Search PubMed⌕ Search

Biomedical subjects

A Stolz

Publications and source records attributed to A Stolz.

At least 73 records · Page 4Linked to original sources
PubMed1994

Genomic organization and chromosomal localization of a novel human hepatic dihydrodiol dehydrogenase with high affinity bile acid binding.

We previously characterized and cloned a unique human hepatic dihydrodiol dehydrogenase (DDH) that exhibits high affinity binding for bile acids (Stolz, A., Hammond, L., Lou, H., Takikawa, H., Ronk, M., and Shively, J. E. (1993) J. Biol. Chem. 268, 10448-10457). This hepatic dihydrodiol dehydrogenase demonstrates significant sequence homology with the cytosolic rat bile acid binder 3 alpha-hydroxysteroid dehydrogenase and other members of the monomeric oxidoreductase gene family. We now report the genomic organization and chromosomal localization of the human hepatic DDH in order to further define its physiological role and provide additional insight into the development of this gene family. The 15-kilobase human hepatic DDH gene was contained in an overlapping cosmid and lambda genomic clones and is composed of nine exons. A major transcriptional start site was determined to be 30 base pairs upstream from the ATG initiation methionine by both primer extension and S1 nuclease mapping studies. The human hepatic DDH gene was mapped by chromosomal in situ hybridization and analysis of human-mouse somatic cell hybrids to the tip of the short arm of chromosome 10 at p14. Strict conservation of the intron-exon junctions in the human hepatic DDH and two other members of the monomeric oxidoreductase gene family, aldose reductase and mouse major vas deferens protein suggests evolution from a common ancestral gene. Human hepatic DDH mRNA was identified in both human hepatoma Hep G2 and human lung carcinoma cell line NCI-H322 by RN'ase protection; thus, these cell lines will be useful in examining the regulation of the gene.

Alcohol Oxidoreductases↗
PubMed1994

Distribution of 3 alpha-hydroxysteroid dehydrogenase (bile acid binder) in rat small intestine: comparison with glutathione S-transferase subunits.

We identified and quantitated the Y' bile acid binder, i.e., 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), in rat small intestinal mucosa, and compared its longitudinal distribution with that of glutathione S-transferases (GST). The enzyme activity of 3 alpha-HSD in intestinal mucosa was approximately one-third of that in liver, and it had similar activity in the proximal, middle, and distal portions of the intestine. Immunoreactive protein corresponding to hepatic bile acid binder was detected in all segments of rat small intestine mucosal cytosol by Western blot analysis. There was no significant difference in the concentration of bile acid binder, assayed by enzyme-linked immunosorbent assay (ELISA), between the proximal and the distal intestine, this being 2.93 +/- 0.03 and 3.29 +/- 0.95 nmol/g tissue, respectively (mean +/- SD of four animals). On the other hand, the concentration of GST 1-1 showed sharp longitudinal decline and that of GST 3-4 was negligible in the small intestine, as we previously reported, indicating that bile acid binder was a prominent cytosol binding protein in the distal intestine. These results suggested the possible role of bile acid binder in the intracellular transport of bile acids in the ileum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Regulation of rat hepatic 3 alpha-hydroxysteroid dehydrogenase in vivo and in primary cultures of rat hepatocytes.

In the bile acid biosynthetic pathways of humans and the rat, hepatic 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH) catalyzes the stereospecific reduction of the 3-oxo group of bile acid precursors. In addition, 3 alpha-HSDH may serve to shuttle bile acids from sinusoidal to apical (cannalicular) membranes of the rat hepatocyte. The objective of the present study was to define the molecular regulation of rat hepatic 3 alpha-HSDH in response to the key effectors of cholesterol 7 alpha-hydroxylase, the rate-determining enzyme in bile acid biosynthesis. Steady-state 3 alpha-HSDH mRNA levels in primary cultures of rat hepatocytes fell to 16 +/- 1% of whole liver levels after 72 h in culture, indicating that the gene is not spontaneously expressed in isolated hepatocytes. However, the addition of thyroxine (1.0 microM) or dexamethasone (1.0 microM) to the culture medium resulted in steady-state mRNA levels of 34 +/- 4% and 102 +/- 20% of whole liver levels, respectively. Moreover, the combination of thyroxine and dexamethasone (each 1.0 microM) induced mRNA to levels 2-fold higher than whole liver. 3 alpha-HSDH specific activity in cultured hepatocyte cytosol increased from 3.0 +/- 0.7 to 10.4 +/- 1.3 nmol/min per mg protein in no-addition and thyroxine plus dexamethasone-treated cultures, respectively; protein mass underwent similar changes. Whole liver 3 alpha-HSDH mRNA levels decreased in thyroidectomized, adrenalectomized, and hypophysectomized rats, to 60 +/- 6%, 51 +/- 4%, and 29 +/- 5% of sham-operated rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

cDNA cloning and expression of the human hepatic bile acid-binding protein. A member of the monomeric reductase gene family.

