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Biomedical subjects

B Ketterer

Publications and source records attributed to B Ketterer.

At least 109 records · Page 6Linked to original sources
PubMed1985

Purification and characterization of fatty acid binding protein in mammalian lung.

A fatty acid-binding protein (FABP) has been isolated and characterized from rat lung tissue. Rat lung FABP has a slightly higher molecular weight than liver FABP, but immunologically, lung FABP is similar to that of liver FABP. Long chain acyl CoA synthetase, a key enzyme in fatty acid metabolism is stimulated by partially purified lung FABP, suggesting a physiologic role of the protein in the activation of long chain fatty acids in pulmonary tissue.

Animals↗
PubMed1984

Isolation, properties and tissue distribution of rat glutathione transferase E.

A simple small-scale purification procedure is described for GSH transferase E. This enzyme is shown to be a dimer of subunits of apparent Mr 28 500, to have an isoelectric point of pH 7.0, GSH transferase activity towards certain alkyl epoxides and alkyl halides, and to be the most active Se-independent GSH peroxidase so far described. It is present in a number of tissues, although at a low concentration. It is relatively abundant in the epididymis and the adrenal gland, but undetectable in lactating mammary gland and skeletal muscle. Its previously observed lability is confirmed.

Animals↗
PubMed1984

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.

Acetophenones↗
PubMed1984

Construction and characterization of a plasmid containing complementary DNA to mRNA encoding the N-terminal amino acid sequence of the rat glutathione transferase Ya subunit.

Free polyribosomal poly(A)-containing RNA isolated from normal rat liver was used to prepare a complementary DNA plasmid library in the Pst1 site of the plasmid pAT153 . A plasmid pGSTr155 complementary to mRNA coding for a glutathione transferase Ya subunit was selected by differential hybridization in situ and preliminary characterization was performed by hybrid-selected mRNA translation, immunoprecipitation and polyacrylamide-gel electrophoresis of the product synthesized in vitro. The nucleotide sequence of the complementary DNA contained within pGSTr155 was determined and shown to contain a single open reading frame corresponding to the first 129 amino acids of the N-terminus of the Ya subunit and a further 63 nucleotides upstream of the initiating methionine codon.

Amino Acid Sequence↗
PubMed1984

Glutathione conjugate formation in the detoxification of ultimate and proximate carcinogens of N-methyl-4-aminoazobenzene.

The presumed ultimate carcinogenic metabolite of the rat hepatocarcinogen N-methyl-4-aminoazobenzene (MAB), N-sulphonyloxy-MAB, was synthesized and reacted with glutathione (GSH) to yield the stable ring-substituted conjugates, 3-, 2'- and 4'-glutathion-S-yl-MAB (3-, 2'- and 4'-GSMAB) and the unstable methylene-substituted conjugate, N-(glutathion-S-methylene)-4-aminoazobenzene. Reaction of the model ultimate carcinogen, N-benzoyloxy-MAB, or the proximate carcinogen, N-hydroxy-MAB, with GSH also gave the same four conjugates. The ratio of ring-substitution relative to methylene conjugate formation increased with stronger leaving groups; i.e., ring-substitution increased in the order: N-hydroxy: N-benzoyloxy: N-sulphonyloxy. The ratio of 3-GSMAB to 2'- plus 4'-GSMAB was constant for all three MAB derivatives.

Azo Compounds↗
PubMed1984

Acetaminophen-induced alterations in blood glucose and blood insulin levels in mice.

Three hours following administration of a toxic dose of the analgesic acetaminophen (500 mg/kg) to mice, blood glucose levels increased to 225 per cent. By six hours blood glucose levels decreased to approximately control values and by 24 hours glucose levels were 45 percent saline-treated control values. Immunoprecipitable blood insulin levels increased dramatically following acetaminophen treatment and were 300 per cent at 3 hours, 1100 per cent at 8 hours and 800 per cent at 24 hours. Saline treatment did not appreciably alter blood insulin levels. These observations indicate that acetaminophen may selectively alter pancreatic beta cells.

Acetaminophen↗
PubMed1983

Evidence that the Yb subunits of hepatic glutathione transferases represent two different but related families of polypeptides.

