Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hepatotoxicity of CCl4 is mediated through its initial reduction by cytochrome P-450 to the CCl3.radical. This radical then damages important metabolic systems such as the ATP-dependent microsomal Ca2+ pump. Previous studies from our laboratory on isolated microsomes have shown that NADPH in the absence of toxic agents inhibits this pump. We have now found in in vitro incubations that CCl4 (0.5-2.5 mM) enhanced the NADPH-dependent inhibition of Ca2+ uptake from 28% without CCl4 to a maximum of 68%. These concentrations are in the range found in the livers and blood of lethally intoxicated animals (Dambrauskas, T., and Cornish, H. H. (1970) Toxicol. Appl. Pharmacol. 17, 83-97; Long, R.M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306) and are toxic to cultured hepatocytes (Long, R. M., and Moore, L. (1988) Toxicol. Appl. Pharmacol. 92, 295-306). The inhibition of Ca2+ uptake was due both to a decrease in the Ca2(+)-dependent ATPase and to an enhanced release of Ca2+ from the microsomes. The NADPH-dependent CCl4 inhibition was greater under N2 and was totally prevented by CO. GSH (1-10 mM) added during the incubation with CCl4 prevented the inhibition. This protection was also seen when the incubations were performed under nitrogen. When samples were preincubated with CCl4, the CCl4 metabolism was stopped, and then the Ca2+ uptake was determined; GSH reversed the CCl4 inhibition of Ca2+ uptake. This reversal showed saturation kinetics for GSH with two Km values of 0.315 and 93 microM when both the preincubation and the Ca2+ uptake were performed under air, and 0.512 and 31 microM when both were performed under nitrogen. Cysteine did not prevent the NADPH-dependent CCl4 inhibition of Ca2+ uptake. CCl4 increased lipid peroxidation in air, but no lipid peroxidation was seen under nitrogen. Lipid peroxidation was only modestly reversed by GSH. GSH did not remove 14C bound to samples preincubated with the 14CCl4. Although EDTA (100 microM) decreased the CCl4 inhibition, the metal-complexing agents deferoxamine (100 microM) and diethyldithiocarbamate (100 microM) had no effect on the inhibition of the pump. Similarly, the reactive oxygen scavengers catalase (65 micrograms/ml), superoxide dismutase (15 micrograms/ml), mannitol (10 mM), and dimethyl sulfoxide (50 mM) also had no effect. Our results suggest that the initial toxicity of CCl4 for the Ca2+ pump results from the metabolism of CCl4 to the CCl3. radical. This radical then directly oxidizes the Ca2+ pump, leading to decreased Ca2+ uptake.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The in vitro NADPH-dependent inhibition by CCl4 of the ATP-dependent calcium uptake of hepatic microsomes from male rats. Studies on the mechanism of the inactivation of the hepatic microsomal calcium pump by the CCl3.radical. 214 Mar 58

An ATP-dependent transport process for S-(2,4-dinitrophenyl) glutathione (Dnp-SG) mediated by a novel ATPase designated as Dnp-SG ATPase has been demonstrated in human erythrocytes (LaBelle et al., FEBS Lett. 228, 53-51, 1988). In order to investigate whether the Dnp-SG ATPase system represents a generalized mechanism for the transport of xenobiotic conjugates of glutathione (GSH), stimulation of this ATPase by different GSH conjugates was studied in membrane vesicles prepared from human erythrocytes. Kinetic parameters for several GSH conjugates including S-(methyl)glutathione, S-(n-propyl)glutathione, S-(n-pentyl)glutathione, S-(n-decyl)glutathione, S-(p-chlorophenacyl)glutathione, S-(p-nitrobenzyl)glutathione, and the GSH conjugate of 9,10-epoxystearic acid were determined in order to evaluate their affinity for Dnp-SG ATPase. These studies reveal that all these conjugates stimulated Dnp-SG ATPase of human erythrocyte membrane. The apparent Km values of Dnp-SG ATPase for different conjugates were found to be in the range of 0.26-0.66 mM with Vmax values ranging from 0.55 to 4.44 nmol/min/mg protein. The results of these studies indicate that erythrocyte membrane Dnp-SG ATPase represents a generalized mechanism for the transport of GSH conjugates formed with xenobiotics as well as with the endogenously generated electrophilic compounds such as epoxystearic acid. It is suggested that Dnp-SG ATPase in conjunction with GSH and GSH S-transferase may play an important role in the protection of erythrocytes from exogenous as well as endogenous electrophilic toxicants.
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PMID:Stimulation of a human erythrocyte membrane ATPase by glutathione conjugates. 214 5

