Five Decades with Glutathione and the GSTome
2012; Elsevier BV; Volume: 287; Issue: 9 Linguagem: Inglês
10.1074/jbc.x112.342675
ISSN1083-351X
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoUncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept "molecular quasi-species," we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution. Uncle Folke inspired me to become a biochemist by demonstrating electrophoresis experiments on butterfly hemolymph in his kitchen. Glutathione became the subject for my undergraduate project in 1964 and has remained a focal point in my research owing to its multifarious roles in the cell. Since the 1960s, the multiple forms of glutathione transferase (GST), the GSTome, were isolated and characterized, some of which were discovered in our laboratory. Products of oxidative processes were found to be natural GST substrates. Examples of toxic compounds against which particular GSTs provide protection include 4-hydroxynonenal and ortho-quinones, with possible links to the etiology of Alzheimer and Parkinson diseases and other degenerative conditions. The role of thioltransferase and glutathione reductase in the cellular reduction of disulfides and other oxidized forms of thiols was clarified. Glyoxalase I catalyzes still another glutathione-dependent detoxication reaction. The unusual steady-state kinetics of this zinc-containing enzyme initiated model discrimination by regression analysis. Functional properties of the enzymes have been altered by stochastic mutations based on DNA shuffling and rationally tailored by structure-based redesign. We found it useful to represent promiscuous enzymes by vectors or points in multidimensional substrate-activity space and visualize them by multivariate analysis. Adopting the concept "molecular quasi-species," we describe clusters of functionally related enzyme variants that may emerge in natural as well as directed evolution. I learned about the scientific discipline of biochemistry as a young teenager. My uncle in Uppsala, Folke Fridén, married to my father's sister Sigrid, was the sage. Uncle Folke was an alumnus of Uppsala University but never became a member of any academic institution. Instead, he privately followed his own interests in science and arts, which he cultivated by weekly visits to the university library, Carolina Rediviva. Since childhood, Uncle Folke had a special interest in insects, and he taught me and my younger brother, Gunnar, how to find caterpillars as well as eggs of moths and butterflies on plants. The eggs were placed in cotton-plugged glass tubes, in which the hatched first-instar larvae were fed fresh leaves daily. Larger caterpillars were reared in gauze-covered glass jars, and we could watch them pupate and eventually emerge as imagos in the form of butterflies and moths. Uncle Folke entrusted us to deliver pupae of numerous species of moths, and he stored them in his refrigerator to undergo diapause for subsequent biochemical experiments. He paid us at the rate of one Swedish crown for each large sphingid pupa and less for smaller specimens. Uncle Folke was extremely meticulous and advised us to collect the larval food from plants of the same clones and biotopes to minimize nutritional variations. Some leaves derived from bushes growing three miles away from the vacation house where we spent the summers, and we went by bicycle to the site to collect twigs several times a week. The kitchen in the small Uppsala apartment at Vaksalagatan was used as a laboratory by Uncle Folke. He showed us how amino acids in insect tissues could be separated by two-dimensional paper chromatography and be detected with ninhydrin. Shortly after publication of the new method of starch gel electrophoresis (1.Smithies O. