Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms.

Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.     

The major isozyme of rat cardiac glutathione transferases. Its correspondence to hepatic transferase X

A major isozyme of rat heart glutathione transferase was purified to homogeneity by Sephadex G-200 gel filtration, ammonium sulfate precipitation, CM-cellulose chromatography and affinity chromatography on S-hexylglutathione-linked Sepharose 6B. The purified isozyme was a dimer with an apparent relative molecular mass of 50 000 composed of two Yb-size subunits (Mr = 26 500). The isozyme is immunologically related to rat liver glutathione transferase X and 3-3, especially closely to transferase X, and no immunological cross-reactivity with subunits 1 and 2 of hepatic glutathione transferases was observed. The isoelectric point (pI = 6.9) of the isozyme was identical with and the substrate specificity was very similar to transferase X. Thus, the cardiac near-neutral isozyme is considered to be identical to glutathione transferase X recognized in rat liver. The amount of this near-neutral isozyme estimated to be present in heart tissue is 70 micrograms/g. The isozyme has relatively high activities towards alpha, beta-unsaturated carbonyl compounds such as trans-4-phenyl-3-buten-2-one and trans-4-hydroxynon-2-enal. The latter is a cytotoxic product resulting from lipid peroxidation of polyunsaturated fatty acids, and the cardiac isozyme may play a physiologically significant role with glutathione conjugation of this compound. In addition to the near-neutral isozyme, acidic forms with isoelectric points of 4.9, 5.2 and 5.5 were partially purified; some of them are considered to consist of subunits immunologically related to transferase X.     

Identification of [1-14C]pantothenic-acid-mediated modified mitochondrial proteins

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.     

Mitochondrial acetyl-CoA acetyltransferase in various organs from rat: form patterns and coenzyme-A-mediated modification

The mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9), which is involved in the biosynthesis or degradation of ketone bodies, was directly demonstrated in organ extracts applying a two-step chromatography-immunoelectrophoresis method. In liver, the enzyme can be shown in at least three forms: in an unmodified state, designated as AAT, and in the CoASH-modified forms A1 and A2, in amounts of 51.5 +/- 5.0%, 39.4 +/- 4.8% and 9.1 +/- 2.7% (areas of immunoprecipitation), respectively. This pattern, which could not be altered by a treatment with glutathione, resembles that of mitochondrial acetyl-CoA acetyltransferase in extrahepatic tissues. However, the proportion of the unmodified enzyme (AAT) is lower as compared to those in other tissues such as brain (81.5 +/- 4.4%). CoASH-modification and transformation into modified forms, which equal naturally occurring forms, can be demonstrated in vitro with acetyl-CoA acetyltransferase from both liver and brain. Thus CoASH-modification of mitochondrial acetyl-CoA acetyltransferase seems to be a process of general importance.     

Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification of major basic glutathione transferase isoenzymes from rat liver by use of affinity chromatography and fast protein liquid chromatofocusing

Seven major isoenzymes of glutathione transferase with isoelectric points ranging from pH 6.9 to 10 were isolated from rat liver cytosol. The purification procedure included affinity chromatography on immobilized S-hexylglutathione followed by high-performance liquid chromatofocusing. Characteristics, such as physical properties, reactions with antibodies, specific activities with various substrates, kinetic constants, and sensitivities to a set of inhibitors, are given for discrimination and identification of the different isoenzymes. The multiple forms of the enzyme correspond to glutathione transferases 1-1, 1-2, 2-2, 3-3, 3-4, and 4-4 in the recently introduced nomenclature [W.B. Jakoby et al. (1984) Biochem. Pharmacol. 33, 2539-2540]. A seventh form appears to be a heterodimeric protein composed of subunit 3 and an as yet unidentified subunit.     

Identification of a basic hybrid glutathione S-transferase from human liver. Glutathione S-transferase delta is composed of two distinct subunits (B1 and B2).

The purification of a hybrid glutathione S-transferase (B1 B2) from human liver is described. This enzyme has an isoelectric point of 8.75 and the B1 and B2 subunits are distinguishable immunologically and are ionically distinct. Hybridization experiments demonstrated that B1 B1 and B2 B2 could be resolved by CM-cellulose chromatography and have pI values of 8.9 and 8.4 respectively. Transferase B1 B2, and the two homodimers from which it is formed, are electrophoretically and immunochemically distinct from the neutral enzyme (transferase mu) and two acidic enzymes (transferases rho and lambda). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis demonstrated that B1 and B2 both have an Mr of 26 000, whereas, in contrast, transferase mu comprises subunits of Mr 27 000 and transferases rho and lambda both comprise subunits of Mr 24 500. Antisera raised against B1 or B2 monomers did not cross-react with the neutral or acidic glutathione S-transferases. The identity of transferase B1 B2 with glutathione S-transferase delta prepared by the method of Kamisaka, Habig, Ketley, Arias & Jakoby [(1975) Eur. J. Biochem. 60, 153-161] has been demonstrated, as well as its relationship to other previously described transferases.     