The half maximal inhibitory concentrations (IC50) of substituted benzimidazoles for the H+, K(+)-ATPase in hog gastric vesicles were measured by using the pyruvate kinase-lactate dehydrogenase-linked system in which hydrolysis of ATP was coupled with the oxidation of NADH. The vesicles were incubated in a solution containing a high concentration of KCl, valinomycin and Mg-ATP, and the intravesicular medium was acidified. The inhibitor was activated in the acidic medium and reacted with SH groups on the luminal (intravesicular) side of the ATPase. The active compound formed in the extravesicular medium (pH 6.11) was quenched by GSH. Under these conditions, IC50 of new compound E3810, 2[(4-(3-methoxypropoxy)-3-methylpyridine-2-yl)methyl-sulfinyl]-1H- benzimidazole sodium salt, was 0.072 microM and that of omeprazole was 0.47 microM at 25 degrees. On the other hand, the rates of formation of active compounds, tetracyclic sulfenamide derivatives, from original substituted benzimidazoles in 0.1 N HCl (k) were determined by measuring optical density at the characteristic wavelengths of the active compounds. There was a good correlation between IC50 and k for various substituted benzimidazoles including E3810, methoxy derivative of E3810, omeprazole, Ro 18-5364, H compound, picoprazole and timoprazole. This fact suggest that the rate of the formation of the acid-activated compound is a main factor determining the potency of the inhibitor.
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PMID:The potency of substituted benzimidazoles such as E3810, omeprazole, Ro 18-5364 to inhibit gastric H+, K(+)-ATPase is correlatedwith the rate of acid-activation of the inhibitor. 215 89

Incubation of rabbit heart microsomes with Adriamycin and NADPH resulted in the oxidation of approximately 25% of protein thiols and 66% inhibition of Ca-ATPase activity. Thiol oxidation and Ca-ATPase inactivation were iron-dependent and could be catalysed by ferritin. Removal of contaminating catalase revealed that both processes required H2O2 which could be supplied by O2 under aerobic conditions. However, O2- was not involved. Butylated hydroxytoluene (BHT), alpha-tocopherol and beta-carotene inhibited lipid peroxidation of microsomes, but did not inhibit thiol oxidation or the inactivation of Ca-ATPase. Likewise, the hydroxyl radical scavengers benzoate, formate and mannitol were not inhibitory. Glutathione (GSH), however, prevented inactivation of Ca-ATPase. It is concluded that Adriamycin-enhanced redox reactions involving iron and H2O2 are responsible for oxidizing microsomal thiol groups and inhibition of Ca-ATPase. Disruption of Ca transport within the myocyte by this process could contribute to the cardiotoxicity of Adriamycin.
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PMID:Thiol oxidation and inhibition of Ca-ATPase by adriamycin in rabbit heart microsomes. 215 95

Lead (Pb) inhibited K(+)-stimulated para-nitrophenyl phosphatase (K(+)-PNPPase) of rat brain P2 fraction in a concentration-dependent manner with IC50 3.5 microM. Altered pH versus activity demonstrated comparable inhibitions by Pb in buffered acidic, neutral and alkaline pH ranges. Inhibition of enzyme activity was higher at lower temperatures (17-27 degrees C) compared to 37 degrees C. Preincubation of enzyme with sulfhydryl (-SH) agents such as cysteine (Cyst) and dithiothreitol (DTT) but not glutathione (GSH) protected against Pb-inhibition. Uncompetitive type of inhibition with respect to the activation of K+ was indicated by a decrease in Vmax from 16.2 to 8.37 mumoles of para-nitrophenol (PNP)/mg protein/hr and Km from 18.99 to 12.39 mM. Kinetic studies on substrate (p-nitrophenyl phosphate) activation in the presence of Pb (3.5 microM) indicated a significant decrease in Vmax from 8.94 to 4.69 mumoles of PNP/mg protein/hr with no change in Km. Cyst (3 microM) and DTT (10 microM) reversed the Pb-inhibited Vmax from 4.69 to 8.38 and 7.24 mumoles of PNP/mg protein/hr respectively. These results suggest that the critical conformational property of K(+)-PNPPase is sensitive to Pb. The data also indicates that the Pb inhibits Na(+)-K+ ATPase system by interacting with dephosphorylation of the enzyme-phosphoryl complex, while Cyst and DTT protected against Pb-inhibition.
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PMID:Effects of lead on K(+)-para-nitrophenyl phosphatase activity and protection by thiol reagents. 216 61