In human liver, we previously identified one isoform of dihydrodiol dehydrogenase activity that expresses high affinity bile acid binding (HBAB) with minimal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) activity for bile acids. This protein may assist in the rapid intracellular transport of bile acids from the sinusoidal to the canalicular pole of the cell. We now report the cDNA cloning and bacterial expression of this novel, multifunctional protein. A 1252-base pair HBAB cDNA was cloned from a HepG2 lambda GT11 library using a rat hepatic bile acid binder cDNA probe. Bacterial expressed recombinant HBAB oxidized racemic trans dihydrodiol benzene (0.455 mumol NADPH/mg/min) with minimal 3 alpha-HSD activity for bile acids (< 0.003 mumol NADPH/mg/min). Lithocholic acid and chenodeoxycholic acid dissociation constants as determined by displacement of the fluorescent probe, bis-1-anilino-8 sulfonate, were higher than those previously reported for the native protein (1 microM versus 10 nM). Significant amino acid sequence homology was found with the human chlordecone reductase, bovine prostaglandin F synthetase, and rat hepatic-3 alpha-HSD suggesting, that HBAB is also a member of the recently identified, monomeric oxidoreductase gene family. Future studies will define the physiologic significance of this novel, multifunctional protein in bile acid transport and xenobiotic metabolism.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Purification and properties of 2'-hydroxybenzalpyruvate aldolase from a bacterium that degrades naphthalenesulfonates.

2'-Hydroxybenzalpyruvate aldolase catalyzes the cleavage of 2'-hydroxybenzalpyruvate to salicylaldehyde and pyruvate. This reaction is part of the degradative pathways for naphthalene and naphthalenesulfonates by bacteria. 2'-Hydroxybenzalpyruvate aldolase has been purified to homogeneity from a bacterium that degrades naphthalenesulfonates (strain BN6). The enzyme has a molecular weight of about 120,000 and is composed of identical subunits with a molecular weight of about 38,500. Thus the enzyme appears to exist as a trimeric oligomer. The NH2-terminal amino acid sequence did not show significant homology to other published amino acid sequences. Extensive loss of enzyme activity occurred when the enzyme was incubated with 2'-hydroxybenzalpyruvate in the presence of sodium borhydride. This suggested the intermediate formation of a stable Schiff base between enzyme and substrate. 2'-Hydroxybenzalpyruvate aldolase was inhibited by p-chloromercuribenzoate and by the reaction product salicylaldehyde. The enzyme converted 2'-hydroxybenzalpyruvate, 2',4'- and 2',6'-dihydroxybenzalpyruvate.

Aldehyde-Lyases↗
PubMed1993

Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9.

The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9 was studied. Intact cells of Pseudomonas sp. BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate. Cell-free extracts of Pseudomonas sp. BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H. A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene. 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate. 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp. BN9 after growth with 2,4-dihydroxybenzoate.

Aerobiosis↗
PubMed1993

Identification and purification of a human liver cytosolic tocopherol binding protein.

We recently purified rat tocopherol binding protein (TBP), a 32-kDa cytosol protein which specifically binds alpha-tocopherol, exists as two charge isoforms, and is expressed exclusively in liver and only in hepatocytes. For the present work, we sought to identify the human hepatic tocopherol binding protein from normal human livers harvested from organ donors. Gel filtration of hepatic cytosol identified a peak of [alpha-3H]-tocopherol binding in the 30- to 40-kDa fractions displaceable by excess unlabeled alpha-tocopherol. The fractions exhibiting this binding were pooled and run on Affi-Gel Blue affinity chromatography eluted with a salt gradient. A single major peak of tocopherol binding activity eluted at 22 mS/cm. This peak was further purified by FPLC chromatofocusing. A single protein peak of specific alpha-tocopherol binding eluted at pH 5.9. Finally, the peak from chromatofocusing was purified to apparent homogeneity by reversed-phase microbore HPLC chromatography. Two closely eluting protein peaks were separated and each was homogeneous, had identical migration on a SDS-PAGE (36 kDa), and had the same amino acid composition. The purified human TBP exhibited displaceable, specific alpha-tocopherol binding in the gel filtration assay of tocopherol binding. Laser desorption time-of-flight mass spectroscopy revealed a molecular weight of 36.6 kDa. Both forms of human TBP reacted in Western blot with polyclonal rabbit anti-rat TBP. Identification of the human tocopherol binding protein will allow future studies on its physiological function in human alpha-tocopherol metabolism.