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.     

Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases.

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.     

Purification and characterization of three forms of glutathione S-transferase A. A comparative study of the major YaYa-, YbYb- and YcYc-containing glutathione S-transferases.

Rat liver glutathione S-transferases have previously been defined by their elution behaviour from DEAE-cellulose and CM-cellulose as M, E, D, C, B, A and AA. These enzymes are dimeric proteins which comprise subunits of mol.wt. 22 000 (Ya), 23 500 (Yb) or 25 000 (Yc). Evidence is presented that YaYa protein, one of two previously described lithocholate-binding proteins which exhibit transferase activity, is an additional enzyme which is not included in the M, E, D, C, B, A and AA nomenclature. We therefore propose that this enzyme is designated transferase YaYa. Transferases YaYa, C, A and AA have molecular weights of 44 000, 47 000, 47 000 and 50 000 respectively and each comprises two subunits of identical size. These enzymes were purified to allow a study of their structural and functional relationships. In addition, transferase A was further resolved into three forms (A1, A2 and A3) which possess identical activities and structures and appear to be the product of a single gene. Transferases YaYa, C, A and AA each had distinct enzymic properties and were inhibited by cholate. The recently proposed proteolytic model, which attributes the presence of multiple forms of glutathione S-transferase activity to partial proteolysis of transferase AA, was tested and shown to be highly improbable. Peptide maps showed significant differences between transferases YaYa, C, A and AA. Immunotitration studies demonstrated that antisera raised against transferases YaYa and C did not precipitate transferase AA.     

Purification and properties of carnitine acetyltransferase from human liver

Carnitine acetyltransferase was purified from the supernatant obtained after centrifugation of human liver homogenate to a final specific activity of 78.75 unit.mg-1 with acetyl-CoA as a substrate. Human carnitine acetyltransferase is a monomer of 60.5 kDa with maximum activity in the presence of propionyl-CoA and a pH optimum of 8.7. Apparent Km values for acetyl-CoA are three times lower than for decanoyl-CoA. Km values for L-carnitine in the presence of acetyl-CoA are six times lower than in the presence of decanoyl-CoA. Km values for acetylcarnitine are three times lower than for octanoylcarnitine. The polyclonal antibodies against human carnitine acetyltransferase recognize a 60.5-kDa peptide in the purified preparation of human liver and brain homogenates and in immunoblots of mitochondrial and peroxisomal fractions from human liver. Immunoprecipitation and SDS/PAGE analysis of 35S-labelled proteins produced by human fibroblasts indicate that mitochondrial carnitine acetyltransferase is synthesized as a precursor of 65 kDa. We also purified carnitine acetyltransferase from the pellet obtained after centrifugation of liver homogenate. The pellet was extracted by sonication in the presence of 0.5% Tween 20. The chromatographic procedures for the purification and the kinetic, physical and immunological properties of pellet-extracted carnitine acetyltransferase are similar to those of carnitine acetyltransferase purified from the supernatant of human liver homogenate.     

Comparison of the carnitine acyltransferase activities from rat liver peroxisomes and microsomes

Carnitine acyltransferase activities for acetyl- and octanoyl-CoA (coenzyme A) occur in isolated peroxisomal, mitochondrial, and microsomal fractions from rat and pig liver. Solubility studies indicated that both peroxisomal carnitine acyltransferases were in the soluble matrix. In contrast, the microsomal carnitine acyltransferases were tightly associated with their membrane. The microsomal short-chain transferase, carnitine acetyltransferase, was solubilized and stabilized by extensive treatment of the membrane with 0.4 m KCl or 0.3 m sucrose in 0.1 m pyrophosphate at pH 7.5. The same treatment only partially solubilized the microsomal medium-chain transferase, carnitine octanoyltransferase.Although half of the total carnitine acetyltransferase activity in rat liver resides in peroxisomes and microsomes, previous reports have only investigated the mitochondrial activity. Transferase activity for acetyl- and octanoyl-CoA were about equal in peroxisomal and in microsomal fractions. A 200-fold purification of peroxisomal and microsomal carnitine acetyltransferases was achieved using O-(diethylaminoethyl)-cellulose and cellulose phosphate chromatography. This short-chain transferase preparation contained less than 5% as much carnitine octanoyltransferase and acyl-CoA deacylase activities. This fact, plus differences in solubility and stability of the microsomal transferase system for acetyl- and octanoyl-CoA indicate the existence of two separate enzymes: a carnitine acetyltransferase and a carnitine octanoyltransferase in peroxisomes and in microsomes.Peroxisomal and microsomal carnitine acetyltransferases had similar properties and could be the same protein. They showed identical chromatographic behavior and had the same pH activity profiles and major isoelectric points. They also had the same apparent molecular weight by gel filtration (59,000) and the same relative velocities and K_m values for several short-chain acyl-CoA substrates. Both were active with propionyl-, acetyl-, malonyl-, and acetyacetyl-CoA, but not with succinyl- and β-hydroxy-β-methylglutaryl-CoA as substrates.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

