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PubMed1986

Identification of two activities of (ADP-ribose)n glycohydrolase in HeLa S3 cells.

HeLa S3 cells contained two activities (form I and II) that degrade (ADP-ribose)n exo-glycosidically. Form I was extracted from nuclei only by sonication in high ionic strength, while form II was soluble in cytosol. The two active forms differed in chromatographic behaviors, in their Km values for (ADP-ribose)n, and in their pH and salt requirements for optimal activity, although both forms exhibited properties characteristic of (ADP-ribose)n glycohydrolase such as requirement of sulfhydryl compounds and sensitivity of ADP-ribose and cAMP. Form I and II had apparent molecular weights of 72,000 and 53,000, respectively, as determined by gel filtration on Sepharose CL-6B.

Adenosine Diphosphate Ribose↗
PubMed1990

Interaction of the catalytic subunits of protein phosphatase-1 and 2A with inhibitor-1 and 2: a fluorescent study with sulfhydryl-specific pyrene maleimide.

The catalytic subunits of protein phosphatase-1 and 2A were covalently modified in their reactive sulfhydryl groups with N-(3-Pyrene) maleimide resulting in fluorescent labeling of the proteins to an extent of 0.85 and 0.9 mole dye/mole enzyme, respectively. The reaction of the sulfhydryl group led to the partial inactivation of both phosphatase-1 and 2A. Inhibitor-1 and inhibitor-2 increased markedly the fluorescence intensity of the dye-phosphatase-1 conjugate implying that the labeled enzyme retained its ability to bind these proteins. In contrast, inhibitor-1 or inhibitor-2 had no influence on the fluorescence of the dye-phosphatase-2A conjugate. No change in either the fluorescence intensity or polarization of labeled phosphatase-1 and 2A was observed in the presence of thiophosphorylase a, suggesting a lack of interaction of these enzyme forms with the substrate after modification of the reactive sulfhydryl group.

Carrier Proteins↗
PubMed1982

Involvement of sulfhydryl groups in the oxidative modulation of particulate lung guanylate cyclase by nitric oxide and N-methyl-N'nitro-N-nitrosoguanidine.

Particulate guanylate cyclase from rat lung was activated by nitric oxide or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a dose-dependent manner that was enhanced by dithiothreitol. Nitric oxide-stimulated guanylate cyclase activity decayed during a 60-min preincubation at 37 degrees, but did not decay at 24 degrees or 4 degrees. Dithiothreitol enhanced the decay of nitric oxide-stimulated enzyme at all temperatures by potentiating the reversal of nitric oxide activation. Following the reversal of nitric oxide activation at 24 degrees by dithiothreitol, the particulate enzyme could be reactivated by a second exposure to nitric oxide. Preincubation of basal particulate guanylate cyclase activity at 37 degrees resulted in the loss of enzyme responsiveness to activation by nitric oxide or MNNG that was potentiated by diamide or oxidized glutathione. The inhibitory effects of the thiol oxidants on enzyme responsiveness to activation by MNNG were prevented by dithiothreitol. The results suggest that activation of particulate guanylate cyclase by nitric oxide or MNNG involves the oxidation of key enzyme sulfhydryl groups.

Animals↗
PubMed1983

Metabolic activation of Trp-P-2, a tryptophan-pyrolysis mutagen, by isolated rat hepatocytes.

Metabolic activation of a tryptophan-pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by isolated rat hepatocytes was studied. The substrate (Trp-P-2) disappearance by hepatocytes from untreated rats was slow, but enhanced by 3-methylcholanthrene (MC) pretreatment of rats. The covalent binding of Trp-P-2 to cellular macromolecules was detected in hepatocytes from untreated rats. The amount of covalent binding of Trp-P-2 to protein and RNA was greater than that to DNA. The covalent binding to Trp-P-2 to DNA, RNA and protein in hepatocytes from untreated rats was about 5-10 times less than that in hepatocytes from MC-pretreated rats. 7,8-Benzoflavone strongly inhibited the substrate disappearance and the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. These results indicate that Trp-P-2 is metabolically activated by the P-448 type of cytochrome P-450 which is induced by MC. Diethylmaleate enhanced by about 50% the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. On the other hand, cysteine inhibited the binding of Trp-P-2 to DNA with a concomitant reduction in the accumulation of the active metabolite, N-hydroxy-Trp-P-2 (N-OH-Trp-P-2). Sulfhydryl compounds seemed to play important roles in the detoxification of Trp-P-2.

Animals↗
PubMed1984

Differences in the uptake of cadmium and mercury by rat hepatocyte primary cultures. Role of a sulfhydryl carrier.

