Rat lung glutathione S-transferases: subunit structure and the interrelationship with the liver enzymes

Six forms of glutathione S-transferases designated as GSH S-transferase I (pI 8.8), II (pI 7.2), III (pI 6.8), IV (pI 6.0), V (pI 5.3) and VI (pI 4.8) have been purified from rat lung. GSH S-transferase I (pI 8.8) is a homodimer of Mr 25,000 subunits; GSH S-transferases II (pI 7.2) and VI (pI 4.8) are homodimers of Mr 22,000 subunits; and GSH S-transferases III (pI 6.8), IV (pI 6.0) and V (pI 5.3) are dimers composed of Mr 23,500 and 22,000 subunits. Immunological properties, peptide fragmentation analysis, and substrate specificity data indicate that Mr 22,000, 23,500 and 25,000, are distinct from each other and correspond to Ya, Yb, and Yc subunits, respectively, of rat liver.     

Identification of multiple glutathione S-transferases from Daphnia magna

1. Six anionic glutathione S-transferases (GST) were purified from the crustacean, Daphnia magna, by means of affinity chromatography, that are responsible for ca. 40% of cytosolic GST activity. 2. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed the presence of three proteins, with molecular weights of 27,500, 28,000, and 30,200. 3. Separation under nondenaturing conditions revealed six proteins, all of which exhibited GST activity, with molecular weights ranging from 55,000 to 61,700. 4. Ethacrynic acid is a competitive inhibitor of activity towards CDNB of all six GSTs, binding each with similar affinities. 5. Chlorinated phenols are also competitive inhibitors of the enzyme, with the degree of inhibition being directly correlated with the lipophilicity of the compounds. 6. Analysis of inhibition of separated isoforms reveals that form 4 is most strongly inhibited by these chlorinated phenols, with forms 5 and 6 being inhibited to a lesser degree.     

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.     

Immunological and sequence interrelationships between multiple human liver and rat glutathione S-transferases

The 13 forms of human liver glutathione S-transferases (GST) (Vander Jagt, D. L., Hunsaker, L. A., Garcia, K. B., and Royer, R. E. (1985) J. Biol. Chem. 260, 11603-11610) are composed of subunits in two electrophoretic mobility groups: Mr = 26,000 (Ha) and Mr = 27,500 (Hb). Preparations purified from the S-hexyl GSH-linked Sepharose 4B affinity column revealed three additional peptides at Mr = 30,800, Mr = 31,200, and Mr = 32,200. Immunoprecipitation of human liver poly(A) RNAs in vitro translation products revealed three classes of GST subunits and related peptides at Mr = 26,000, Mr = 27,500, and Mr = 31,000. The Mr = 26,000 species (Ha) can be precipitated with antisera against a variety of rat liver GSTs containing Ya, Yb, and Yc subunits, whereas the Mr = 27,500 species (Hb) can be immunoprecipitated most efficiently by antiserum against the anionic isozymes as well as a second Yb-containing isozyme (peak V) from the rat liver. The Mr = 31,000 band can be immunoprecipitated by antisera preparations against sheep liver, rat liver, and rat testis isozymes. Human liver GSTs do not have any subunits of the rat liver Yc mobility. Antiserum against the human liver GSTs did not cross-react with the Yc subunits of rat livers or brains in immunoblotting experiments. The human liver GST cDNA clone, pGTH1, selected human liver poly(A) RNAs for the Ha subunit(s) in the hybrid-selected in vitro translation experiments. Southern blot hybridization results revealed cross-hybridization of pGTH1 with the Ya, Yb, and Yc subunit cDNA clones of rat liver GSTs. This sequence homology was substantiated further in that immobilized pGTH1 DNA selected rat liver poly(A) RNAs for the Ya, Yb, and Yc subunits with different efficiency as assayed by in vitro translation and immunoprecipitation. Therefore, we have demonstrated convincingly that sequence homology as well as immunological cross-reactivity exist between GST subunits from several rat tissues and the human liver. Also, the multiple forms of human liver GSTs are most likely encoded by a minimum of three different classes of mRNAs. These results suggest a genetic basis for the subunit heterogeneity of human liver GSTs.     

Trialkyl phosphorothioates and glutathione S-transferases

Using a rat liver cytosol source of enzyme trialkyl phosphorothioates have been shown to be substrates of glutathione S-transferases. Using OSS-trimethyl phosphorodithioate (OSS-Me(O] and OOS-trimethyl phosphorothioate (OOS-Me(O] the methyl transferred to the sulphydryl of glutathione is that attached to phosphorus via an oxygen atom. Fractionation of liver cytosol has shown that although the bulk activity is due to the three isozymes (1-1; 3-4; 1.2), OSS-Me(O) is a general substrate for glutathione S-transferases. The specific activity is low compared with the substrates 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene.     

Interaction of the mycotoxin penicillic acid with glutathione and rat liver glutathione S-transferases

The in vitro interaction of the mycotoxin penicillic acid (PA) with rat liver glutathione S-transferase (GST) was studied using reduced glutathione and 1-chloro-2,4-dinitrobenzene as substrates. The inhibition of the GST activity by PA in crude extracts was dose dependent. Each of the different GST isoenzymes was inhibited, albeit at different degrees. Kinetic studies never revealed competitive inhibition kinetics. The conjugation of PA with GSH occurred spontaneously; it was not enzymatically catalyzed by GST, indicating that an epoxide intermediate is not involved in conjugation. The direct binding of PA to GST provides an additional detoxication mechanism.     

Plant SCPL acyltransferases: multiplicity of enzymes with various functions in secondary metabolism

Phytochemistry Reviews - Enzymes of acyltransferase families transfer an acyl moiety from energy-rich donor molecules to various acceptors, contributing to the vast diversification of plant...     