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults.Biochem. J. 1955; 61: 629-641Crossref PubMed Scopus (925) Google Scholar), Uncle Folke separated the proteins in the hemolymph of larvae and pupae. He identified pigmented proteins in all species analyzed. Some moths had several colored proteins, but the predominant component appeared to be blue-green. Inspired by the nomenclature for human globulins coined by Nobel Laureate Arne Tiselius of Uppsala University, Uncle Folke designated the main pigmented protein by the Greek letters χρ (chi rho) to indicate its colored nature (Greek, chroma). Approximately thirty years later, in a seminar at the Uppsala Biomedical Center, I witnessed with astonishment Robert Huber describe the structure of a colored bilin-binding protein from Pieris brassicae (the large white butterfly). I told Huber that, no doubt, he and his co-workers had analyzed a protein that I had seen as a distinct band in starch gels in my uncle's kitchen. Much later, I found out that the protein had also been described in the hemolymph of the sphingid moth Manduca sexta (the tobacco hornworm) and given the name "insecticyanin" (see Ref. 2.Holden H.M. Rypniewski W.R. Law J.H. Rayment I. The molecular structure of insecticyanin from the tobacco hornworm Mandica sexta L. at 2.6 Å resolution.EMBO J. 1987; 6: 1565-1570Crossref PubMed Scopus (172) Google Scholar). In 1958, my parents, Lisbeth and Tage Eriksson, received a thesis on the energy metabolism of caterpillars written by my uncle (3.Fridén F. Frass-Drop Frequency in Lepidoptera. Almqvist & Wiksell, Uppsala1958Google Scholar). They appreciated the gift, but, not being scientists, they could not digest its contents. However, I was intrigued by the simple but clever idea of using the defecation frequency of larvae as a measure of their food consumption, and with my drive as a 15-year-old, I read the thesis voraciously. Many scientific terms such as "cytochromes" and "endocrine secretion" were unknown to me, and I had to consult major textbooks of biochemistry in the City Library of Stockholm for enlightenment. My parents were very supportive and provided me with scientific monographs as Christmas gifts and birthday presents in the ensuing years. I read texts by Linus Pauling, Albert Szent-Györgyi, Joe Neilands, and Paul Stumpf, and other scientists, whom I got to meet personally later in my scientific life. By 1962, I had decided to aim for a career as a biochemist and enrolled as a student majoring in chemistry at Stockholm University. During my undergraduate studies, I wanted to take advantage of the summer vacation to obtain hands-on experience of biochemical research in an academic setting. The summer of 1963 was spent in the Nobel Medical Institute of the Karolinska Institutet at the Department of Biochemistry, headed by Hugo Theorell. I got to work with Göran Eriksson, a graduate student synthesizing isotope-substituted flavin molecules to elucidate the electron spin density distribution in radicals of the cofactor. His supervisor was Anders Ehrenberg, one of the pioneers in using paramagnetic susceptibility and electron spin resonance for studies of biomolecules. In the following summer, before enrolling in the biochemistry course at Stockholm University, I was permitted to carry out my undergraduate degree project with Bo Sörbo at the Swedish Research Institute of National Defense. Sörbo was a devoted sulfur biochemist who had obtained his Ph.D. degree with Theorell on the enzyme rhodanese, which catalyzes the formation of rhodanide (thiocyanate) from thiosulfate and cyanide. Rhodanese is the only enzyme with a name ending in "ese," the reason being that it was considered a synthetic enzyme, in distinction from the "ase" enzymes involved in degradation of carbohydrates, proteins, and other substrates. My assignment was to prepare a new derivative of glutathione, the thiosulfonate that could potentially be formed in biological tissues subjected to irradiation. In thiosulfonates, one of the oxygens in a sulfonate is replaced by a labile bivalent sulfur (similar to that in inorganic thiosulfate). I demonstrated that rhodanese catalyzes transsulfuration from glutathione thiosulfonate to cyanide (4.Eriksson B. Sörbo B. The synthesis and some properties of the thiosulfonate analogue of glutathione (γ-l-glutamyl-l-3-thiosulfoalanylglycine).Acta Chem. Scand. 