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W B Jakoby

Publications and source records attributed to W B Jakoby.

At least 19 recordsLinked to original sources
PubMed1992

Tyrosine-ester sulfotransferase from rat liver: bacterial expression and identification.

A nucleotide sequence that had been proposed for, but not identified as, rat liver aryl sulfotransferase (EC 2.8.2.1) was prepared in an appropriate vector and transformed into Escherichia coli. The protein, expressed in large amounts, was not aryl sulfotransferase (EC 2.8.2.1) but rather tyrosine-ester sulfotransferase (EC 2.8.2.9), a sulfotransferase also active with phenols but having a much wider substrate range that includes hydroxylamines and esters of tyrosine. The recombinant tyrosine-ester sulfotransferase was identified by its unique substrate spectrum, by comparison with three peptides that were sequenced from homogeneous tyrosine-ester sulfotransferase isolated directly from rat liver, and by the specificity of antibody raised to the rat liver enzyme. Two isoforms were obtained, each of which was difficult to solubilize upon sonication of E. coli. Both forms were solubilized with a solution of polyols (glycerol and sucrose) and subsequently purified to homogeneity.

Amino Acid Sequence↗
PubMed1979

Phenol sulfotransferases.

Two phenol sulfotransferases have been purified from rat liver by conventional techniques coupled with affinity chromatography on Affi-Gel blue and ATP-agarose. Both enzymes are homogeneous by the criterion of sodium dodecyl sulfate gel electrophoresis. Each enzyme has a molecular weight of approximately 65,000 and consists of two subunits of apparently equal size. The enzymes are also similar in specificity and in their kinetic parameters but differ in amino acid composition and in their elution from DEAE-cellulose. With adenosine 3'-phosphate 5'-phosphosulfate as donor, a large variety of phenolic compounds serve as sulfate acceptor; sterols, simple alcohols, bile acids, and hydroxamates do not serve as substrates. The transferases may be considered as detoxification enzymes which catalyze the conjugation of xenobiotics containing a phenol group or of phenolic compounds generated by endogenous oxidation. The enzymes act on 3-hydroxyindole to yield indican, suggesting that their in vivo function may include the production of this normal tryptophan metabolite.

Amino Acids↗
PubMed1978

Glutathione transferases. Catalysis of nucleophilic reactions of glutathione.

Homogeneous preparations of the glutathione transferases from rat liver have been tested for their ability to catalyze a number of diverse nucleophilic reactions of GSH. Although disulfide interchange with GSSG or L-cystine, and cis-trans isomerization of maleic acid, are clearly promoted by thiols in solution, the reactions were not catalyzed by the glutathione transferases. In contrast, certain more hydrophobic analogs of these compounds were found to serve as substrates. The transferases also catalyze the glutathione-dependent release of p-nitrophenol from p-nitrophenyl acetate and p-nitrophenyl trimethylacetate. These observations are consistent with the formulation that catalysis may result from close juxtaposition of sufficiently electrophilic, nonpolar compounds with GSH on the enzyme surface.

Animals↗
PubMed1978

The glutathione S-transferases: a group of multifunctional detoxification proteins.

The physiological roles of the glutathione S-transferases, by whatever name, seem to result in detoxification. As is true of albumin, members of this group of proteins bind an enormous number of compounds that appear to have in common only hydrophobic topography; the binding of bilirubin is an example of a major function common to all higher species. If the ligand bears a sufficiently electrophilic center, it will be attacked by the nucleophile GSH; such compounds would be the substrates of the enzyme. And should such a ligand be extraordinarily reactive--as, for example, some of the epoxide carcinogens generated by the cytochrome P450-linked, mixed-function oxidases, or even 1-chloro-2,4-dinitrobenzene--then reaction may occur either with GSH or irreversibly with the transferase itself. By reason of the wide distribution and high intracellular concentration of these proteins, there appears to be sufficient enzyme for all three roles in detoxification.

Amino Acids↗
PubMed1978

Hepatic accumulation and intracellular binding of conjugated bilirubin.

After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.

Animals↗
PubMed1977

Relationship between the soluble glutathione-dependent delta 5-3-ketosteroid isomerase and the glutathione S-transferases of the liver.

