Purification and characterization of glutathione S-transferases from rat pancreas

Glutathione S-transferases (GSTs) of rat pancreas have been characterized and their interrelationship with fatty acid ethyl ester synthase (FAEES) has been studied. Seven GST isozymes with pI values of 9.2, 8.15, 7.8, 7.0, 6.3, 5.9 and 5.4 have been isolated and designated as rat pancreas GST suffixed by their pI values. Structural, immunological and kinetic properties of these isozymes indicated that GST 9.2 belonged to the alpha class, GST 7.8, 7.0, 6.3 and 5.9 belonged to the mu class, whereas GST 8.15 and 5.4 belong to pi class. The N-terminal sequences and pI values of the mu class isozymes suggested that rat GST subunits 3, 4 and 6 may be expressed in pancreas. N-Terminal sequences of both the pi class isozymes, GST 8.15 and 5.4, were similar to that of GST-P, but there were significant differences in the substrate specificities of these two enzymes. Results of peptide finger print studies also indicated minor structural differences between these two isozymes. None of the GST isozymes of rat pancreas expressed FAEES activity. Rat pancreas had a significant amount of FAEES activity, but it segregated independently during the purification of GST indicating that these two activities are expressed by different proteins and are not related as suggested previously.     

Purification and characterization of glutathione S-transferases from guinea pig liver

Four types of glutathione S-transferase were purified to homogeneity from guinea pig liver by DEAE-cellulose, Sephadex G-75, CM-cellulose, and affinity chromatography. These isozymes were named a, b, c, and d based on the reverse order of elution from a CM-cellulose column, and had specific activities of 89.6, 92.2, 99.0, and 44.0 units/mg, respectively, when assayed with 1 mM each of 1-chloro-2,4-dinitrobenzene and reduced glutathione. All four transferases of guinea pig liver were homodimers. The transferases b, c, and d had a similar molecular weight of 50,000 and their subunit sizes were 25,000, but the corresponding values for transferase a were 45,000 and 23,500, respectively. Transferase a was notably different in the activities towards organic hydroperoxides and 1,2-dichloro-4-nitrobenzene from the other isozymes. Transferases a and b, the major forms in guinea pig liver, were studied with respect to their biochemical properties, including kinetic parameters, absorption and fluorescence spectra, and bilirubin binding. Glutathione peroxidase activity of the transferase a was about 100 times higher than that of other isozymes. In guinea pig liver, it is estimated that transferase a is the major glutathione peroxidase, accounting for about 75% of the total organic hydroperoxide reduction.     

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

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.     

Rat liver glutathione S-transferases. Nucleotide sequence analysis of a Yb1 cDNA clone and prediction of the complete amino acid sequence of the Yb1 subunit

We have constructed a nearly full length cDNA clone, pGTA/C44, complementary to the rat liver glutathione S-transferase Yb1 mRNA. The nucleotide sequence of pGTA/C44 has been determined, and the complete amino acid sequence of the Yb1 subunit has been deduced. The cDNA clone contains an open reading frame of 654 nucleotides encoding a polypeptide comprising 218 amino acids with Mr = 25,919. The NH2-terminal sequence deduced from DNA sequence analysis of pGTA/C44 is in agreement with the first 19 amino acids determined for purified glutathione S-transferase A, a Yb1 homodimer, by Frey et al. (Frey, A. B., Friedberg, T., Oesch, F., and Kreibich, G. (1983) J. Biol. Chem. 258, 11321-11325). The DNA sequence of pGTA/C44 shares significant sequence homology with a cDNA clone, pGT55, which is complementary to a mouse liver glutathione S-transferase (Pearson, W. R., Windle, J. J., Morrow, J. F., Benson, A. M., and Talalay, P. (1983) J. Biol. Chem. 258, 2052-2062). We have also determined 37 nucleotides of the 5'-untranslated region and 348 nucleotides of the 3'-untranslated region of the Yb1 mRNA. The Yb1 mRNA and subunit do not share any sequence homology with the rat liver glutathione S-transferase Ya or Yc mRNAs or their corresponding subunits. These data provide the first direct evidence that the Yb1 subunit is derived from a gene or gene family which is distinct from the Ya-Yc gene family.     

