Structural studies on human glutathione S-transferase pi. Family of native-specific monoclonal antibodies used to block catalysis.

The glutathione S-transferases are a family of related detoxification enzymes that have been shown to conjugate numerous electrophiles to the common cellular thiol glutathione. We have generated a panel of monoclonal antibodies against the human pi class isozyme of this enzyme, and, in this report, we characterize the binding of these antibodies to the glutathione S-transferase antigen. Of the 10 monoclonal antibodies that we have isolated, 7 are able to recognize the native form of the enzyme while the remaining 3 are only able to bind to glutathione S-transferase pi in assays that partially denature the antigen, such as an enzyme-linked immunosorbent assay or a Western blot. We synthesized seven partial protein fragments and asked whether the monoclonal antibodies could bind to these fragments in an immunoprecipitation reaction. The antibodies that can bind the native form of the enzyme all bind to the carboxyl-terminal domain of the protein. Two antibodies are able to inhibit the glutathione S-transferase-catalyzed reaction noncompetitively against glutathione. Incubation of a 10-fold molar excess of either antibody over enzyme can inhibit the reaction by 50%. We have also used the same protein fragments of glutathione S-transferase pi to show that amino acids 1-77 retain the capacity to bind glutathione in a glutathione-agarose binding assay.     

Immunochemistry, genuine size and tissue localization of collagen VI

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple-helical and globular structures. Antisera prepared against a constituent-chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross-react with other collagen types I-V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide-bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers. Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross-striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

Biochemical and immunological analysis of an abundant form of glutathione S-transferase,in mouse testis

One of the major forms of glutathione S-transferase (designated as Ft transferase) has been identified and purified to near homogeneity from mouse testis. The purification was achieved by ammonium sulfate fractionation, DEAE cellulose chromatography, hydroxylapatite chromatography and the preparative isoelectric focusing. Purified Ft transferase has an isoelectric point of 4.9 ± 0.3 and was shown to be a homodimer with a native molecular weight of about 50 000.Immunologically, antisera to Ft transferase do not crossreact with F2 or F3 transferase. However, a weak cross reactivity was observed between the antisera to F3 transferase and Ft transferase. Biochemical properties of purified Ft transferase are similar to those transferases isolated from mouse liver. Tissue distributions of the multiple forms of glutathione S-transferase were examined by column isoelectric focusing of various mouse tissue homogenates. It was found that mouse Ft transferase is present only in testis as a major form and in brain as a minor form, but not in other tissues that were examined.     

Isolation and characterization of the multiple glutathione S-transferases from human liver. Evidence for unique heme-binding sites

Thirteen forms of glutathione S-transferase were isolated from human liver in high yields by glutathione-affinity chromatography and chromatofocusing. Apparent isoelectric points ranged from 4.9 to 8.9 and included neutral forms. All 13 forms appeared to be identical immunochemically in a quantitative enzyme-linked immunosorbent assay. These forms were immunochemically distinct from the major acidic glutathione S-transferase found in placenta and erythrocyte and were immunochemically distinct from two forms of higher molecular weight glutathione S-transferase found in some but not all liver samples. The 13 forms exhibited similar activities with 1-chloro-2,4-dinitro-benzene as substrate, specific activities of 33-94 mumol/min/mg. Likewise, these forms all exhibited glutathione peroxidase activity with cumene hydroperoxide, specific activities of 1.5-8.3 mumol/min/mg. All 13 forms bound bilirubin with subsequent conformational changes leading to states devoid of transferase activity, a process prevented by the presence of foreign proteins. As hematin-binding proteins, however, these multiple transferases exhibited a very broad range of binding extending from nonbinding to high-affinity binding (KD approximately 10(-8) M). Hematin binding was noncompetitive with transferase activity and did not involve the bilirubin-binding site, suggesting the existence of unique heme-binding sites on these proteins. The two forms of the immunochemically distinct glutathione S-transferases transferases found in some liver samples also exhibited both transferase and peroxidase activities. In addition, they also have separate sites for binding bilirubin and hematin.     

Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases.

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.     

Immunological Studies of Human Monoamine Oxidases

Antisera have been raised against monoamine oxidase preparations from human placenta and platelets. These antisera have been employed to characterize membrane-bound enzyme from a variety of human sources including liver, heart, and brain. The comparisons were based on a displacement radioimmunoassay system with soluble placental monoamine oxidase, previously labelled specifically with [3H]pargyline, as antigen. All forms of enzyme investigated demonstrated immunological cross reaction; however, the placental enzyme appeared to possess determinants not exhibited by the enzyme from the platelets or other tissues examined.     