1967; 21: 958-960Crossref Google Scholar). The stay in Sörbo's laboratory also allowed time for additional enzymology, and I purified glutathione reductase from bovine liver for assays with my new compound. That summer, I became acquainted with the literature describing the multifarious roles of glutathione. A few years later, I read the Nature paper "Lest I Forget Thee, Glutathione … " by Edward and Nechama Kosower (5.Kosower E.M. Kosower N.S. Lest I forget thee, glutathione ….Nature. 1969; 224: 117-120Crossref PubMed Scopus (146) Google Scholar), and I have certainly not forgotten glutathione since the summer of 1964. In the fall of 1964, after completing my course of biochemistry, I sought admission as a graduate student to the Department of Biochemistry of Stockholm University. I was known to Klas-Bertil Augustinsson since I had authored an article on the biochemical origin of life in one of the major daily newspapers, "Svenska Dagbladet." My sources were books by Oparin, Calvin, Darwin, Chardin, and others, as well as the classic paper by Miller and Urey, and I attempted to put them all in perspective for a general readership. Augustinsson provided bench space in his laboratory, and I was given a teaching assistantship in the department at the beginning of 1965. Once admitted to the laboratory, I was asked what I wanted to work on, and I suggested the flavoenzyme glutathione reductase, in line with my prior experience with flavins and glutathione. Augustinsson, himself an authority on cholinesterase, found the proposal excellent because "nobody in the department was working on oxidoreductases." I faced the challenge, surprised that he did not want me to investigate esterases. In theory as well as in practice, I was thus left to be my own mentor, even though Augustinsson generously allowed me to use equipment and chemicals in his laboratory. As a teaching assistant, I came in contact with students of biochemistry, and I made some of them interested in my research. In the fall of 1965, Viveka Schalén started to work with me for her degree project. I had been authorized by Karl Myrbäck to serve as a supervisor. Myrbäck was the first professor of biochemistry at Stockholm University. His mentor was Hans von Euler-Chelpin, professor of general and organic chemistry in Stockholm, actively conducting biochemical research in the neighboring building until the age of 91. Myrbäck was best known to biochemists as one of the editors of the standard treatise "The Enzymes," with Paul Boyer and Henry Lardy, a follow-up of the first edition by James Sumner and Myrbäck. Myrbäck was also the founder and editor-in-chief of Acta Chemica Scandinavica. In this journal, I published my first paper (6.Eriksson B. On the synthesis and enzymatic reduction of the coenzyme A-glutathione mixed disulfide.Acta Chem. Scand. 1966; 20: 1178-1179Crossref PubMed Google Scholar) on the synthesis of the mixed disulfide of glutathione and coenzyme A. (At the time, my name was Bengt Eriksson; the surname Mannervik was adopted from my maternal grandparents by me and my brothers in 1968.) By the time of my Ph.D. dissertation (7.Mannervik, B., (1969) Syntheses, Quantitative Analyses, and Enzymatic Reactions of Some Naturally Occurring Glutathione Sulfenyl Derivatives. Ph.D. dissertation, Stockholm University,.Google Scholar), I had already formed a small research group in the department. Funding for the subsequent work was obtained from the Swedish Natural Science Research Council and the Swedish Cancer Society. Glyoxalase I was used in an enzymatic assay specific for reduced glutathione in our research. We started to characterize the commercially available yeast enzyme and proceeded with studies of the enzyme purified from other sources. We developed an affinity gel by linking S-hexylglutathione to a matrix (8.Aronsson A.C. Mannervik B. Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography.Biochem. J. 1977; 165: 503-509Crossref PubMed Scopus (53) Google Scholar), purified the enzyme, and demonstrated that the protein was a zinc-dependent metalloenzyme (9.Aronsson A.C. Marmstål E. Mannervik B. Glyoxalase I, a zinc metalloenzyme of mammals and yeast.Biochem. Biophys. Res. Commun. 