Newly identified bile acid binders in rat liver cytosol. Purification and comparison with glutathione S-transferases

Gel filtration of male rat liver cytosol preincubated with radiolabeled lithocholic, chenodeoxycholic, and glycochenodeoxycholic acids, and taurocholic acid revealed two major peaks of radioactivity, one co-eluting with the glutathione S-transferases and the other with a separate fraction, respectively. Chromatofocusing of the pooled fractions containing the new bile acid binding activity resulted in a separation of bile acid binding from the previously described organic anion binding activity in this fraction. Two binding peaks for lithocholic acid (pI 5.6, Binder I, and pI 5.5, Binder II) were identified on chromatofocusing and were further purified to apparent homogeneity by hydroxyapatite chromatography. The two Binders were monomers having identical molecular weight (33,000) and similar amino acid compositions. Bile acid binding to purified Binders I and II and glutathione S-transferases A, B, and C was studied by inhibition of the fluorescence of bound 1-anilino-8-naphthalenesulfonate (ANS). Confirmatory experiments using equilibrium dialysis produced comparable results. Glutathione S-transferase B had greater affinity for bile acids than transferases A or C. Binder II, which had greater affinity than Binder I for most bile acids, had greater affinity for chenodeoxycholic acid than transferase B but comparable or lower affinities for the other bile acids. All bile acids studied diminished ANS fluorescence with Binder II. Taurocholic and cholic acids increased ANS fluorescence with Binder I without affecting KANS, whereas lithocholic and chenodeoxycholic acids diminished ANS fluorescence with Binder I. In summary, we have identified and isolated two proteins (Binders I and II) which, along with glutathione S-transferase B, are the major hepatic cytosol bile acid binding proteins; these proteins have overlapping but distinct specificities for various bile acids.     

Purification and characterization of the flavoenzyme glutathione reductase from rat liver.

Glutathione reductase from rat liver has been purified greater than 5000-fold in a yield of 20%. The molecular weights of the enzyme and its subunits were estimated to be 125,000 and 60,000, respectively, indicating that the native enzyme is a dimer. The enzyme molecular contains 2 FAD molecules, which are reducible by NADPH, GSH or dithioerythritol. The reduced flavin is instantaneously reoxidized by addition of GSSG. The steady state kinetic data are consistent with a branching reaction mechanism previously proposed for glutathione reductase from yeast (MANNERVIK, B. (1973) Biochem. Biophy. Res. Commun. 53, 1151-1158). This mechanism is also favored by the nonlinear inhibition pattern produced by NADP-+. However, at low GSSG concentrations the rate equation can be approximated by that of a simple ping pong mechanism. NADPH and the mixed disulfide of coenzyme A and GSH were about 10% as active as NADPH and GSSG, respectively, whereas some sulfenyl derivatives related to GSSG were less active as substrates. The pH activity profiles of these substrates differed from that of the NADPH-GSSG substrate pair.     

Purification and characterization of two glutathione S-aryltransferase activities from rat liver.

Two forms of glutathione S-aryltransferase were purified from rat liver. The only differences noted between the two forms were in the chromatographic and electrophoretic properties, which permitted the separation of the two species. The molecular weights of the enzyme and its subunits were estimated as about 50000 and 23000 respectively. The steady-state kinetics did no follow Michaelis-Menten kinetics when one substrate concentration was kept constant while the second substrate concentration was varied. Several S-substituted GSH derivatives were tested as inhibitors of the enzymic reaction. The enzyme was inactivated by thiol-group reagents.     

A comparison of the glutathione S-transferases of trout and rat liver.

1. Cytosol from trout liver, gills and intestinal caeca has substantial glutathione S-transferase activity. 2. Gel-exclusion and ion-exchange chromatography suggest that trout liver has several glutathione S-transferases with different molecular weights and ionic charges. 3. A component capable of binding lithocholic acid eluted together with glutathione S-transferase activity. Some of the transferase activity did not elute together with binding activity. 4. The enzymic activity from trout liver was less stable at 37 degrees C than that from rat liver. 5. The glutathione S-transferases of fish liver have a similar specific activity to those of rat liver but different molecular properties.     

Cellular retinol-binding protein from rat liver. Purification and characterization

Cellular retinol-binding protein has been purified to homogeneity from rat liver. The procedures utilized in the purification included acid precipitation, gel filtration on Sephadex G-75 and G-50, and chromatography on DEAE-cellulose. The binding protein was purified approximately 3,500-fold, based on total soluble liver protein. The protein is a single polypeptide chain with a molecular weight of 14,600 based on information obtained by the techniques of sedimentation equilibrium analysis, gel filtration, and sodium dodecyl sulfate-polyacrylamide electrophoresis. The protein binds retinol with high affinity; the appparent dissociation constant was determined by fluorometric titration to be 1.6 X 10(-8) M. Retinol bound to the protein has an absorption spectrum (lambdamax, 350 nm) considerably altered from the spectrum of retinol in ethanol (lambdamax, 325 nm).     

Purification of glutathione S-transferases from rat lung by affinity chromatography. Evidence for an enzyme form absent in rat liver.

Glutathione $\text{S}$-transferases from rat lung cytosol were purified about 200-fold in one step by chromatography on $\text{S}$-hexylglutathione bound to epoxy-activated Sepharose 6B. Further purification on hydroxyapatite resolved the lung transferases into five peaks of activity as measured with 1-chloro-2,4-dinitrobenzene as substrate. Three of the peaks were identified with transferases A, B, and C of rat liver on the basis of chromatographic properties, immunochemical reactivity, and substrate specificity. The other two activity peaks were not detectable in liver: one originated from the lung tissue and one appeared to result from blood in the lung.     