Three soluble rat liver glutathione (GSH) transferases A, C and one referred to as 'D', all of which are dimers of Yb subunits [Bass et al. (1977) Biochim. Biophys. Acta, 492, 163-175], have been compared with respect to C-terminal amino acids and tryptic peptide maps. GSH transferases A and 'D' gave different tryptic peptide maps and different C-terminal amino acids, lysine and proline respectively. In each case the number of tryptic peptides is about half of that expected from their lysine and arginine content, and there are 2 mol C-terminal amino acid/mol enzyme. This indicates that GSH transferases A and 'D' represent two different Yb homodimers, which we refer to here as Y1bY1b and Y2bY2b respectively. GSH transferase C is the corresponding heterodimer Y1bY2b since it gives all the tryptic peptides which arise from GSH transferase A and GSH transferase 'D' and also contains both C-terminal lysine and proline. These results provide a structural basis to similar conclusions drawn by Mannervik and Jensson [(1980) J. Biol. Chem. 257, 9909-9912] based on enzymic and immunological comparisons. Tryptic peptide maps show that GSH transferases A and 'D' have considerable homology since there are 23 peptides common to both, 12 peptides unique to A and 8 peptides unique to 'D'. Even so GSH transferase A is selectively induced by a phenobarbitone regime. It is, therefore, concluded that Y1b and Y2b are derived from separate but related genes. A similar conclusion has been drawn concerning the Ya and Yc subunits [Beale et al. (1982) Eur. J. Biochem. 126, 459-463], and a comparison of amino acid compositions, presented here, further suggests a genetic relationship between both pairs of subunits.

Amino Acids↗
PubMed1983

The major role of glutathione in the metabolism and excretion of N,N-dimethyl-4-aminoazobenzene in the rat.

In the normal rat given a single dose of one mg N,N-dimethyl-4-aminoazobenzene (DAB) via the hepatic portal vein the following biliary metabolites reached their maximal rates of excretion in the sequence: 4'-sulphonyloxy-DAB, N-(glutathione-S-methylene)-4-aminoazobenzene (GSCH2AB), 4'-sulphonyloxy-N-methyl-4-aminoazobenzene (4'-sulphonyloxy-MAB) 4'-sulphonyloxy-GSCH2AB and MAB-4'-beta-glucuronide. The unusual and relatively unstable N-methylene glutathione conjugates were major metabolites accounting for up to 70% of the whole. It was shown that all the 4-aminoazobenzene (AB) and perhaps all of the 4'-sulphonyloxy-AB, which may be observed in bile, are artefacts due to decomposition of GSCH2AB and 4'-sulphonyloxy-GSCH2AB respectively and that biliary excretion of N-methyl oxidised products of MAB and 4'-hydroxy-MAB is dependent on their conversion to the GSH conjugates, GSCH2AB and 4'-hydroxy-GSCH2AB respectively. Sulphotransferase inhibition by pentachlorophenol caused a reduction in the excretion of all sulphate conjugates, but biliary excretion as a whole was not reduced significantly due to a compensatory increase in the excretion of MAB-4'-beta-glucuronide and the appearance of 4'-OH-GSCH2AB. Glutathione (GSH) depletion by diethylmaleate caused a reduction in biliary metabolites of DAB by lowering the levels of GSH conjugates. This was because the amount of N-methyl oxidation of MAB and 4'-hydroxy-MAB were proportional to the amount of GSH present. The fall in N-methyl oxidation was not compensated for by an increase in 4'-hydroxylation and was accompanied by a delay in the appearance of 4'-hydroxylated metabolites. The administration of potential precursors of 4'-sulphonyloxy-GSCH2AB establishes the sequence of reactions resulting in its formation to be 4'-hydroxylation, N-methyl oxidation, GSH conjugation and O-sulphation.

Animals↗
PubMed1983

The role of glutathione in detoxication.

