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N Kaplowitz

Publications and source records attributed to N Kaplowitz.

At least 109 records · Page 6Linked to original sources
PubMed1987

A serologic follow-up of the 1942 epidemic of post-vaccination hepatitis in the United States Army.

An epidemic of icteric hepatitis in 1942 affected approximately 50,000 U.S. Army personnel. This outbreak was linked to specific lots of yellow-fever vaccine stabilized with human serum. To identify the responsible virus and the consequences of the epidemic, during 1985 we interviewed and serologically screened 597 veterans who had been in the army in 1942. These subjects were selected from three groups. Group I consisted of patients who had received the implicated vaccine and had jaundice; Group II had received the implicated vaccine but remained well; Group III had received a new, serum-free vaccine, with no subsequent jaundice. Ninety-seven percent of Group I, 76 percent of Group II, and 13 percent of Group III were positive for antibodies to hepatitis B virus. Only one subject had hepatitis B surface antigen, for a carrier rate of 0.26 percent among recipients of the implicated vaccine. The prevalence of hepatitis A antibody was similar in all three groups, and no subject had antibody to hepatitis delta virus. We conclude that hepatitis B caused the outbreak, that about 330,000 persons may have been infected, that the hepatitis B virus carrier state was a rare consequence, and that the outbreak induced hepatitis B antibodies that appear to persist for life.

Aged↗
PubMed1987

Trans-stimulation and driving forces for GSH transport in sinusoidal membrane vesicles from rat liver.

Sinusoidal membrane vesicles from rat liver were employed to study the characteristics of GSH transport. Saturable concentration dependent uptake was best described by the sum of a high and low Km transport. Preloading with GSH markedly stimulated the initial uptake of GSH. GSH transport was electrogenic; uptake was enhanced by an inwardly directed K+ gradient which could be blocked by the K+-channel blocker, Ba2+. The other cations such as Na+, Li+ were poor substitutes for K+. These results therefore show that net GSH transport involves movement of K+.

Animals↗
PubMed1987

Subunit heterogeneity of cationic human hepatic glutathione S-transferases.

We have purified the major reduced glutathione (GSH) S-transferases from 3 apparently normal human livers: two obtained at surgery and one at autopsy. Purification was by sequential gel filtration. GSH-affinity chromatography, and chromatofocusing. All three livers exhibited the same two major transferase peaks from chromatofocusing at pH 9.0 and 8.7 (designated C1 and C2, respectively) and several (2-4) minor peaks. Another major form (designated A1) from two livers eluted from chromatofocusing at pH 5.4, whereas the major form from the third liver (designated N1) eluted near neutral (pH 6.8). The transferase from erythrocytes eluted at pH 4.6. Isoelectric focusing revealed that the true pI of A1 was pH 7.1 indicating that C1, C2 and A1 are all cationic. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, C1, C2 and A1 exhibited the same single subunit (25,000) whereas N1 was different (26,000). The erythrocyte enzyme had a smaller subunit (23,500). Urea/SDS-polyacrylamide gel electrophoresis resolved the apparent single subunit of A1, C1 and C2 into two distinct subunits. C1 from all 3 livers was a homodimer of the faster migrating subunit (designated subunit I); C2 was a heterodimer (designated I-II); and A1 was a homodimer of the slower migrating subunit (designated subunit II). Hybridization experiments demonstrated that by mixing C1 and A1 we could produce C2 whereas dissociation and reassociation of the subunits of C2 generated C1 and A1 as well as C2. Rabbit antiserum to C1 recognized C1 and C2, but not A1. Thus, the cationic human hepatic transferases are dimers of two distinct subunits.

Amino Acids↗
PubMed1987

3 alpha-hydroxysteroid dehydrogenase activity of the Y' bile acid binders in rat liver cytosol. Identification, kinetics, and physiologic significance.

