to the glutathione S-transferases.

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.     

Purification and characterization of hepatic glutathione S-transferases of rhesus monkeys. A family of enzymes similar to the human hepatic glutathione S-transferases.

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.     

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.     

Acidic glutathione S-transferases of rat testis.

In most organs of the rat the predominant forms of glutathione S-transferase have alkaline (greater than 7.0) pI values. In contrast, in the cytosol from rat testes almost 50% of the transferase activity is due to isoenzymes with acidic (less than 7.0) pI values. We have purified three acidic forms of glutathione S-transferase from rat testis cytosol. One form accounted for more than 90% of the enzymic activity in the acidic fraction. This major form was a homodimer of a new subunit, termed Yt. This subunit had an electrophoretic mobility that was different from the subunits that form the alkaline transferases. In addition, functional and immunological studies were consistent with the unique nature of the Yt subunit. The two minor acidic enzymes of rat testis appeared to be heterodimers of the Yt subunit and a subunit with an electrophoretic mobility identical with that of the Yb subunit present in some alkaline enzymes.     

Quantification of human hepatic glutathione S-transferases.

Human hepatic glutathione S-transferase (GST) subunits were characterized and quantified with the aid of a recently developed h.p.l.c. method. In 20 hepatic tissue specimens the absolute amounts of the basic Class Alpha subunits B1 and B2, the near-neutral Class Mu subunits mu and psi and the acidic subunit pi were determined. The average total amount of GST was 37 micrograms/mg of cytosolic protein, with the Class Alpha GST being the predominant class (84% of total GSTs), and pi as the sole representative of the Class Pi GSTs present in the lowest concentration (4% of total GSTs). Large interindividual differences were observed for all subunits, with variations up to 27-fold, depending on the subunit. For the Class Alpha GST-subunits B1 and B2, a biphasic ratio was observed. The genetic polymorphism of the subunits mu and psi was confirmed by h.p.l.c. analysis, and correlated with the enzymic glutathione conjugation of trans-stilbene oxide and with Western blotting of cytosols, using a monoclonal anti-(Class Mu GST) antibody. Of the 20 livers examined, ten contained only mu, whereas the occurrence of psi alone, and the combination of mu and psi, were found in only one liver each.     

Comparison of the binding sites of GSH S-transferases of the Ya- and Yb-subunit classes: effect of glutathione on the binding of bile acids

We have previously observed that the Ya subunit-containing glutathione (GSH) S-transferases from rat liver exhibit a common high affinity binding site for lithocholic acid, bilirubin, and sulfobromophthalein (BSP) (1984. J. Lipid Res. 25: 1177-1183). Subsequently we found that cholic acid and its amidates bound to a site on the Ya subunit separate for the lithocholic acid/bilirubin site (1986. J. Lipid Res. 27: 955-966). We now have extended this work by showing that amidates of lithocholic acid as well as chenodeoxycholic acid and its amidates competitively displace [14C]lithocholic acid from the Ya subunit. GSH did not inhibit binding of any of the ligands to the high affinity Ya site, but did inhibit binding to the cholic acid site on the Ya subunit. We have also defined the binding sites and effects of GSH on the Yb class of subunits. Lithocholic, chenodeoxycholic, and cholic acids (and amidates) shared a common site on the Yb or Y'b subunit, whereas BSP and bilirubin were bound at a different site. Both the bile acid and organic anion sites on the Yb subunit were inhibited by GSH. The inhibition by GSH in all cases (Ya cholic acid site or Yb bile acid or bilirubin sites) was saturable, of the competitive type, and incomplete at maximal GSH concentrations, suggesting that when GSH binds to its distinct substrate site, it induces a conformational change in the proteins affecting the other binding sites.     

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.     

Identification of the fatty acid binding site on glutathione S-transferase P.