Glutathione (GSH) is a strong nucleophile which reacts well with soft electrophiles, but poorly with both weak and strong electrophiles. Weak electrophiles have low reactivity with all nucleophiles while strong electrophiles react well with weak nucleophiles including superabundant H(2)O. There are enzymes, the GSH transferases, which catalyze GSH conjugation with all the types of electrophiles described above. In order to deal with the wide variety of potential substrates, a multiplicity of GSH transferases exists-each tissue having its own collection and each enzyme having a different substrate specificity. These enzymes are often very abundant, e.g., in the rat liver cytosol, their concentration is 0.2 mM. THE FOLLOWING SUBSTRATES ARE CONSIDERED IN SOME DETAIL: 1-chloro-2,4-dinitrobenzene, the electrophile derived metabolically from paracetamol N-acetyliminoquinone?), benzo(a)pyrene-4-5-oxide, cholesterol-5alpha,6alpha-oxide, benzo(a)pyrene-7,8-diol-9,10-oxide and the electrophiles derived metabolically from aflatoxin B(1) (the 2,3-oxide?). According to the substrate, optimal enzyme rates vary over seven orders of magnitude from 10(-5) to 10(-12) mole/min/mg. Despite the wide embrace of the GSH transferases, not all metabolically produced electrophiles are substrates. We know of the following examples: N-methylol-4-aminoazobenzene and its 4'-hydroxy derivative (these are soft electrophiles and react well with GSH noncatalytically), N-sulfonyloxy-N-methyl-4-aminoazobenzene, N-sulfonyloxy-N-acetyl-2-aminofluorene (these are strong electrophiles which do not react selectively with GSH) and N-hydroxy-2-aminofluorene which appears to react only slowly with GSH. It is of interest in the present context that all these compounds are derived from either arylamine or arylamide carcinogens. Whether the reaction be enzymic or nonenzymic, conjugation with GSH is a very important means of detoxication accounting in some cases for up to 60% of the biliary metabolites. As seen in the example of aflatoxin B(1), very low enzymic rates observed in vitro are sufficient to account for apparently high rates of biliary excretion of GSH conjugates.GSH transferases have evolved other functions apart from the catalysis of GSH conjugation. GSH transferase B participates in the hepatic uptake of bilirubin and the intracellular distribution of the heme prosthetic group. It also has GSH peroxidase activity which suggests that it might participate in the detoxication of by-products of oxygen utilization including those produced by the action of cytochrome P-450. It is shown that GSH transferase B inhibits lipid peroxidation in vitro.

Animals↗
PubMed1982

5 alpha,6 alpha-Epoxy-cholestan-3 beta-ol (cholesterol alpha-oxide): A specific substrate for rat liver glutathione transferase B.

A semi-micro assay was developed for the conjugation of 5 alpha,6 alpha-epoxy-cholestan-3 beta-ol (cholesterol alpha-oxide) with glutathione. The soluble supernatant of rat liver homogenate catalysed the reaction at a rate of 0.2-0.5 pmol . min-1 . mg protein-1 with 4 microM cholesterol alpha-oxide, while the reaction in the presence of GSH alone was barely detectable. Enzymic activity in the soluble supernatant was due equally to the two forms of glutathione transferase B (approximately 100 pmol . min-1 . mg protein-1), glutathione transferases AA, A, C and E being unreactive. The activity of purified glutathione transferase B was about 5-times that expected from the activity of the soluble supernatant. Complex enzyme kinetics were obtained suggestive of substrate inhibition.

Animals↗
PubMed1982

Characterization and purification of fatty acid-binding protein in rat and human adipose tissue.

A protein with properties similar to fatty acid-binding protein has been isolated from rat and human adipose tissue. Comparison of fatty acid-binding protein from rat liver and adipose tissue and human adipose tissue shows that all have approximately similar molecular weights. Immunologically, rat liver fatty acid-binding protein is similar to the protein characterized from rat adipose tissue. In isolated rat fat cells the fatty acid-binding protein was demonstrated to be involved in the uptake and esterification of long-chain fatty acids. These observations constitute evidence for a potential role of this protein in the fatty acid metabolism of adipocytes.

Adipose Tissue↗
PubMed1982

Evidence that the Ya and Yc subunits of glutathione transferase B (ligandin) are the products of separate genes.