Rat Y' bile acid binders (33 kD) have been previously recognized as cytosolic bile acid binding proteins (Sugiyama, Y., T. Yamada, and N. Kaplowitz, 1983, J. Biol. Chem., 258:3602-3607). We have now determined that these Y' binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD), bile acid-metabolizing enzymes. 3 alpha-HSD activity copurified with lithocholic acid-binding activity after sequential gel filtration, chromatofocusing, and affinity chromatography. Three peaks of 3 alpha-HSD activity (I, II, III) were observed in chromatofocusing and all were identified on Western blot by a specific Y' binder antiserum. 3 alpha-HSD-I, the predominant form, was purified and functioned best as a reductase at pH 7.0 with a marked preference for NADPH. Michaelis constant values for mono- and dihydroxy bile acids were 1-2 microM, and cholic acid competitively inhibited the reduction of 3-oxo-cholic acid. Under normal redox conditions, partially purified 3 alpha-HSD-I and freshly isolated hepatocytes catalyzed the rapid reduction of 3-oxo-cholic to cholic acid without formation of isocholic acid, whereas the reverse reaction was negligible. The Y' bile acid binders are therefore 3 alpha-HSD, which preferentially and stereospecifically catalyze the reduction of 3-oxo-bile acids to 3 alpha-hydroxy bile acids.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Effect of chronic ethanol feeding on rat hepatocytic glutathione. Compartmentation, efflux, and response to incubation with ethanol.

Hepatocytes from rats that were fed ethanol chronically for 6-8 wk were found to have a modest decrease in cytosolic GSH (24%) and a marked decrease in mitochondrial GSH (65%) as compared with pair-fed controls. Incubation of hepatocytes from ethanol-fed rats for 4 h in modified Fisher's medium revealed a greater absolute and fractional GSH efflux rate than controls with maintenance of constant cellular GSH, indicating increased net GSH synthesis. Inhibition of gamma-glutamyltransferase had no effect on these results, which indicates that no degradation of GSH had occurred during these studies. Enhanced fractional efflux was also noted in the perfused livers from ethanol-fed rats. Incubation of hepatocytes in medium containing up to 50 mM ethanol had no effect on cellular GSH, accumulation of GSH in the medium, or cell viability. Thus, chronic ethanol feeding causes a modest fall in cytosolic and a marked fall in mitochondrial GSH. Fractional GSH efflux and therefore synthesis are increased under basal conditions by chronic ethanol feeding, whereas the cellular concentration of GSH drops to a lower steady state level. Incubation of hepatocytes with ethanol indicates that it has no direct, acute effect on hepatic GSH homeostasis.

Animals↗
PubMed1987

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by rat hepatic 3 alpha-hydroxysteroid dehydrogenase. I. Studies with the purified enzyme, isolated rat hepatocytes, and inhibition by indomethacin.

We recently identified that the Y' bile acid binders are 3 alpha-hydroxysteroid dehydrogenases (3 alpha-HSD). In the present studies, purified 3 alpha-HSD catalyzed rapid 3H loss from [3 beta-3H, C24-14C]lithocholic and chenodeoxycholic acids without net conversion to 3-oxo bile acids under physiologic pH and redox conditions. [3 beta-3H]Cholic acid was a poor substrate. The Y' fraction of hepatic cytosol was exclusively responsible for this activity and 3H was transferred selectively to NADP+. Time-dependent 3H loss was also seen in isolated hepatocytes. Further hydroxylation products of lithocholic and chenodeoxycholic acids lost 3H at the same rate, whereas 3H loss from lithocholic acid rapidly ceased, which suggests compartmentation of this bile acid in hepatocytes. Indomethacin inhibited 3H loss from bile acids either in incubations with the pure enzyme or in isolated hepatocytes. Indomethacin did not alter the initial uptake rate of bile acids by hepatocytes, but caused a redistribution of unconjugated bile acids into the medium at early time points (2.5 and 5.0 min) and that of conjugated bile acids at later time intervals (30 min). 3H loss from the 3 beta position therefore can be used to probe the interaction between bile acids and cytosolic 3 alpha-HSD in intact cells, and indomethacin is capable of inhibiting this interaction.

3-Hydroxysteroid Dehydrogenases↗
PubMed1987

Cyclical oxidation-reduction of the C3 position on bile acids catalyzed by 3 alpha-hydroxysteroid dehydrogenase. II. Studies in the prograde and retrograde single-pass, perfused rat liver and inhibition by indomethacin.