Animals↗
PubMed1993

Bacterial metabolism of 5-aminosalicylic acid: enzymic conversion to L-malate, pyruvate and ammonia.

5-Aminosalicylate (5AS) was converted to L-malate, pyruvate and ammonia by cell-free extracts from Pseudomonas sp. BN9 in the presence of glutathione. In the absence of glutathione, 5AS was oxidized to the ring-fission product cis-4-amino-6-carboxy-2-oxo-hexa-3,5-dienoate (cis-ACOHDA). Glutathione catalysed the spontaneous isomerization of cis-ACOHDA to its trans-isomer. The same reaction was catalysed by light and by acidic conditions. trans-ACOHDA was enzymically deaminated to fumarylpyruvate (trans-2,4-dioxo-5-hexenoate). The trans-ACOHDA hydrolase was induced after growth of Pseudomonas sp. BN9 with 5AS, but not after growth with acetate or nutrient broth. At the fumarylpyruvate stage, the metabolism of 5AS converged with a pathway described for the degradation of gentisate. Fumarylpyruvate was cleaved by Pseudomonas sp. BN9 to fumarate and pyruvate.

Acids↗
PubMed1992

Bacterial metabolism of 5-aminosalicylic acid. Initial ring cleavage.

The metabolism of 5-aminosalicylate (5AS) by a bacterial strain, Pseudomonas sp. BN9, was studied. Intact cells of Pseudomonas sp. BN9 grown with 5AS oxidized 5AS and 2,5-dihydroxybenzoate (gentisate), whereas cells grown with gentisate oxidized only the growth substrate of all substituted salicylates tested. Cell extracts from Pseudomonas sp. BN9 catalysed the stoichiometric reaction of 1 mol of oxygen with 1 mol of 5AS to a metabolite with an intense u.v.-absorption maximum at 352 nm (pH 8.0). This metabolite was accumulated under neutral conditions, but was rapidly destroyed at acid pH. It was identified by m.s. and acid-catalysed deamination to fumarylpyruvate (trans-2,4-dioxohept-5-enedioic acid) as cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, thus demonstrating direct cleavage of the monohydroxylated substrate 5AS to a non-aromatic ring-fission product. The enzyme responsible for conversion of 5AS was shown to be Fe(II)-dependent and to be distinct from gentisate 1,2-dioxygenase in strain BN9.

Aminosalicylic Acids↗
PubMed1992

Identification, purification, and immunochemical characterization of a tocopherol-binding protein in rat liver cytosol.

Tocopherol binding activity accompanying a rat liver cytosolic protein with molecular weight of 30-36 kDa has been demonstrated previously, although the isolation of the protein has not been reported. We now report the purification of an alpha-tocopherol-binding protein (TBP) from rat liver cytosol utilizing three chromatographic procedures: gel filtration, Affi-Gel Blue affinity chromatography, and chromatofocusing. Three peaks of specific alpha-tocopherol-binding activity were resolved on Affi-Gel Blue, referred to as AFB-1A, 1B, and 2. A 32-kDa homogeneous form was obtained after chromatofocusing of AFB-1B. D-alpha-[3H]tocopherol was displaced from homogeneous TBP in the presence of 500-fold excess of nonlabeled alpha-tocopherol, indicating the specificity of the binding. Anti-TBP rabbit antisera identified only one protein in rat hepatic cytosol on Western blotting. TBP immunoreactivity was found in the cytosol of rat liver and the lysate of fractionated hepatocytes, but not in the cytosol of other organs (including the heart, spleen, testes, and lung) nor in the lysate of fractioned Ito cells, endothelial cells, or Kupffer cells isolated from rat liver. Semi-quantitative ELISA demonstrated that rat liver cytosol contained approximately 2 mg TBP/g of cytosol protein. This immunoreactivity was associated with only the 30-36 kDa gel filtration fractions of rat liver cytosol and with both AFB-1A and -1B but not with AFB-2.

Amino Acids↗
PubMed1991

Molecular structure of rat hepatic 3 alpha-hydroxysteroid dehydrogenase. A member of the oxidoreductase gene family.