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (YaYa) and a heterodimer (YaYc) in which the monomers are defined by their apparent molecular weights, that of Ya being 22000 and Yc 25000. Soluble tryptic peptides from the native homodimer YaYa have been compared with those from an artificial homodimer YcYc produced by rehybridization of native YaYc. Approximately 10 peptides specific to YaYa, 12 specific to YcYc and 21 common to both have been detected. Some of the above peptides are derived from variants of the monomers themselves. YaYa and YcYc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to YaYa and YcYc and one specific to each. These results suggest that Ya and Yc are derived from at least two different but related genes.

Chemical Phenomena↗
PubMed1982

Identification of glutathione conjugates formed from N-hydroxy-2-acetylaminofluorene in the rat.

Rats which had received N-hydroxy-2-acetylaminofluorene (N-OH-AAF) excreted two glutathione conjugates in the bile. Pretreatment with the sulphotransferase inhibitor pentachlorophenol reduced the amounts excreted by 50% suggesting that these conjugates were formed in vivo from the highly reactive N-O-sulphate ester of N-OH-AAF (AAF-N-sulphate). In order to identify these biliary conjugates synthetic GSH conjugates were prepared by the reaction of N-acetoxy-AAF, (a model compound for AAF-N-sulphate), with GSH. Four conjugates were isolated and characterized by ultraviolet, proton magnetic resonance, mass spectroscopy and by the determination of their amino acid composition. These conjugates were identified as 1-, 3-, 4- and 7-(glutathion-S-yl)-N-acetyl-2-aminofluorene, respectively. The two biliary conjugates were identified as 1- and 3-(glutathion-S-yl)-N-acetyl-2-aminofluorene.

2-Acetylaminofluorene↗
PubMed1982

Formation and identification of glutathione conjugates from 2-nitrosofluorene and N-hydroxy-2-aminofluorene.

2-Nitrosofluorene (NOF) and N-hydroxy-2-aminofluorene (N-HO-AF) are potent direct-acting mutagens, derived from metabolic activation of the carcinogen, N-acetyl-2-aminofluorene (AAF). To assess the ability of cellular glutathione (GSH) to detoxify these electrophilic derivatives, we examined the reaction of NOF and N-HO-AF with GSH in vitro. Two reaction products were isolated and identified as glutathionyl derivatives of 2-aminofluorene (AF) containing an N-S linkage. Amino acid analysis, infrared and NMR (500 MHz) spectroscopy, fast atom bombardment mass spectrometry and analysis of reaction characteristics and hydrolysis products established their structures as N-(glutathion-S-yl)-2-aminofluorene S-oxide (GS-AFI) and N-(glutathion-S-yl)-2-aminofluorene (GS-AFII). Ascorbic acid, which reduces NOF to N-HO-AF, was used to modify reaction yields. These results indicated that GS-AFI was derived from reaction with NOF and that GS-AFII could be formed from both NOF and N-HO-AF. A reaction scheme is proposed in which NOF reacts with GSH to form an intermediate addition product that can rearrange either to GS-AFI or be reduced to GS-AFII. The latter could also be formed by direct reaction with N-HO-AF.

Amino Acids↗
PubMed1982

Formation of N-(glutathion-S-methylene)-4-aminoazobenzene following metabolic oxidation of the N-methyl group of the carcinogen, N-methyl-4-aminoazobenzene.

A major biliary metabolite of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), in the rat was identified as N-(glutathion-S-methylene)-4-aminoazobenzene (GS-CH2-AB). This conjugate was prepared synthetically by a Mannich condensation of 4-aminoazobenzene (AB), formaldehyde (CH2O) and glutathione (GSH) and has been characterized by chemical analysis and by ultraviolet, visible and 13C-NMR spectroscopy. The same conjugate was also formed in vitro by incubating N-methyl-4-aminoazobenzene (MAB), NADPH, NADH and GSH with rat hepatic microsomes. Evidence is presented that GSH reacted with an intermediate resulting from a cytochrome P-450-dependent oxidation of the N-methyl substituent. This reactive intermediate is presumed to be either an N-methylol or a methimine derivative of AB. The significance of this detoxification mechanism is discussed. The presence of an additional major aminoazo-dye GSH conjugate is also noted.

Animals↗