1978; 81: 1235-1240Crossref PubMed Scopus (91) Google Scholar) that was active also with several other divalent metal ions. The mammalian enzyme is a homodimer with two active sites at the interface between the subunits (10.Cameron A.D. Olin B. Ridderström M. Mannervik B. Jones T.A. Crystal structure of human glyoxalase I: evidence for gene duplication and three-dimensional domain swapping.EMBO J. 1997; 16: 3386-3395Crossref PubMed Scopus (214) Google Scholar). By contrast, we subsequently found that glyoxalase I from Saccharomyces cerevisiae is a monomer with two active sites in the same polypeptide chain (11.Frickel E.M. Jemth P. Widersten M. Mannervik B. Yeast glyoxalase I is a monomeric enzyme with two active sites.J. Biol. Chem. 2001; 276: 1845-1849Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Detailed steady-state kinetic studies of glyoxalase I demonstrated unconventional rate saturation behavior. The analysis prompted the development of procedures for discrimination between alternative rate equations. We started out using algorithms published by Wallace W. Cleland (12.Cleland W.W. Statistical analysis of enzyme kinetic data.Adv. Enzymol. 1967; 29: 1-32PubMed Google Scholar). However, my newly recruited student, Tamas Bártfai, refined these methods in his thesis work (13.Bártfai T. Mannervik B. A procedure based on statistical criteria for discrimination between steady-state kinetic models.FEBS Lett. 1972; 26: 252-256Crossref PubMed Scopus (37) Google Scholar) using glyoxalase I as the test case (14.Mannervik B. Górna-Hall B. Bártfai T. The steady-state kinetics of glyoxalase I from porcine erythrocytes. Evidence for a random-pathway mechanism involving one- and two-substrate branches.Eur. J. Biochem. 1973; 37: 270-281Crossref PubMed Scopus (29) Google Scholar). Our main emphasis was the discrimination between alternative mathematical models rather than parameter estimation (15.Mannervik B. Purich D. Contemporary Enzyme Kinetics and Mechanism. Elsevier, Amsterdam2009: 73-94Google Scholar). Tamas, who graduated in 1973, became my first student to receive a Ph.D. degree. Leopold Flohé had taken notice of our research and invited me as a speaker to an international conference on glutathione in March 1973 in Tübingen, Germany. I had the unusual privilege of being asked to give three full lectures: one on glutathione reductase, one on glyoxalase I, and one on glutathione-dependent transferase reactions. At the conference, our findings of isoenzymes of what was called glutathione S-aryltransferase were first presented to an international audience (16.Mannervik B. Eriksson S.A. Flohé L. Benöhr H.C. Sies H. Waller H.D. Wendel A. Glutathione. Georg Thieme Publishers, Stuttgart, Germany1974: 120-131Google Scholar). Enzymes present in the cytosolic fraction of rat liver were separated by isoelectric focusing, and activities were determined with distinguishing substrates in the different fractions. My interest in naturally occurring mixed disulfides and their reversible thiol-disulfide interchange led to the clarification of the intermediacy of a low-Mr enzyme catalyzing the reactions. We purified the enzyme from rat liver and human placenta and demonstrated that it catalyzes the reduction of mixed disulfides of glutathione, but also disulfides in general, as well as thiosulfate esters such as S-sulfoglutathione and S-sulfocysteine (17.Axelsson K. Eriksson S. Mannervik B. Purification and characterization of cytoplasmic thioltransferase (glutathione:disulfide oxidoreductase) from rat liver.Biochemistry. 