Isoelectric focusing of glutathione S-transferases from rat liver and kidney

The glutathione S-transferases that were purified to homogeneity from liver cytosol have overlapping but distinct substrate specificities and different isoelectric points. This report explores the possibility of using preparative electrofocusing to compare the composition of the transferases in liver and kidney cytosol. Hepatic cytosol from adult male Sprague–Dawley rats was resolved by isoelectric focusing on Sephadex columns into five peaks of transferase activity, each with characteristic substrate specificity. The first four peaks of transferase activity (in order of decreasing basicity) are identified as transferases AA, B, A and C respectively, on the basis of substrate specificity, but the fifth peak (pI6.6) does not correspond to a previously described transferase. Isoelectric focusing of renal cytosol resolves only three major peaks of transferase activity, each with narrow substrate specificity. In the kidney, peak 1 (pI9.0) has most of the activity toward 1-chloro-2,4-dinitrobenzene, peak 2 (pI8.5) toward p-nitrobenzyl chloride, and peak 3 (pI7.0) toward trans-4-phenylbut-3-en-2-one. Renal transferase peak 1 (pI9.0) appears to correspond to transferase B on the basis of pI, substrate specificity and antigenicity. Kidney transferase peaks 2 (pI8.5) and 3 (pI7.0) do not correspond to previously described glutathione S-transferases, although kidney transferase peak 3 is similar to the transferase peak 5 from focused hepatic cytosol. Transferases A and C were not found in kidney cytosol, and transferase AA was detected in only one out of six replicates. Thus it is important to recognize the contribution of individual transferases to total transferase activity in that each transferase may be regulated independently.     

Acetyl-coenzyme A hydrolase,an artifact? The conversion of acetyl-coenzyme A into acetate by the combined action of carnitine acetyltransferase and acetylcarnitine hydrolase

1. The nature of the acetyl-CoA hydrolase (EC 3.1.2.1) reaction in rat and sheep liver homogenates was investigated. 2. The activity determined in an incubated system was 5.10 and 3.28nmol/min per mg of protein for rat and sheep liver homogenate respectively. This activity was not affected by the addition of l-carnitine, but was decreased by the addition of d-carnitine. 3. No acetyl-CoA hydrolase activity could be detected in rat or sheep liver homogenates first treated with Sephadex G-25. This treatment decreased the carnitine concentrations of the homogenates to about one-twentieth. Subsequent addition of l-carnitine, but not d-carnitine, restored the apparent acetyl-CoA hydrolase activity. 4. Sephadex treatment did not affect acetyl-carnitine hydrolase activity of the homogenates, which was 5.8 and 8.1nmol/min per mg of protein respectively for rat and sheep liver. 5. Direct spectrophotometric assay of acetyl-CoA hydrolase, based on the reaction of CoA released with 5,5'-dithiobis-(2-nitrobenzoic acid), clearly demonstrated that after Sephadex treatment no activity could be measured. 6. Carnitine acetyltransferase (EC 2.3.1.7) activity measured in the same assay system in response to added l-carnitine was very low in normal rat liver homogenates, owing to the apparent high acetyl-CoA hydrolase activity, but was increased markedly after Sephadex treatment. The V(max.) for this enzyme in rat liver homogenates was increased from 3.4 to 14.8nmol/min per mg of protein whereas the K(m) for l-carnitine was decreased from 936 to 32mum after Sephadex treatment. 7. Acetyl-CoA hydrolase activity could be demonstrated in disrupted rat liver mitochondria but not in separated outer or inner mitochondrial membrane fractions. Activity could be demonstrated after recombination of outer and inner mitochondrial membrane fractions. The outer mitochondrial membrane fraction showed acetylcarnitine hydrolase activity and the inner mitochondrial membrane fraction showed carnitine acetyltransferase activity. 8. The results presented here demonstrate that acetyl-CoA hydrolase activity in rat and sheep liver is an artifact and the activity is due to the combined activity of carnitine acetyltransferase and acetylcarnitine hydrolase.     