Anionic glutathione S-transferases of human erythrocytes, placenta, and lung: evidence for structural differences

Studies were undertaken to elucidate the structural interrelationships among glutathione S-transferase (GST) isozymes of human placenta, lung, and erythrocytes. Results of the high-performance liquid chromatography of the trypsin digests of the three isozymes indicate minor but significant differences in their elution profiles. Although a number of peptides generated by proteolysis were common for either 2 or 3 of the isozymes, significant differences were observed in elution profiles of other peptides. Qualitative as well as quantitative differences were also observed in the electrophoretic peptide maps of these isozymes. These studies suggest that there may be fine structural differences among the pi class GST isozymes of human tissues.     

Purification of a fluoroacetate-specific defluorinase from mouse liver cytosol

Fluoraocetate-specific defluorinase, an enzyme which catalyzes the release of fluoride ion from the rodenticide fluoroacetate, has been purified 347-fold from mouse liver cytosol and shown to be distinct from multiple cationic and anionic glutathione S-transferase isozymes. Fluoroacetate-specific defluorinase was obtained at a final specific activity of 659 nmol of F-/min/mg of protein and was prepared in an overall yield of 12%. The isoelectric point of this hepatic enzyme was acidic, at pH 6.4, as determined by column chromatofocusing. The molecular weight of the active species was estimated at 41,000, and sodium dodecyl sulfate-polyacrylamide gels of the purified defluorinase demonstrated a predominant subunit, Mr = 27,000. Chromatofocusing completely partitioned the fluoroacetate-specific defluorinase from two separate peaks of murine anionic glutathione S-transferase activity. Rabbit antibodies prepared against the purified hepatic defluorinase quantitatively precipitated native defluorinase from mouse and rat liver, but were unable to immunoprecipitate cationic or anionic glutathione S-transferase enzymes from the same preparation. The evidence presented suggests that fluoroacetate-specific defluorinase and glutathione S-transferase activities are catalyzed by separate proteins present in the cytosol of mouse liver.     

Raised plasma glutathione S-transferase values in hyperthyroidism and in hypothyroid patients receiving thyroxine replacement: evidence for hepatic damage.

Using plasma glutathione S-transferase measurements hepatocellular integrity was assessed in groups of hyperthyroid and hypothyroid patients before and after treatment. Ten of 14 hyperthyroid patients had clearly raised plasma glutathione S-transferase values at presentation and in each patient treatment with either iodine-131 or carbimazole resulted in a significant fall in glutathione S-transferase. The eight hypothyroid patients had normal glutathione S-transferase values at presentation and all showed a significant increase in these after thyroxine replacement therapy. In three of these patients in whom standard doses of replacement therapy were associated with a raised free thyroxine (T4) concentration but normal total and free triiodothyronine (T3) values glutathione S-transferase was increased. Similar though less consistent changes were seen in the results of standard chemical tests of liver function. It is concluded that hyperthyroidism may produce subclinical liver damage in a high proportion of patients and that this resolves with effective treatment. More important, the data suggest that hypothyroid patients receiving thyroxine replacement therapy may have similar subclinical liver damage. Patients receiving thyroxine should be monitored by the measurement of free, not total hormone concentrations, and in those in whom free T4 is raised the dose of thyroxine should be reduced. It would also be expedient to include periodic biochemical assessment of liver function in patients receiving thyroxine.     

The effects of in vitro lipid peroxidation on the activity of rat liver microsomal glutathione S-transferase from rats supplemented or deficient in antioxidants

Glutathione S-transferases are a group of multifunctional isozymes that play a central role in the detoxification of hydrophobic xenobiotics with electrophilic centers (1). In this study we investigated the effects of in vitro lipid peroxidation on the activity of liver microsomal glutathione S-transferases from rats either supplemented or deficient in both vitamin E and selenium. Increased formation of malondialdehyde (MDA), a by-product of lipid peroxidation, was associated with a decreased activity of rat liver microsomal glutathione S-transferase. The inhibition of glutathione S-transferase occurred rapidly in microsomes from rats fed a diet deficient in both vitamin E and selenium (the B diet) but was delayed for 15 minutes in microsomes from rats fed the same diet but supplemented with these micro-nutrients (B+E+Se diet). Lipid peroxidation inhibits microsomal glutathione S-transferase and this inhibition is modulated by dietary antioxidants.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Glutathione S-transferases in human prostate