Soluble, glutathione-stimulated delta 5-3-ketosteroid isomerase (EC 5.3.3.A) activity of human and rat liver resides in very basic proteins with molecular weights of about 45,000 which are present in high concentrations in these tissues. Physiochemical and immunological evidence is presented for the identity of the proteins responsible for this enzymatic activity with the glutathione S-transferases (RX:glutathione R-transferase, EC 2.5.1.18) that conjugate glutathione with a variety of electrophilic compounds. In the rat, the steroid isomerase is associated principally with the major transferase (B), which is also known as ligandin, and has the versatility to bind various hydrophobic compounds such as bilirubin, corticosteroids, and metabolites of a number of carcinogens. Other rat liver-glutathione S-transferase species are far less active in the steroid isomerization reaction. The delta 5-3-ketosteroid isomerase activity of human liver is more uniformly distributed among the five glutathione S-transferases that have been described. Steroid isomerization differs fundamentally from other reactions promoted by glutathione S-transferases in that glutathione is not consumed in the reaction. However, because the transferase enzymes promote nucleophilic attack by glutathione on a variety of largely foreign organic substrates, a similar mechanism may be involved in the isomerase reaction. Delta 5-3-ketosteroids are among the few known naturally occurring substrates for these enzymes.

Animals↗
PubMed1976

Mechanism for the several activities of the glutathione S-transferases.

The catalyzed reactions of GSH with organic nitrate and thiocyanate esters and with a series of chloronitrobenzene substrates have been investigated and the results used to formulate a mechanism for glutathione S-transferase catalysis. All the homogeneous preparations of the glutathione transferases that have been tested catalyze the reaction of GSH with organic nitrates and thiocyanates. The nature of the reaction with nitrate esters, resulting in the formation of GSSG rather than a thioether, has been investigated further. The presence of an additional nonsubstrate thiol decreased the formation of GSSG to an extent that cannot be explained by disulfide interchange. These results are interpreted to reflect the enzymatic formation of an unstable glutathione sulfenyl nitrite that undergoes subsequent non-enzymatic decomposition. Hammett plots of the catalytic constants of rat liver transferases B and C obtained with a series of 4-substituted 1-chloro-2-nitrobenzene substrates demonstrate a linear relationship with sigma- substituent constants, reflecting the nucleophilic nature of the enzymatic reactions and their strong dependence on the electrophilicity of the nonthiol substrate. These data suggest that the many diverse reactions catalyzed by the glutathione transferases may be formulated as a nucleophilic attack of enzyme-bound GSH on the electrophilic center of the second substrate. The final products observed reflect this primary event and the existence of subsequent nonenzymatic reactions.

Animals↗
PubMed1975

Multiple forms of human glutathione S-transferase and their affinity for bilirubin.

The initial enzymic step in mercapturic acid formation is catalyzed by glutathione S-transferase. Several species of this enzyme, designated as transferases alpha, beta, gamma, delta and epsilon on the basis of increasing isoelectric points, were isolated from human liver. Evidence is presented that each of the purified species is homogeneous with respect to sodium dodecylsulfate-gel electrophoresis. Transferases alpha, beta and epsilon each appear as a single band on gel electrofocusing; transferases gamma and delta are present as two and three bands, respectively, with each band catalytically active. Amino acid analysis indicated the five transferases to be either very closely related or identical in this respect. All enzyme species have a molecular weight of about 48500 and consist of two apparently identical subunits. The spectrum of substrates is the same for each although the enzymes differ slightly in specific activity. As is the case for the rat liver enzymes, each of the human transferases binds bilirubin although this compound is not a substrate.

Amino Acids↗
PubMed1975

Binding of nonsubstrate ligands to the glutathione S-transferases.

Fluorescence spectroscopy and inhibition kinetics were used to quantitate the affinity of nonsubstrate ligands for the rat liver glutathione S-transferases AA, A, B, and C in the presence of glutahione. The dissociation constants KD, for ligands such as bilirubin, indocyanine green, and hematin were determined by measuring the decrease in the intrinsic fluorescence of the proteins attendant on the addition of ligand. A second technique, used for compounds which absorb strongly at the excitation maxima of tryptophan, was to utilize 8-anilinonaphthalen sulfonate in the formation of protein complex fluorescing at a higher wavelength. The quenching of this complex allowed the determination of the dissociation constants for ligands such as 3,6-dibromosulfophthalein and cephalothin. These data indicate that all four proteins bind these ligands but do so with different affinities. The bilirubin-induced decrease in fluorescence was used to estimate the stoichiometry of binding as 1.2 mol of bilirubin bound/mol of transferase B and 0.7 mol/mol of transferase C. All of the ligands examine are inhibitors of catalytic activity, as tested in a standard assay with GSH and 1-chloro-2,4-dinitrobenzene as substrates. From these studies we conclude that these proteins have a broad specificity not only for their substrates, but for the binding of nonsubstrate ligands as well.

Anilino Naphthalenesulfonates↗