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.     

Purification and physical characterization of glutathione S-transferase K. Differential use of S-hexylglutathione and glutathione affinity matrices to isolate a novel glutathione S-transferase from rat liver.

A novel hepatic enzyme, glutathione S-transferase K, is described that, unlike previously characterized transferases, possesses little affinity for S-hexylglutathione-Sepharose 6B but can be isolated because it binds to a glutathione affinity matrix. A purification scheme for this new enzyme was devised, with the use of DEAE-cellulose, S-hexylglutathione-Sepharose 6B, glutathione-Sepharose 6B and hydroxyapatite chromatography. The final hydroxyapatite step results in the elution of three chromatographically interconvertible forms, K1, K2 and K3. The purified protein has an isoelectric point of 6.1 and comprises subunits that are designated Yk (Mr 25,000); during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, it migrates marginally faster than the Ya subunit but slower than the pulmonary Yf monomer (Mr 24,500). Transferase K displays catalytic, immunochemical and physical properties that are distinct from those of other liver transferases. Tryptic peptide maps suggest that transferase K is a homodimer, or comprises closely homologous subunits. The tryptic fingerprints also demonstrate that, although transferase K is structurally separate from previously described hepatic forms, a limited sequence homology exists between the Yk, Ya and Yc polypeptides. These structural data are in accord with the immunochemical results presented in the accompanying paper [Hayes & Mantle (1986) Biochem. J. 233, 779-788].     

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.     

The presence and longitudinal distribution of the glutathione S-transferase in rat epididymis and vas deferens

The presence of the glutathione S-transferases, enzymes that catalyse the conjugation of glutathione with a variety of compounds, is reported here, for the first time, in the mammalian epididymis–vas deferens. These glutathione S-transferases, approx. 50% of those from rat liver on a per-mg-of-protein basis, are resolved by isoelectric focusing into six peaks, each with a characteristic isoelectric point and substrate specificity. By these same criteria, the first three peaks (pI 8.9, 8.2 and 7.8) can be identified as transferases B, A and C respectively. The fifth peak (pI7.2) may correspond to transferase M; the fourth (pI7.5) and sixth (pI7.0) peaks do not correspond to previously described transferases. The distribution of transferase activity towards any one substrate studied differs in sequential sections of the epididymis and vas deferens; in addition, the longitudinal-distribution pattern differs for each of the three substrates studied. Isoelectric focusing of the cytosol fractions of the different sections further substantiates these observations. The potential significance of these enzymes and of their distribution in terms of epididymal function, maturation of spermatozoa, is discussed.     

Multiple Mass Isotopomer Tracing of Acetyl-CoA Metabolism in Langendorff-perfused Rat Hearts: CHANNELING OF ACETYL-CoA FROM PYRUVATE DEHYDROGENASE TO CARNITINE ACETYLTRANSFERASE*

We developed an isotopic technique to assess mitochondrial acetyl-CoA turnover (≈citric acid flux) in perfused rat hearts. Hearts are perfused with buffer containing tracer [¹³C₂,²H₃]acetate, which forms M5 + M4 + M3 acetyl-CoA. The buffer may also contain one or two labeled substrates, which generate M2 acetyl-CoA (e.g. [¹³C₆]glucose or [1,2-¹³C₂]palmitate) or/and M1 acetyl-CoA (e.g. [1-¹³C]octanoate). The total acetyl-CoA turnover and the contributions of fuels to acetyl-CoA are calculated from the uptake of the acetate tracer and the mass isotopomer distribution of acetyl-CoA. The method was applied to measurements of acetyl-CoA turnover under different conditions (glucose ± palmitate ± insulin ± dichloroacetate). The data revealed (i) substrate cycling between glycogen and glucose-6-P and between glucose-6-P and triose phosphates, (ii) the release of small excess acetyl groups as acetylcarnitine and ketone bodies, and (iii) the channeling of mitochondrial acetyl-CoA from pyruvate dehydrogenase to carnitine acetyltransferase. Because of this channeling, the labeling of acetylcarnitine and ketone bodies released by the heart are not proxies of the labeling of mitochondrial acetyl-CoA.