Expression of glutathione S-transferases in rat brains

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.     

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.     

Functional promiscuity correlates with conformational heterogeneity in A-class glutathione S-transferases

The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability.     

Plant defensins: Common fold,multiple functions

Plant defensins represent a large class of structurally similar peptides found throughout the plant kingdom. Despite a conserved cysteine spacing pattern and three-dimensional structure, their sequences are highly divergent and they display a range of activities including antifungal and antibacterial activities, enzyme inhibitory activities as well as roles in heavy metal tolerance and development. The vast number of sequences along with their diverse range of activities makes it impossible to test the activity and assign function to all plant defensins. However, as the number of characterized defensins increases, in depth sequence analysis may allow us to predict the function of newly identified peptides. In this review, we analyze the sequences of defensins whose activities have been described and group these based on similarity using a maximum-likelihood phylogenetic tree. We also compare the amino acids that have been described as essential for the activity of various plant defensins between these groups. While many more plant defensins will need to be characterized before we can develop rules to predict the activity of novel sequences, this approach may prove useful in identifying structure–function relationships.     

Plant defensins: Common fold,multiple functions

Plant defensins represent a large class of structurally similar peptides found throughout the plant kingdom. Despite a conserved cysteine spacing pattern and three-dimensional structure, their sequences are highly divergent and they display a range of activities including antifungal and antibacterial activities, enzyme inhibitory activities as well as roles in heavy metal tolerance and development. The vast number of sequences along with their diverse range of activities makes it impossible to test the activity and assign function to all plant defensins. However, as the number of characterized defensins increases, in depth sequence analysis may allow us to predict the function of newly identified peptides. In this review, we analyze the sequences of defensins whose activities have been described and group these based on similarity using a maximum-likelihood phylogenetic tree. We also compare the amino acids that have been described as essential for the activity of various plant defensins between these groups. While many more plant defensins will need to be characterized before we can develop rules to predict the activity of novel sequences, this approach may prove useful in identifying structure–function relationships.     

Rat liver glutathione S-transferases. A study of the structure of the basic YbYb-containing enzymes.

A purification scheme was devised that resulted in the resolution of a number of basic glutathione S-transferases from rat liver, three of which contained two subunits of molecular mass 23500 Da (i.e. Yb monomers). These were identified as transferases D, C and A by their elution positions from CM-cellulose and their specific activities towards a variety of substrates. Hybridization, immunotitration and peptide 'mapping' experiments demonstrated that transferases D, C and A comprise Yb2Yb2, Yb1Yb2 and Yb1Yb1 subunits.     

Subunit structure of human and rat glutathione S-transferases

In rat tissues different forms of glutathione (GSH) S-transferases represent various dimeric combinations of at least four different classes of subunits categorized on the basis of their Mr values as seen on polyacrylamide gels. These subunit types represent heterogeneous populations and the actual number of subunits in rat GSH S-transferases may be far more than is known at present. Human GSH S-transferases arise from dimeric combinations of at least four immunologically and functionally distinct subunits which can be classified into three types, A (Mr 26,500), B (Mr 24,500) and C (Mr 22,500). There is evidence for considerable charge heterogeneity in each of these subunit types.     

Polymorphisms in glutathione S-transferases in French vinyl chloride workers

The authors have recently demonstrated a significant gene–environment interaction between vinyl chloride exposure and polymorphisms in the DNA repair protein XRCC1 on the occurrence of mutant p53 biomarkers of vinyl chloride-induced genetic damage. The aim of this study was to examine the polymorphisms in the glutathione S-transferases (GSTs) as potential modifiers of this relationship, since these enzymes may be involved in the phase II metabolism of the reactive intermediates of vinyl chloride. A cohort of 211 French vinyl chloride workers was genotyped for common polymorphisms in GSTM1, GSTT1 and GSTP1. Although no independent, statistically significant effect of these polymorphisms on the occurrence of the mutant p53 biomarker was found, the null GSTM1 and null GSTT1 polymorphisms were found to interact with the XRCC1 polymorphism to increase the occurrence of the biomarker such that, for example, workers with at least one variant XRCC1 allele who were null for both GSTM1 and GSTT1 had a significant odds ratio for the biomarker (OR=8.4, 95% CI=1.3–54.0) compared with workers who were wild-type for all alleles, controlling for potential confounders including cumulative vinyl chloride exposure.     

Autophagy: in sickness and in health

The degradation of intracellular components in lysosomes (autophagy) has recaptured the attention of cell biologists in recent years. The main reason for this renewed interest is the dissection of the molecular machinery that participates in this process, because the identification of new intracellular elements involved in autophagy has provided new tools to trace, quantify and manipulate autophagy in a growing number of organisms. As a result, a better understanding of the physiological roles of autophagy, the consequences of its malfunctioning and its participation in different pathological processes has emerged. This article reviews our current knowledge of the role of autophagy in disease and the efforts to reconcile its proposed dual function as both a cell protector and a cell killer.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

Mitochondria: in sickness and in health

Mitochondria perform diverse yet interconnected functions, producing ATP and many biosynthetic intermediates while also contributing to cellular stress responses such as autophagy and apoptosis. Mitochondria form a dynamic, interconnected network that is intimately integrated with other cellular compartments. In addition, mitochondrial functions extend beyond the boundaries of the cell and influence an organism's physiology by regulating communication between cells and tissues. It is therefore not surprising that mitochondrial dysfunction has emerged as a key factor in a myriad of diseases, including neurodegenerative and metabolic disorders. We provide a current view of how mitochondrial functions impinge on health and disease.