Studies measuring the uptake of cadmium or mercury in isolated hepatocytes demonstrated that hepatocytes accumulated more cadmium than mercury in serum-containing medium, serum-free medium, or balanced salt solution. The preferential hepatocellular accumulation of cadmium, independent of medium composition, suggested that the uptake mechanism for cadmium and mercury might be different in hepatocytes. Pretreatment of hepatocytes with 50 microM N-ethylmaleimide decreased cadmium uptake by 23% while having no effect on the uptake of mercury was inhibited in a concentration-dependent manner. The uptake of cadmium was maximally inhibited (80%) with 75 microM parachloromercuribenzenesulfonate or 20 microM mercury respectively. Cadmium had no effect on mercury. Hepatocytes treated with parachloromercuribenzenesulfonate or mercury accumulated cadmium at a rate closely resembling the rate of mercury uptake in untreated hepatocytes. These results suggested that an SH-containing carrier may be operative in the uptake of cadmium by hepatocytes. Mercury can interact with this carrier to inhibit cadmium uptake; however, this carrier does not appear to facilitate mercury uptake.

Biological Transport, Active↗
PubMed1984

Inhibitory action of hexavalent chromium (Cr(VI)) on the mitochondrial respiration and a possible coupling to the reduction of Cr(VI).

The reduction of hexavalent chromium (Cr(VI] in isolated liver mitochondria was studied under different redox conditions in the respiratory chain. With 25 microM Na2CrO4 the rates were 1.6 +/- 0.7, 13.9 +/- 0.6 and 12.7 +/- 0.7 nmole Cr(VI) reduced 15 min/mg protein with the electron transport chain oxidized, reduced and only complex 1 reduced respectively. Electrons from succinate, bypassing complex 1, were apparently unavailable for Cr(VI)-reduction. The kinetics of chromate reduction was studied with only complex 1 in a reduced state. A rapid and a slow phase were found, probably corresponding to different electron donors in the mitochondria. Blocking the free thiols with N-ethylmaleimide lead to less than 10% decrease in the rapid initial Cr(VI)-reduction and to about 20% decrease during the whole incubation period (15 min). The amounts of free thiols were moderately decreased (15%) in chromate treated mitochondria during the slow reduction of Cr(VI) only. SH-groups may thus participate as reductants during the slow reduction phase. The respiration rate was inhibited about 50% by 25 microM Na2CrO4 when the mitochondria oxidized NAD-linked substrates. In contrast, succinate stimulated respiration was inhibited 50% by 3.6 mM Na2CrO4. The observed inhibition was Na2CrO4 in the micromolar range was therefore probably localized at complex 1 and may be coupled to the reduction of Cr(VI) at the same place. The respiration of isolated hepatocytes was also affected by Na2CrO4. Five micromolar chromate caused 5-10% inhibition. The inhibitory action of chromate on the mitochondrial respiration may thus constitute an important cytotoxic mechanism.

Animals↗
PubMed1986

Effects of glutathione and phenobarbital on the toxicity of codeinone.

The ability of sulfhydryl compounds to provide protection against the acute toxicity of codeinone, a toxic metabolite of codeine, was investigated in mice. Subcutaneous administration of codeinone produced a slight reduction in hepatic glutathione concentration. Pretreatment of the mice with glutathione or cysteine significantly increased the survival rate for mice given a lethal dose of codeinone (10 mg/kg). The lethality of codeine was lowered by naloxone, whereas that of codeinone was not blocked by naloxone. The strychnine-like convulsant action of codeinone could be prevented by phenobarbital pretreatment. Glutathione pretreatment reduced the amounts of radioactivity in tissues of mice injected with [N-methyl-3-H]codeinone. A possible explanation for these observations is that glutathione reacts in vivo with codeinone and plays a role as a scavenger of this compound. This assumption is supported by the observation that codeinone reacts non-enzymatically with glutathione under physiological conditions.

Animals↗
PubMed1990

Mechanisms of in vitro immunosuppression by hepatocyte-generated cyclophosphamide metabolites and 4-hydroperoxycyclophosphamide.