[3 beta-3H, 24-14C]Lithocholic, chenodeoxycholic, and cholic acids were administered in tracer bolus doses either prograde or retrograde in the isolated perfused rat liver. Little 3H loss from cholic acid was observed, whereas with the other bile acids, 20-40% of the administered 3H was lost in a single pass from perfusate to bile. Most of the 3H loss occurred rapidly (5 min) and was recovered as [3H]water in perfusate. Excretion of bile acids was delayed with retrograde administration, and 3H loss was more extensive. In both prograde and retrograde studies, indomethacin markedly inhibited the excretion of the bolus of bile acid into bile. Indomethacin inhibited the extraction of glycocholate (50 microM) during steady state perfusion without affecting transport maximum for excretion. At lower glycocholate concentration (5 microM), indomethacin inhibited both extraction and excretion. A greater effect was seen on excretion in the latter case, which suggests that displacement of bile acid from the cytosolic protein lead to redistribution in the hepatocyte as well as reflux into the sinusoid. These data suggest that binding of bile acids to cytosolic 3 alpha-hydroxysteroid dehydrogenases occurs extensively during hepatic transit and is important in mediating the translocation of bile acids from the sinusoidal to canalicular pole of the cell.

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Binding of bile acids, oleic acid, and organic anions by rat and human hepatic Z protein.

Binding affinities of purified Z proteins from rat and human liver for bile acids, oleic acid, and organic anions were studied. Purification of Z protein from both rat and human hepatic cytosol was performed by gel filtration, chromatofocusing, and hydroxyapatite chromatography. Both purified proteins showed the same molecular weight (Mr = 14,000) and isoelectric points were 6.9 and 6.5 for rat and human proteins, respectively. Binding studies were performed by the competitive displacement of 1-anilino-8-naphthalene sulfonate. Rat and human Z proteins exhibited similar binding affinities for bile acids, oleic acid, and organic anions. Among various bile acids, both proteins bound monohydroxy bile acids with high affinity and trihydroxy bile acids with low affinity; sulfates were bound with higher and glucuronides with lower affinity than their parent bile acids. In comparison with GSH S-transferases, rat Z protein had lower affinity for bile acids than rat GSH S-transferase B and human Z protein had higher affinity for bile acids than human cationic GSH S-transferase. The role for Z protein in the intracellular binding of bile acids may be particularly important in human liver.

Animals↗
PubMed1986

Purification of a 32.5 kDa monomeric sulfotransferase from rat liver with activity for bile acids and phenolic steroids.

Both bile acid and phenolic steroid sulfotransferase activities in rat liver cytosol have previously been identified in fractions corresponding to apparent molecular masses of 60-70 and 30-35 kDa. We purified the latter activity corresponding to a monomeric protein. Activity for bile acids and phenolic steroids co-eluted on sequential chromatography on Sephadex G-75 sf, Affigel blue, chromatofocusing and hydroxyapatite. The protein was homogeneous on SDS-PAGE (32.5 kDa).

3-Hydroxysteroid Dehydrogenases↗
PubMed1986

Kinetics of glutathione efflux from isolated rat hepatocytes.

The characteristics and kinetics of glutathione (GSH) efflux were examined in homogeneous suspensions of freshly isolated rat hepatocytes. GSH efflux was measured as its linear accumulation in the suspension medium. Appearance of GSH extracellularly was reflected in a quantitative loss in cellular GSH. However, the total GSH remained essentially unchanged, indicating minimal net synthesis of GSH under these experimental conditions. GSH efflux was sensitive to temperature, with a calculated Q10 value of 2.3. A wide range of cellular GSH concentration ranging from near complete and moderate depletion to severalfold the control values was achieved by treatment of animals or cells with various GSH depletors or inducers. At physiological (fed) and elevated (3-methylcholanthrene- and CoCl2-induced) cellular GSH, the rate of GSH efflux was near maximum. The rate fell dramatically to 50% maximum at a GSH concentration equaling 35 nmol/10(6) cells. A 48-h fast resulted in a 40% loss of cellular GSH, with a corresponding decrease in efflux rates. Addition of GSH to the incubation medium had no effect on efflux rates. The relationship of GSH efflux to cellular GSH concentration was characterized by apparent sigmoidal saturation kinetics. The data were fitted well by the Hill model with the following kinetic parameters: Vmax = 0.25 nmol X 10(6) cells-1 X min-1, Km = 3.5 mM, and n = 3. These results correspond very closely to our previous findings in the perfused liver.