Glutathione S-transferase P (GST-P) bound a series of endogenous fatty acids (C12-C18). To clarify the function and the binding site of the fatty acids, interaction between fatty acids and GST-P was investigated by using 12-(9-anthroyloxy) stearic acid conjugated with Woodward's reagent K. The fluorescence-conjugated fatty acid noncompetitively inhibited GST activity. After GST-P was covalently labeled with the fatty acid, the enzyme was digested with Lysyl Endopeptidase. From the peptide mapping, a single fluorescence-labeled peptide was obtained. By the sequence analysis, the peptide binding fatty acid was determined as the residues of 141-188 from the amino terminus.     

The binding of substrates and a product of the enzymatic reaction to glutathione S-transferase A.

The binding of substrates and a product to glutathione S-transferase A from rat liver was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrates glutathione and bromosulfophthalein as well as a product of glutathione and 3,4-dichloro-1-nitrobenzene, S-(2-chloro-4-nitrophenyl)glutathione, gave hyperbolic binding isotherms with a stoichiometry of 2 mol per mol of enzyme (i.e. 1 molecule per subunit). Glutathione (and glutathione disulfide) had an equilibrium (dissociation) constant for the binding of about 10 microM, whereas bromosulfophthalein and the product had equilibrium constants of about 0.5 microM. All ligands showed the same binding stoichiometry, and competition experiments involving unlabeled ligands indicated that glutathione and the glutathione derivatives were binding to the same site. Low affinity sites appeared to exist in addition to the specific high affinity sites (one per subunit) for all ligands tested. The binding studies are fully consistent with a steady state random kinetic mechanism for the enzyme.     

槲皮素对B型烟粉虱羧酸酯酶和谷胱甘肽S-转移酶活性的影响

采用人工饲料添加法,研究植物防御性次生物质槲皮素对B型烟粉虱Bemisiatabaci(Gennadius)成虫主要解毒酶系羧酸酯酶(CarE)和谷胱甘肽S-转移酶(GSTs)影响的剂量效应和时间效应。用低剂量槲皮素(0.01%,wv)处理B型烟粉虱成虫24h后,其羧酸酯酶(CarE)和谷胱甘肽S-转移酶(GSTs)比活力分别为51.09mOD(min·头)和2.249OD(mgpro.min),是对照的1.233倍和2.20倍;而高剂量的槲皮素对2种解毒酶没有诱导增加作用,甚至还有抑制作用。低剂量的槲皮素短时间处理烟粉虱后,可诱导2种酶活性增加,谷胱甘肽S-转移酶(GSTs)在0.005%的槲皮素处理30min后,比活力值达到8.454OD(mgpro·min),为对照的8.30倍。     

Identification of the binding site of methylglyoxal on glutathione peroxidase: methylglyoxal inhibits glutathione peroxidase activity via binding to glutathione binding sites Arg 184 and 185

Methylglyoxal (MG), a physiological alpha-dicarbonyl compound is derived from glycolytic intermediates and produced during the Maillard reaction. The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino group of proteins, contributes to the aging of proteins and to complications associated with diabetes. In our previous studies (Che, et al. (1997) "Selective induction of heparin-binding epidermal growth factor-like growth factor by MG and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes". J. Biol. Chem., 272, 18453-18459), we reported that MG elevates intracellular peroxide levels, but the mechanisms for this remain unclear. Here, we report that MG inactivates bovine glutathione peroxidase (GPx), a major antioxidant enzyme, in a dose- and time-dependent manner. The use of BIAM labeling, it was showed that the selenocysteine residue in the active site was intact when GPx was incubated with MG. MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) and protein sequencing examined the possibility that MG modifies arginine residues in GPx. The results show that Arg 184 and Arg 185, located in the glutathione binding site of GPx was irreversively modified by treatment with MG. Reactive dicarbonyl compounds such as 3-deoxyglucosone, glyoxal and phenylglyoxal also inactivated GPx, although the rates for this inactivation varied widely. These data suggest that dicarbonyl compounds are able to directly inactivate GPx, resulting in an increase in intracellular peroxides which are responsible for oxidative cellular damage.     