3-alpha-Hydroxysteroid dehydrogenase (3 alpha-HSD) (EC 1.1.1.50) is an important multifunctional oxidoreductase capable of metabolizing steroid hormones, polycyclic aromatic hydrocarbons, and prostaglandins. 3 alpha-HSD is also required for bile acid synthesis and has been suggested to play an important role in net bile acid transport across the hepatocyte (Stolz, A., Takikawa, H., Ookhtens, M., and Kaplowitz, N. (1989) Annu. Rev. Physiol. 51, 166-177). In order to characterize molecular forms and begin to determine its regulation, we now report the nucleotide sequence, tissue distribution, and homology to other members of the oxidoreductase superfamily. Rat hepatic 3 alpha-HSD cDNA encodes for a 322-amino acid protein with a predicted molecular weight of 37,022 expressed in a 2.4-kilobase (kb) message size. Northern blot analysis of total RNA revealed equivalent steady-state levels in liver and intestine in male rats with lower levels of expression in the colon and minimal expression in stomach, lung, and testis. Female liver contained approximately 2-3-fold greater steady-state levels of mRNA as compared to the male liver with equivalent intestinal expression. Two hybridizing bands, 2.4 and 1.4 kb, were identified in total RNA from the ovary. 3 alpha-HSD exhibits 75% amino acid sequence homology with bovine lung prostaglandin F synthetase and 50% homology with human aldose reductases. Amino acid sequence analysis with short chain alcohol dehydrogenases identified a possible NADP(H) cofactor-binding site at the amino terminus. The significant homology of 3 alpha-HSD with both prostaglandin F synthetase and aldose reductases suggest a subdivision of monomeric, NADPH reductases within the larger oxidoreductases superfamily.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Effect of indomethacin on the uptake, metabolism and excretion of 3-oxocholic acid: studies in isolated hepatocytes and perfused rat liver.

3 alpha-Hydroxysteroid dehydrogenase catalyzes the reduction of 3-oxo-bile acids and binds 3 alpha-hydroxy bile acids. Indomethacin is a competitive inhibitor of the enzyme. In incubations of isolated rat hepatocytes, indomethacin delayed the intracellular reduction and the initial uptake of 3-oxocholic acid. Following a tracer dose of 3-oxocholic acid in perfused rat liver, rapid biliary excretion was observed mainly as taurocholic acid. Only 1.1% of the dose was recovered in the caval outflow and nearly all appeared in the first 5 min collection. When the tracer dose was given after initiating a constant infusion of indomethacin (50 microM), a dramatic decrease in biliary excretion was observed, still mainly as taurocholic acid, and 14% of the dose was recovered in the caval effluent: 10% in the first 5 min collection, mainly as 3-oxocholic acid, followed by a steady, slow release of mainly taurocholic acid. The increased intrahepatic retention of bile acids and slow release into perfusate and bile in response to indomethacin are consistent with displacement of bile acids from cytosolic protein.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Metabolism of naphthalene by the biphenyl-degrading bacterium Pseudomonas paucimobilis Q1.

Pseudomonas paucimobilis Q1 originally isolated as biphenyl degrading organism (Furukawa et al. 1983), was shown to grow with naphthalene. After growth with biphenyl or naphthalene the strain synthesized the same enzyme for the ring cleavage of 2,3-dihydroxybiphenyl or 1,2-dihydroxynaphthalene. The enzyme, although characterized as 2,3-dihydroxybiphenyl dioxygenase (Taira et al. 1988), exhibited considerably higher relative activity with 1,2-dihydroxynaphthalene. These results demonstrate that this enzyme can function both in the naphthalene and biphenyl degradative pathway.

Biodegradation, Environmental↗
PubMed1991

Mineralization of the sulfonated azo dye Mordant Yellow 3 by a 6-aminonaphthalene-2-sulfonate-degrading bacterial consortium.

Under anaerobic conditions the sulfonated azo dye Mordant Yellow 3 was reduced by the biomass of a bacterial consortium grown aerobically with 6-aminonaphthalene-2-sulfonic acid. Stoichiometric amounts of the aromatic amines 6-aminonaphthalene-2-sulfonate and 5-aminosalicylate were generated and excreted into the medium. After re-aeration of the culture, these amines were mineralized by different members of the bacterial culture. Thus, total degradation of a sulfonated azo dye was achieved by using an alternating anaerobic-aerobic treatment. The ability of the mixed bacterial culture to reduce the azo dye was correlated with the presence of strain BN6, which possessed the ability to oxidize various naphthalenesulfonic acids. It is suggested that strain BN6 has a transport system for naphthalenesulfonic acids which also catalyzes uptake of sulfonated azo dyes. These dyes are then gratuitously reduced in the cytoplasm by unspecific reductases.