Animals↗
PubMed1986

Mechanism of inhibition of glutathione efflux by methionine from isolated rat hepatocytes.

We studied mechanism of inhibition of glutathione (GSH) efflux by methionine with freshly isolated rat hepatocytes. Inhibition was specific for L-methionine and was not due to changes in membrane potential or cell volume. Methionine (100 microM) inhibited GSH efflux from cells having 20-60 nmol GSH/10(6) cells. Inhibition was overcome in cells with greater than 75 nmol GSH/10(6) cells. Kinetics of control and inhibited efflux were sigmoidal saturable and were fitted well with the Hill model having three cooperative binding per transport sites. Vmax was the same for both cases (0.24 +/- 0.013 nmol X min-1 X 10(6) cells-1), implying that the inhibition was competitive. Apparent Km of efflux was 3.3 +/- 0.20 mM for controls but was shifted to 5.6 +/- 0.14 mM (P less than 0.01) in the presence of 100 microM methionine. Kinetic analysis of the inhibition by varying concentrations of methionine estimated Ki = 61.3 +/- 6.0 microM and n = 1.2 +/- 0.07, suggesting involvement of a single inhibition site. Methionine uptake was independent of GSH concentration, and blocking its uptake with 2-amino-2-norbornanecarboxylic acid did not affect inhibition. When methionine-preloaded cells were resuspended in methionine-free buffer, GSH efflux rapidly returned to control levels, whereas digitonin-releasable cellular methionine remained nearly constant. Thus, inhibition appeared to be exerted from outside the cell, possibly through an allosteric mechanism. A consequence of inhibition of GSH efflux by methionine was decreased uptake of cysteine equivalents from extracellular cystine.

Animals↗
PubMed1986

Drug-induced hepatotoxicity.

Drug-induced injury to the liver can mimic any form of acute or chronic liver disease. Acute injury to the liver frequently is due to the action of cytochrome P450, which breaks down drugs into electrophiles or free radicals; these reactive metabolites can covalently bind to protein and unsaturated fatty acids or induce lipid peroxidation, respectively. These events may impair vital functions of the cell, such as maintenance of calcium homeostasis, leading to death; or hypothetically they may elicit a hypersensitivity reaction directed mainly at the liver. Glutathione and tocopherol play critical roles in cellular defense. Cholestatic disease caused by drugs results from a selective disturbance in bile secretion. Agents such as estrogens, chlorpromazine, and monohydroxy bile acids alter the chemical and physical properties of membranes, leading to impaired activity of carriers and pumps for bile acids and electrolytes. Certain drugs produce chronic liver disease that is pathologically identical to chronic active hepatitis, biliary cirrhosis, or alcoholic liver disease.

Acetaminophen↗
PubMed1986

Comparison of the affinities of newly identified human bile acid binder and cationic glutathione S-transferase for bile acids.

The bile acid binding properties of the newly identified bile acid binder (Mr = 36,000) (FEBS Lett. 1984. 177: 31-35) and the major cationic glutathione (GSH) S-transferase (Mr = 50,000) in human liver cytosol were compared. Binding affinities were measured by the competitive displacement by bile acids of 1-anilino-8-naphthalene sulfonate (ANS) bound to the proteins and, in some cases, by direct methods of flow dialysis and equilibrium dialysis. The binding affinities for various bile acids by the human bile acid binder were 2-5 orders of magnitude greater than those by human cationic GSH S-transferase. This suggests an important physiologic role for the former protein in intracellular transfer of bile acids in human liver.