1978; 17: 2978-2984Crossref PubMed Scopus (97) Google Scholar). On the basis of its function, we named the enzyme thioltransferase (18.Askelöf P. Axelsson K. Eriksson S. Mannervik B. Mechanism of action of enzymes catalyzing thiol-disulfide interchange. Thioltransferases rather than transhydrogenases.FEBS Lett. 1974; 38: 263-267Crossref PubMed Scopus (51) Google Scholar); the corresponding Escherichia coli enzyme was later renamed as a member of the glutaredoxin family (19.Lillig C.H. Berndt C. Holmgren A. Glutaredoxin systems.Biochim. Biophys. Acta. 2008; 1780: 1304-1317Crossref PubMed Scopus (486) Google Scholar). We showed that thioltransferase catalyzes the reversible formation of mixed disulfides of glutathione and proteins, in addition to small substrates, and our work was summarized in a symposium in 1978 at Schloss Reisensburg (Fig. 1). We proposed that, in addition to its central role in thiol and disulfide metabolism, thioltransferase could mediate redox regulation of cellular functions via sulfhydryl modification of proteins (20.Mannervik B. Axelsson K. Role of cytoplasmic thioltransferase in cellular regulation by thiol-disulfide interchange.Biochem. J. 1980; 190: 125-130Crossref PubMed Scopus (127) Google Scholar). The notion of protein-glutathione mixed disulfide formation gained increased attention in the mid-1990s under the short name of "protein thiolation." I recommended the more accurate name "glutathionylation" to my colleague Ian Cotgreave (21.Lind C. Gerdes R. Schuppe-Koistinen I. Cotgreave I.A. Studies on the mechanism of oxidative modification of human glyceraldehyde-3-phosphate dehydrogenase by glutathione: catalysis by glutaredoxin.Biochem. Biophys. Res. Commun. 1998; 247: 481-486Crossref PubMed Scopus (94) Google Scholar), and this designation is now generally used in the steadily increasing literature on this important protein modification. The thioltransferase reactions generated glutathione disulfide, and my interest in glutathione reductase catalyzing the regeneration of reduced glutathione remained active. The purification of the enzyme from porcine erythrocytes bought cheaply from a local slaughterhouse was only partly successful, and my early attempts in the late 1960s to purify the flavoprotein glutathione reductase on riboflavin immobilized on cellulose did not succeed. Only after introduction of the affinity matrix 2′,5′-ADP-Sepharose, developed by Klaus Mosbach, was a facile purification procedure established (22.Mannervik B. Jacobsson K. Boggaram V. Purification of glutathione reductase from erythrocytes by the use of affinity chromatography on 2′,5′-ADP-Sepharose 4B.FEBS Lett. 1976; 66: 221-224Crossref PubMed Scopus (33) Google Scholar). I formulated a novel "branching mechanism" consisting of a fusion of the well established ping-pong and sequential mechanisms for two-substrate reactions (23.Mannervik B. A branching reaction mechanism of glutathione reductase.Biochem. Biophys. Res. Commun. 1973; 53: 1151-1158Crossref PubMed Scopus (49) Google Scholar) and found an uncoupling of electron transfer from FAD to disulfide by nitroaromatic compounds, converting the enzyme into an oxidase (24.Carlberg I. Mannervik B. Reduction of 2,4,6-trinitrobenzenesulfonate by glutathione reductase and the effect of NADP+ on the electron transfer.J. Biol. Chem. 1986; 261: 1629-1635Abstract Full Text PDF PubMed Google Scholar). The flavoenzyme guru Vincent Massey in Ann Arbor became my friend in 1969 and remained a source of inspiration for many years. GST activity in liver cytosol was first reported in 1961 by Eric Boyland and co-workers (25.Booth J. Boyland E. Sims P. An enzyme from rat liver catalyzing conjugations with glutathione.Biochem. J. 1961; 79: 516-524Crossref PubMed Google Scholar) and by Combes and Stakelum (26.Combes B. Stakelum G.S. A liver enzyme that conjugates sulfobromophthalein sodium with glutathione.J. Clin. Invest. 1961; 40: 981-988Crossref PubMed Scopus (78) Google Scholar). About a decade later, I serendipitously became acquainted with these important enzymes, catalyzing the conjugation of glutathione with electrophiles, when new graduate students called for novel projects. With time, GSTs became a major theme in the laboratory, and the enzymes have remained a vital topic in my research for about forty years. The pivotal role of GSTs in the protection of cells against electrophilic xenobiotics, including carcinogens, had been made clear by Boyland and co-workers (27.Boyland E. Chasseaud L.F. Role of glutathione and glutathione S-transferases in mercapturic acid biosynthesis.Adv. Enzymol. 