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out. Approximately 260-fold purification was achieved using glutathione-Sepharose affinity chromatography, with glutathione S-transferase accounting for approximately 0.19-0.33% of the total protein. Substrate specificity determinations suggested similar, but not identical, glutathione S-transferase subunits in normal prostate and benign prostatic hypertrophy. One- and two-dimensional electrophoresis (isoelectric focusing and 12.5% SDS-polyacrylamide gel electrophoresis) identified at least seven stained polypeptides in the purified glutathione S-transferase preparations. These ranged in Mr from approximately 24,000 to 28,500 and in pI from near neutral to basic. Western blot analysis using polyclonal antibodies raised against rat liver glutathione S-transferase suggested crossreactivity with five of the human isoenzymes in both normal prostate and benign prostatic hypertrophy. One of the glutathione S-transferases, present in both normal prostate and benign prostatic hypertrophy, had an Mr of approx. 24,000 and a near-neutral pI and crossreacted immunologically with a polyclonal antibody raised against human placental glutathione S-transferase (Yf, subunit 7 or pi). These data suggest that four glutathione S-transferases are expressed in human prostate, with subunits from each of the major classes alpha, mu and pi. These are characterized as Ya, Yb, Yb' and Yf (analogous alternative nomenclature subunits 1, 3, 4 and 7).     

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.     

Size and stability to sodium dodecyl sulfate of alkaline phosphatases from their three established human genes

Subunit molecular weights of human alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) were dependent upon acrylamide concentration, a reflection of their glycoprotein nature. Molecular weights at a concentration of 7% (w/w) or greater were 68300, 80800 and 79400 for the enzymes from placenta, liver and mucosa of small intestine, respectively. All enzymes were dimers, the respective native Mr values determined by gradient gel electrophoresis being 138000, 186000 and 180000. None of the molecular weights was altered by desialylation. Stability of the catalytic activity of the purified enzymes to SDS varied and was very dependent on pH. SDS at 1% (w/v) rapidly denatured both native and desialylated alkaline phosphatase from placenta at pH 7.5 but had little effect on these at pH 10.3. Compared with placenta, the native enzyme from liver had greater stability at pH 7.5 and both native and desialylated forms had lower stability at pH 10.3. The enzyme from intestinal mucosa was sharply different from the other two isoenzymes: SDS had little effect at pH 7.5 but very rapidly denatured the enzyme at pH 10.3. The size of alkaline phosphatases and their stability to SDS can be used to identify gene products and to recognize heterodimers formed between products of more than one gene.     

Conformational studies of a synthetic peptide corresponding to the repeat motif of C hordein.

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.     

Alkaline phosphatase and 5'-nucleotide phosphodiesterase from bovine intestine are cross-reactive

Polyclonal antibodies to native alkaline phosphatase and to native 5'-nucleotide phosphodiesterase were found to strongly cross-react with both enzymes. The antibodies also cross-react with both denatured enzymes, with glycopeptides from 5'-nucleotide phosphodiesterase, and with the oligosaccharides remaining after Pronase E digestion of the phosphodiesterase. They do not cross-react with either enzyme after their oligosaccharides have been modified or removed by periodate or trifluoromethanesulfonic acid treatment. Antibodies to denatured 5'-nucleotide phosphodiesterase do not bind to the native phosphodiesterase or alkaline phosphatase but do cross-react with denatured alkaline phosphatase even after removal or modification of the carbohydrate moieties. These results suggest that antibodies to denatured 5'-nucleotide phosphodiesterase may recognize amino acid sequence homology between alkaline phosphatase and 5'-nucleotide phosphodiesterase. However, antibodies to native enzymes apparently recognize cross-reactive determinants of the native enzymes which are carbohydrate in nature. This is the first report of antimammalian alkaline phosphatase antibodies which recognize the carbohydrate moieties of the enzyme.     

Interrelationship between cationic and anionic forms of glutathione S-transferases of bovine ocular lens.

Since the eye is constantly exposed to potentially damaging chemical compounds present in the atmosphere and vascular system, we investigated the physiological role of glutathione S-transferase (GSH S-transferase) in detoxification mechanisms operative in the ocular lens. We have purified an anionic and a cationic GSH S-transferase from the bovine lens to homogeneity through a combination of gel filtration, ion-exchange and affinity chromatography. The anionic (pI 5.6) and cationic (pI 7.4) S-transferases were found to have distinct kinetic parameters (apparent Km and Vmax. pH optimum and energy of activation). However, both species were demonstrated to have similar molecular weights and amino acid compositions. Double-immunodiffusion and immunotitration studies showed that both lens S-transferases were immunologically similar. The very close similarity in amino acid compositions and immunological properties strongly indicates that these two transferases either originate from the same gene or at least share common antigenic determinants and originate from similar genes. The bovine lens GSH S-transferases had no glutathione peroxidase activity with either t-butyl hydroperoxide or cumene hydroperoxide as substrate. However, the antibody raised against the homogeneous anionic glutathione S-transferase from the bovine lens was found to precipitate both glutathione S-transferase and glutathione peroxidase activities out of solution in the supernatant of a crude bovine liver homogenate.     

Transforming growth factor beta 1 treatment of AKR-2B cells is coupled through a pertussis-toxin-sensitive G-protein(s).

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.