Purification, characterization, and subunit structure of rat core 1 Beta1,3-galactosyltransferase.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1-->Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.     

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.     

Bile acid inhibition of basic and neutral glutathione S-transferases in rat liver.

A purification scheme is described for the neutral glutathione S-transferases of rat liver. Discontinuous sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that one of these enzymes contains a previously unidentified subunit, which has a molecular mass of 23 000 Da and has been designated Yn. Bile acids inhibited the activity of all the basic and neutral transferases investigated, but marked differences in the effects of bile acids on individual enzymes were observed. The activity of each transferase was inhibited more by lithocholate 3-sulphate than by chenodeoxycholate, which in turn was more inhibitory than cholate. The enzymes that were most sensitive to cholate inhibition were not found to be as readily inhibited as other transferases by chenodeoxycholate or lithocholate 3-sulphate. Conversely, the activity of transferase AA was more resistant to cholate, chenodeoxycholate and lithocholate 3-sulphate inhibition than was any of the other enzymes studied.     

Purification of a new acidic glutathione S-transferase, GST-Yn1Yn1, with a high leukotriene-C4 synthase activity from rat brain

A new acidic form of glutathione S-transferase (GST, pI 6.2) was purified from rat brain by S-hexylglutathione affinity chromatography followed by chromatofocusing. This form occupied 20-25% of the total activity bound to the affinity column. It had a molecular mass (subunit 26 kDa) similar to that of a major GST form of rat testis (MT or 6-6) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. However, it differed from the MT in isoelectric point, activity towards 1,2-dichloro-4-nitrobenzene and immunological properties. On two-dimensional gel electrophoresis the brain form gave a spot which was identical in molecular mass, isoelectric point and immunological properties to a less acidic one (Yn1) of two spots (Yn1 and Yn2) of the testis GST-MT. Therefore, the brain acidic form is a homodimer, and named GST-Yn1Yn1. The activity was inhibited by sulfasalazine, an inhibitor of leukotriene-C4 synthase. This form (GST-Yn1Yn1) showed the highest leukotriene-C4 synthase activity, 496 nmol/mg protein in 5 min, among nine cytosolic GST isoenzymes from the rat. The Km values for leukotriene A4 and glutathione were 26 microM and 3.5 mM respectively. A major GST form of rat brain, occupying about 40% of the total activity, was identical with GST-P (7-7) purified from rat liver bearing preneoplastic hyperplastic nodules and localized at astroglias. GST-P also showed the significant leukotriene-C4 synthase activity, 67.2 nmol/mg protein in 5 min, but the Km for leukotriene A4 was 100 microM, fourfold higher than that of GST-Yn1 Yn1. These results suggest that mainly GST-Yn1 Yn1 may be involved in leukotriene-C4 synthesis in rat brain.     

Purification and characterization of a novel monomeric glutathione peroxidase from rat liver

A novel glutathione peroxidase, which is active toward hydroperoxides of phospholipid in the presence of a detergent, has been purified to homogeneity from a rat liver postmicrosomal supernatant fraction by ammonium sulfate fractionation and three different column chromatographies. From a DE52 column, glutathione peroxidase active toward phosphatidylcholine dilinoleoyl hydroperoxides was eluted in one major and two minor peaks. The enzyme in the major peak was found to be separated from the "classic" glutathione peroxidase and glutathione S-transferases and further purified by Sephacryl S-200 and Mono Q column chromatographies. The purified enzyme was found to be homogeneous on polyacrylamide gel electrophoresis under nondenaturing conditions as well as that in the presence of sodium dodecyl sulfate. The molecular weight of the enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 22,000, and that by gel filtration was comparable, indicating that the enzyme protein is a single polypeptide. The purified enzyme was found to catalyze the reduction of phosphatidylcholine dilinoleoyl hydroperoxides to the corresponding hydroxy derivatives. The isoelectric point of the enzyme was found at pH 6.2, and the optimum pH for the enzyme activity was 8.0. The enzyme was active toward cumene hydroperoxide, H2O2, and 1-monolinolein hydroperoxides in the absence of a detergent. The enzyme activity toward phospholipid hydroperoxides was minute in the absence of a detergent but was remarkably enhanced by the addition of a detergent. From these results, the presently purified enzyme is obviously different from the classic glutathione peroxidase and also from phospholipid hydroperoxide glutathione peroxidase purified from pig heart (Ursini, F., Maiorino, M., and Gregolin, C. (1985) Biochim. Biophys. Acta 839, 62-70), though considerably similar to the latter.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.