1969; 32: 173-219PubMed Google Scholar), but the enzymes had not been separately identified and purified. Using high-resolution methods such as gradient elution from ion-exchange columns and isoelectric focusing, we purified and characterized several GSTs with partially overlapping substrate selectivities from rat liver (28.Askelöf P. Guthenberg C. Jakobson I. Mannervik B. Purification and characterization of two glutathione S-aryltransferase activities from rat liver.Biochem. J. 1975; 147: 513-522Crossref PubMed Scopus (143) Google Scholar). We subsequently purified GSTs from all major organs and found that the "isoenzyme" distribution was different among the tissues (Fig. 2) (29.Mannervik B. Guthenberg C. Jensson H. Warholm M. Ålin P. Larsson A. Orrenius S. Holmgren A. Mannervik B. Functions of Glutathione: Biochemical, Physiological, Toxicological, and Clinical Aspects. Raven Press, New York1983: 75-88Google Scholar). Furthermore, the multiple forms in the rat and in the mouse were shown not only to be differentially expressed in normal tissues but also to respond differentially to inducers of drug and carcinogen metabolism (30.Guthenberg C. Morgenstern R. DePierre J.W. Mannervik B. Induction of glutathione S-transferases A, B, and C in rat liver cytosol by trans-stilbene oxide.Biochim. Biophys. Acta. 1980; 631: 1-10Crossref PubMed Scopus (65) Google Scholar, 31.Di Simplicio P. Jensson H. Mannervik B. Effects of inducers of drug metabolism on basic hepatic forms of mouse glutathione transferase.Biochem. J. 1989; 263: 679-685Crossref PubMed Scopus (50) Google Scholar). Neoplastic transformations also demonstrated major changes in GST expression such that the Pi class GST P1-1 represented 85–90% of the GST activity in ascites hepatoma cells, whereas normal hepatocytes have no detectable GST P1-1 expression (32.Tahir M.K. Guthenberg C. Mannervik B. Glutathione transferases in rat hepatoma cells. Effects of ascites cells on the isoenzyme pattern in liver and induction of glutathione transferases in the tumor cells.Biochem. J. 1989; 257: 215-220Crossref PubMed Scopus (15) Google Scholar). Our finding that human malignant melanoma cells overexpress GST P1-1 suggested a role of the enzyme in tumor drug resistance (33.Mannervik B. Castro V.M. Danielson U.H. Tahir M.K. Hansson J. Ringborg U. Expression of class Pi glutathione transferase in human malignant melanoma cells.Carcinogenesis. 1987; 8: 1929-1932Crossref PubMed Scopus (113) Google Scholar, 34.Hansson J. Berhane K. Castro V.M. Jungnelius U. Mannervik B. Ringborg U. Sensitization of human melanoma cells to the cytotoxic effect of melphalan by the glutathione transferase inhibitor ethacrynic acid.Cancer Res. 1991; 51: 94-98PubMed Google Scholar). The significant role of GSTs in detoxication reactions made it imperative to purify and study the human complement of the enzymes. Full-term placentas were obtained from a maternity ward, and we discovered that the tissue was dominated by a single GST (35.Guthenberg C. Åkerfeldt K. Mannervik B. Purification of glutathione S-transferase from human placenta.Acta Chem. Scand. 1979; B33: 595-596Crossref Scopus (65) Google Scholar), later called GST P1-1 of the Pi class. William Jakoby and associates had previously found a GST in erythrocytes, and we concluded that it was the enzyme also found in placenta (36.Guthenberg C. Mannervik B. Glutathione S-transferase (transferase π) from human placenta is identical or closely related to glutathione S-transferase (transferase ρ) from erythrocytes.Biochim. Biophys. Acta. 1981; 661: 255-260Crossref PubMed Scopus (111) Google Scholar). The same enzyme was found in fetal (but not adult) human liver (37.Guthenberg C. Warholm M. Rane A. Mannervik B. Two distinct forms of glutathione transferase from human fetal liver. Purification and comparison with isoenzymes isolated from adult liver and placenta.Biochem. J. 1986; 235: 741-745Crossref PubMed Scopus (37) Google Scholar). Adult human livers showed individual differences in their expression of GSTs, in particular with respect to a novel enzyme with distinctive catalytic properties (38.Warholm M. Guthenberg C. Mannervik B. von Bahr C. Glaumann H. Identification of a new glutathione S-transferase in human liver.Acta Chem. Scand. 