Cyclophosphamide (CY) is metabolized to 4-hydroxy-CY which spontaneously breaks down to the reactive intermediates, phosphoramide mustard (PAM) and acrolein. The alkylating property of PAM is thought to mediate the anti-proliferative and cytotoxic actions of CY. Acrolein is known to bind sulfhydryl groups of cellular molecules and may contribute to the action of CY. However, the role of acrolein in the CY-induced immunosuppression remains unclear. The results of studies in which a hepatocyte co-culture system was used suggest that acrolein may play an important role in the cytotoxic action of CY, whereas those investigations using activated derivatives of CY indicate that acrolein is not an important factor. Thus, both approaches of CY exposure were utilized in the present study. Splenocytes of B6C3F1 mice were incubated with syngeneic isolated hepatocytes and CY or with 4-hydroperoxycyclophosphamide (4-HC) (which spontaneously decomposes to 4-hydroxy-CY). The in vitro antibody forming cell (AFC) response was found to be suppressed with both methods of exposure to CY metabolites. The addition of DNA to bind extracellular PAM was ineffective in preventing the suppression produced by hepatocyte-activated CY. However, it was also observed that DNA was able to attenuate the PAM-induced suppression. The sulfhydryl compounds 2-mercaptoethanesulfonate (MESNA) (15 microM) or reduced glutathione (GSH) (1 mM) inhibited the suppression of the AFC response of splenocytes incubated with CY and mouse hepatocytes. The suppression produced by 4-HC, however, was not affected by MESNA and only slightly inhibited by GSH. Similarly, the PAM-induced suppression was not affected by MESNA and slightly attenuated with GSH. In contrast, both MESNA and GSH were very effective in abrogating the acrolein-induced suppression, whereas DNA was ineffective. The findings of this study suggest that in the hepatocyte co-culture system, the immunosuppressive actions of CY are mediated by acrolein generated outside of the splenocyte, whereas the 4-HC induced suppression is not mediated by extracellular acrolein. Thus, this difference may explain the contradictory findings of previous studies that used different means of exposing cells to activated CY.

Acrolein↗
PubMed1991

Glutathione-dependent biliary excretion of arsenic.

This study aimed to clarify whether glutathione (GSH) plays a role in the hepatobiliary transport of arsenic. For this purpose, the biliary excretion of 74As was measured in urethane-anaesthetized rats for 2 hr after the administration of labelled sodium arsenite (50 mumol/kg, i.v.) or arsenate (150 mumol/kg, i.v.) and under the influence of sulfobromophthalein (BSP), indocyanine green (ICG) or diethyl maleate (DEM) which are known to diminish hepatobiliary transport of GSH. Although the biliary excretion of arsenic was different after arsenite and arsenate administration in terms of quantity (19% vs 6% of dose in 2 hr, respectively) and time course, arsenic excretion responded similarly to BSP (50 mumol/kg, i.v.), ICG (25 mumol/kg, i.v.) or DEM (4 mumol/kg, i.p.) irrespective of the injected arsenical. Initially the biliary excretion of arsenic in rats with either arsenite or arsenate was significantly reduced, but then moderately increased by BSP and, more lastingly, depressed by ICG, whereas it was virtually abolished by DEM. The responses of arsenic excretion to BSP, ICG and DEM were related, both proportionally and temporally, to the effects exerted by these agents on the hepatobiliary transport of GSH, as assessed by the biliary excretion of non-protein thiols. These findings indicate that the biliary excretion of arsenic after the administration of either arsenite or arsenate is dependent on the hepatobiliary transport of GSH. Transport of arsenic as a GSH complex may account for the GSH dependence of biliary arsenic excretion.

Animals↗
PubMed1992

Human kidney thiopurine methyltransferase. Photoaffinity labeling with S-adenosyl-L-methionine.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of heterocyclic and aromatic sulfhydryl compounds such as the thiopurine drug 6-mercaptopurine (6-MP). TPMT activity in human tissue is regulated by a common genetic polymorphism, and "pharmacogenetic" variation in TPMT activity is an important factor in individual differences in thiopurine drug metabolism, toxicity and therapeutic efficacy. Human renal tissue contains two isozymes of TPMT, Peak I and Peak II, that can be separated by ion exchange chromatography. Our experiments were performed to determine whether S-adenosyl-L-methionine (Ado-Met), the methyl donor for the TPMT reaction, could be used as a photoaffinity ligand for these isozymes as one step in the study of the molecular basis for the TPMT genetic polymorphism. When [3H-methyl]Ado-Met and partially purified preparations of either isozyme of human kidney TPMT were exposed to ultraviolet light at 254 nm, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a 35 kDa protein was the predominant species that was radioactively labeled. The same 35 kDa protein was photoaffinity labeled with [14C-carboxyl]Ado-Met, demonstrating that labeling involved covalent binding of Ado-Met rather than methylation of the protein. TPMT enzymatic activity co-eluted with the 35 kDa protein during sequential DEAE ion exchange, gel filtration and hydroxylapatite chromatography. Inhibitors of TPMT enzymatic activity including S-adenosyl-L-homocysteine, sinefungin, 6-methylmercaptopurine and 3,4-dimethoxy-5-hydroxybenzoic acid inhibited photoaffinity labeling of the 35 kDa protein in preparations of both TPMT Peak I and Peak II isozymes in a concentration-dependent fashion, as did 6-MP, the methyl acceptor substrate for the TPMT reaction. All of these results were compatible with the conclusion that the 35 kDa protein was TPMT. Photoaffinity labeling of TPMT with [3H]Ado-Met should make it possible to purify the enzyme to homogeneity and to study amino acid sequences at or near its active site.

Adenosine↗