Aerobiosis↗
PubMed1991

Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids.

1,2-Dihydroxynaphthalene dioxygenase was purified to homogeneity from a bacterium that degrades naphthalenesulfonic acids (strain BN6). The enzyme requires Fe2+ for maximal activity and consists of eight identical subunits with a molecular weight of about 33,000. Analysis of the NH2-terminal amino acid sequence revealed a high degree of homology (22 of 29 amino acids) with the NH2-terminal amino acid sequence of 2,3-dihydroxybiphenyl dioxygenase from strain Pseudomonas paucimobilis Q1. 1,2-Dihydroxynaphthalene dioxygenase from strain BN6 shows a wide substrate specificity and also cleaves 5-, 6-, and 7-hydroxy-1,2-dihydroxynaphthalene, 2,3- and 3,4-dihydroxybiphenyl, catechol, and 3-methyl- and 4-methylcatechol. Similar activities against the hydroxy-1,2-dihydroxynaphthalenes were also found in cell extracts from naphthalene-degrading bacteria.

Amino Acid Sequence↗
PubMed1990

Oxidation and reduction of bile acid precursors by rat hepatic 3 alpha-hydroxysteroid dehydrogenase and inhibition by bile acids and indomethacin.

Enzyme kinetics of purified rat hepatic 3 alpha-hydroxysteroid dehydrogenase for bile acid precursors and effects of bile acids and indomethacin on those activities were studied. This enzyme catalyzed the oxidoreduction of the C3 position of bile acid precursors. Km for 7 alpha, 12 alpha-dihydroxy-5 beta-cholestan-3-one (1.6 microM) was markedly lower than Km for 7 alpha-hydroxy-5 beta-cholestan-3-one (28 microM) but Vmax was similar. Km for 3 alpha, 7 alpha-dihydroxy-5 beta-cholestane (12 microM) was lower than Km for 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholestane (150 microM) although Vmax/Km values were similar for both compounds. Bile acids and indomethacin inhibited the reduction of 3-oxo bile acid precursors. NADPH inhibited the binding of lithocholic acid (3 alpha-hydroxy-5 beta-cholanic acid) by 3 alpha-hydroxysteroid dehydrogenase. These data suggest that intrahepatic bile acid concentrations may affect the reduction of 3-oxo-bile acid precursors and intrahepatic redox conditions may affect intracellular bile acid transfer.

3-Hydroxysteroid Dehydrogenases↗
PubMed1990

Relationship between the newly identified bile acid binder and bile acid oxidoreductases in human liver.

Gel filtration fractions of human hepatic cytosol obtained from an autopsy liver were examined for elution of bile acid oxidoreductases. Several enzymes including 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 3-ketosteroid reductase, and dihydrodiol dehydrogenase eluted mainly in the 30,000-40,000 Mr fractions known to contain the newly identified bile acid binder (Stolz, A., Sugiyama, Y., Kuhlenkamp, J., and Kaplowitz, N. (1984) FEBS Lett. 177, 31-35). These enzyme activities could be resolved into six peaks of dihydrodiol dehydrogenase activity on chromatofocusing, some of which also had oxidoreductase activity with bile acids. Using equilibrium dialysis, the major lithocholate-binding activity was found to coelute with 3 beta-HSD, completely separate from 3 alpha-HSD. Reexamination of the surgical liver specimen originally used to purify the bile acid binder confirmed these results. The peak fraction from chromatofocusing, which exhibited the bulk of binding activity with bile acids, had 3 beta-HSD activity, whereas other fractions had 3 alpha-HSD. Anti-serum to the previously purified binder identified a single 36-kDa protein in both liver specimens and exclusively in the chromatofocusing fractions containing both the binding and 3 beta-HSD activity. However, upon further purification of the binder from this fraction, 3 beta-HSD activity was separated from the binder, but the homogeneous protein retained dihydrodiol dehydrogenase activity. Thus, in contrast to the rat in which the major bile acid binder is identical to 3 alpha-HSD, in human liver the bile acid binder is distinct from 3 alpha-HSD and copurifies with a different oxidoreductase that has dihydrodiol dehydrogenase activity but no activity with bile acids.

3-Hydroxysteroid Dehydrogenases↗