1980; B34: 607-610Crossref Scopus (57) Google Scholar). This enzyme, now named GST M1-1 of the Mu class, is present only in approximately half of the human population, and our discovery of the GST null phenotype appeared particularly significant in view of a high catalytic activity with carcinogenic epoxides (39.Warholm M. Guthenberg C. Mannervik B. von Bahr C. Purification of a new glutathione S-transferase (transferase μ) from human liver having high activity with benzo(a)pyrene 4,5-oxide.Biochem. Biophys. Res. Commun. 1981; 98: 512-519Crossref PubMed Scopus (119) Google Scholar). Establishment of this first GST polymorphism and the subsequent demonstration of the null allele of the corresponding gene (40.Seidegård J. Vorachek W.R. Pero R.W. Pearson W.R. Hereditary differences in the expression of the human glutathione transferase active on trans-stilbene oxide are due to a gene deletion.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 7293-7297Crossref PubMed Scopus (722) Google Scholar) have spawned numerous epidemiological studies. The physicochemical properties indicated that the soluble GSTs found in the cytosolic fraction of mammalian cells are dimers, and inhibition studies with homologous S-alkylglutathiones demonstrated enhanced affinity with increased chain length of the alkyl group (28.Askelöf P. Guthenberg C. Jakobson I. Mannervik B. Purification and characterization of two glutathione S-aryltransferase activities from rat liver.Biochem. J. 1975; 147: 513-522Crossref PubMed Scopus (143) Google Scholar). Kinetic and equilibrium binding studies established high specificity for glutathione bound at a ratio of 1:1 per subunit, and the electrophilic substrates appeared to bind with similar stoichiometry at a less specific hydrophobic site. We named the two positions the "G-site" and the "H-site," respectively (41.Mannervik B. Guthenberg C. Jakobson I. Warholm M. Aitio A. Conjugation Reactions in Drug Biotransformation. Elsevier North-Holland, Amsterdam1978: 101-110Google Scholar). In the late 1970s, data appeared suggesting that the major rat liver enzyme GST B, also known as "ligandin," was composed of two non-identical subunits. However, SDS-PAGE analysis showed different amounts of the constituent subunits, indicating that the ligandin preparations were not homogeneous. Our characterization of the six major GSTs in rat liver showed that they could be divided into two groups of isoenzymes, each of which contains two subunits in homodimeric and heterodimeric combinations (42.Mannervik B. Jensson H. Binary combinations of four protein subunits with different catalytic specificities explain the relationship between six basic glutathione S-transferases in rat liver cytosol.J. Biol. Chem. 1982; 257: 9909-9912Abstract Full Text PDF PubMed Google Scholar). The functional properties of the heterodimers could be predicted from linear combinations of the properties of the corresponding homodimers (42.Mannervik B. Jensson H. Binary combinations of four protein subunits with different catalytic specificities explain the relationship between six basic glutathione S-transferases in rat liver cytosol.J. Biol. Chem. 1982; 257: 9909-9912Abstract Full Text PDF PubMed Google Scholar, 43.Tahir M.K. Mannervik B. Simple inhibition studies for distinction between homodimeric and heterodimeric isoenzymes of glutathione transferase.J. Biol. Chem. 1986; 261: 1048-1051Abstract Full Text PDF PubMed Google Scholar). We proposed that the enzymes should be named in accord with their subunit composition, a principle still followed (44.Mannervik B. Board P.G. Hayes J.D. Listowsky I. Pearson W.R. Nomenclature for mammalian soluble glutathione transferases.Methods Enzymol. 2005; 401: 1-8Crossref PubMed Scopus (24
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