High-affinity binding of lower-density lipoproteins to chicken oocyte membranes.

Oocyte membrane fragments bind specifically radioiodinated VLD lipoprotein (very-low density lipoprotein) and LD lipoprotein (low-density lipoprotein). Competitive binding assays showed 2-3 times more VLD lipoprotein than LD lipoprotein bound at 4 degrees C. Equilibrium-binding data revealed the presence of one class of non-interacting sites for VLD lipoprotein (kD 12 microgram/ml) and co-operative binding for LD lipoprotein. The binding of VLD lipoprotein showed a distinct pH maximum at 5.3, whereas an indistinct maximum at about pH 7.3 was observed for LD lipoprotein. Unlabelled VLD lipoprotein did compete with 125I-labelled LD lipoprotein binding, but unlabelled LD lipoprotein did not compete with 125I-labelled VLD lipoprotein binding. VLD lipoprotein binding was inhibited by HD lipoprotein (high-density lipoprotein), but not by lysozyme, collagen, poly-L-lysine or poly-L-arginine; LD lipoprotein binding was inhibited by lysozyme and collagen, but not by HD lipoprotein. On the basis of these studies, we suggest that: (1) VLD lipoprotein and LD lipoprotein enter the oocytes by a receptor-mediated transport mechanism; (2) the receptors for VLD lipoprotein and LD lipoprotein are distinct; and (3) the binding of LD lipoprotein to chicken oocyte membranes differs from that to other cell types.     

Purification and characterization of glutathione S-transferases of human kidney.

Several forms of glutathione S-transferase (GST) are present in human kidney, and the overall isoenzyme pattern of kidney differs significantly from those of other human tissues. All the three major classes of GST isoenzymes (alpha, mu and pi) are present in significant amounts in kidney, indicating that GST1, GST2 and GST3 gene loci are expressed in this tissue. More than one form of GST is present in each of these classes of enzymes, and individual variations are observed for these classes. The structural, immunological and functional properties of GST isoenzymes of three classes differ significantly from each other, whereas the isoenzymes belonging to the same class have similar properties. All the cationic GST isoenzymes of human kidney except for GST 9.1 are heterodimers of 26,500-Mr and 24,500-Mr subunits. GST 9.1 is a dimer of 24,500-Mr subunits. All the cationic isoenzymes of kidney GST cross-react with antibodies raised against a mixture of GST alpha, beta, gamma, delta and epsilon isoenzymes of liver. GST 6.6 and GST 5.5 of kidney are dimers of 26,500-Mr subunits and are immunologically similar to GST psi of liver. Unlike other human tissues, kidney has at least two isoenzymes (pI 4.7 and 4.9) associated with the GST3 locus. Both these isoenzymes are dimers of 22,500-Mr subunits and are immunologically similar to GST pi of placenta. Some of the isoenzymes of kidney do not correspond to known GST isoenzymes from other human tissues and may be specific to this tissue.     

Single chain urokinase. Augmentation of enzymatic activity upon binding to monocytes

Cellular migration typically requires cell surface-associated urokinase-type plasminogen activator (u-PA) and plasminogen, both of which are present as proenzymes. Because each active enzyme can activate the other zymogen, the mechanism by which the initial proteolytic event of this two-zymogen system occurs is unclear. A mutant of single chain u-PA that could not be cleaved to the more active two-chain u-PA was used to demonstrate that (i) u-PA in its single-chain form exhibits a reactive active site serine, (ii) the enzymatic activity of this molecule is augmented 100-fold upon binding to the u-PA receptor on monocytes as compared with the enzymatic activity of the same number of molecules in the fluid phase, and (iii) the molecule thus bound and active remains in the single-chain form. This is likely an important mechanism for the initiation and control of cell surface-associated fibrinolysis.