Anilino Naphthalenesulfonates↗
PubMed1986

Binding of bile acids by glutathione S-transferases from rat liver.

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.

Anilino Naphthalenesulfonates↗
PubMed1985

Gamma-glutamylcysteine: a substrate for glutathione S-transferases.

A new high performance liquid chromatography (HPLC) method for the separation of gamma-glutamylcysteine (GC) from glutathione (GSH) following derivatization with 1-chloro-2,4-dinitrobenzene (CDNB) was developed using a Vydac C18 column and an acetonitrile-trifluoroacetic acid gradient. When the derivatization of GC, GSH, cysteine, and cysteinylglycine was performed with GSH S-transferase, peak heights for the GC and GSH derivatives were accentuated markedly, suggesting that GC, like GSH, is an enzyme substrate. Subsequently, GC was found to be a substrate for five purified forms of rat hepatic GSH S-transferase. However, the Km for GC was about 6-20 times higher than that for GSH. GSH was a competitive inhibitor of GC-CDNB conjugation, indicating that GC and GSH share the same binding site on the transferase. However, endogenous hepatic GC content in fed rats was only 5.8 +/- 0.1 nmoles/g, three orders of magnitude lower than GSH. Thus, under normal circumstances, GC would not be expected to contribute to detoxification reactions catalyzed by the GSH S-transferases. Its weak interaction with the GSH site of the GSH S-transferases supports the role of the glycine moiety of GSH in enhancing this interaction.

Animals↗
PubMed1985

Modulating effect of thiol-disulfide status on [14C]aminopyrine accumulation in the isolated parietal cell.

Thiol-oxidizing agents were found to stimulate [14C] aminopyrine accumulation, a reliable index of acid secretory function of isolated canine parietal cells. Glutathione is the predominant intracellular free thiol; thus, its oxidation status largely determines the thiol-disulfide status of the cell by thiol-disulfide interchange reactions. Three agents which alter glutathione oxidation status by different mechanisms were applied to parietal cells in vitro to investigate whether enhanced formation of GSSG alters acid secretory function. The agents studied were diamide (which nonenzymatically oxidizes GSH to GSSG), tert-butyl hydroperoxide (an organic peroxide specifically reduced by glutathione peroxidase, thereby generating GSSG for GSH), and 1,3-bis(2-chloroethyl)-1-nitrosourea (an inhibitor of NADPH:GSSG reductase, which presumably allows the accumulation of GSSG). Each of these agents stimulated aminopyrine accumulation in a dose-dependent fashion. Simple depletion of GSH by diethyl maleate or 2-cyclohexene-1-one did not stimulate aminopyrine accumulation. Likewise, enhanced aminopyrine accumulation occurred at diamide concentrations which did not cause significant depletion of total cellular glutathione. The thiol-reducing agent, dithiothreitol, prevented enhanced aminopyrine accumulation by 1,3-bis(2-chloroethyl)-1-nitrosourea and tert-butyl hydroperoxide. These observations support the hypothesis that thiol-disulfide interchange reactions involving GSSG modulate the acid secretory function of the isolated parietal cell.

Aminopyrine↗
PubMed1985

Acetaldehyde-dependent oxidation of glutathione catalyzed by rat liver cytosol.

We have identified a novel reaction in which acetaldehyde promotes rat hepatic cytosolic catalysis of O2 consumption coupled with glutathione oxidation without apparent release of activated forms of O2. Acetaldehyde is not consumed in the reaction. The reaction (O2 consumption or oxidized glutathione production) is saturable with respect to varying glutathione (K'm congruent to 20-45 microM) but not at high acetaldehyde concentrations. However, activity in the range of acetaldehyde found in liver from alcohol metabolism (10-100 microM) appeared to be saturable (K'm congruent to 25-50 microM). Since neither acetaldehyde-dependent glutathione loss nor O2 consumption is detectable in guinea pig hepatic cytosol or hepatic cytosol from selenium-deficient rats, we propose that acetaldehyde interacts with glutathione peroxidase, converting the enzyme into a glutathione oxidase.

Acetaldehyde↗