When 7,12-dimethylbenz[a]anthracene (DMBA) and aflatoxin B1 (AFB1) were activated by hepatocytes from Fischer 344 rats fed a diet containing 2% butylated hydroxyanisole (BHA), frequencies of mutation to 6-thioguanine resistance (TGR) at the HGPRTase gene locus and to ouabain resistance (OuR) at the Na+,K(+)-ATPase gene locus in V79 cells were 30-70% less than those obtained with hepatocytes from untreated controls. A difference in the mutation frequency did not occur when dimethylnitrosamine (DMN) was activated by BHA induced- rather than control-hepatocytes. Analysis of hepatocytes from rats fed 2% BHA showed a small (1.5-fold), but significant, increase in glutathione levels over that in the controls but no change in activity of cytochrome P450. Cytosolic glutathione S-transferase (GST) activity was increased 2-3-fold in hepatocytes from rats fed the 2% BHA diet. These results suggest that mutagenic response to DMBA and AFB1 is reduced, at least in part, because of BHA-induction of hepatocyte GST activity; while activation of DMN can occur by pathway(s) unaffected by BHA-induction of these liver enzymes. In contrast to mutation frequencies, significant differences between BHA- and control-activation in the production of sister-chromatid exchange (SCE) and micronucleus formation (MN) were not detected with any of the genotoxins. It was concluded that the mechanism(s) by which SCE and MN occur are likely unrelated to the capacity of BHA to induced activity of hepatic enzymes, e.g. the GSH S-transferases, that directly or indirectly affect mutation end-points.
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PMID:Comparative genotoxicity of 3 procarcinogens in V79 cells as related to glutathione S-transferase activity of hepatocytes from untreated rats and those fed 2% butylated hydroxyanisole. 216 83

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.
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PMID:Hormonal regulation of glutathione efflux. 216 79

The cellular mechanisms by which nephrotoxic heavy metals injure the proximal tubule are incompletely defined. We used extracellular electrodes to measure the early effects of heavy metals and other sulfhydryl reagents on net K+ and Ca2+ transport and respiration (QO2) of proximal tubule suspensions. Hg2+, Cu2+, and Au3+ (10(-4)M) each caused a rapid net K+ efflux and a delayed inhibition of QO2. The Hg2(+)-induced net K+ release represented passive K+ transport and was not inhibited by barium, tetraethylammonium, or furosemide. Both Hg2+ and Ag+ promoted a net Ca2+ uptake that was nearly coincident with the onset of the net K+ efflux. A delayed inhibition of ouabain-sensitive QO2 and nystatin-stimulated QO2, indicative of Na+, K(+)-ATPase inhibition, was observed after 30 sec of exposure to Hg2+. More prolonged treatment (2 min) of the tubules with Hg2+ resulted in a 40% reduction in the CCCP-uncoupled QO2, indicating delayed injury to the mitochondria. The net K+ efflux was mimicked by the sulfhydryl reagents pCMBS and N-ethylmale-imide (10(-4) M) and prevented by dithiothreitol (DTT) or reduced glutathione (GSH) (10(-4) M). In addition, both DTT and GSH immediately reversed the Ag(+)-induced net Ca2+ uptake. Thus, sulfhydryl-reactive heavy metals cause rapid, dramatic changes in the membrane ionic permeability of the proximal tubule before disrupting Na+, K(+)-ATPase activity or mitochondrial function. These alterations appear to be the result of an interaction of the metal ions with sulfhydryl groups of cell membrane proteins responsible for the modulation of cation permeability.
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PMID:Sulfhydryl-reactive heavy metals increase cell membrane K+ and Ca2+ transport in renal proximal tubule. 230 68

Nitroimidazole compounds are effective radiosensitizers, but neurotoxic side effects prevent their clinical use. Studying the effect of misonidazole, metronidazole and two of its derivatives, 4.5-NO2-METRO and 4-NO2-METRO, on red blood cell, it was recently demonstrated that these compounds inhibit the red cell membrane (Na+-K+) ATPase and decrease the fluidity of the membrane bilayer. In order to extend these observations and to achieve a more complete interpretation, four additional investigations were selected: the (Ca++-Mg++)ATPase activity, the anion channel (band 3 protein) kinetics, the susceptibility of the phospholipids to peroxidation, and their influence on the concentration of reduced glutathione (GSH). The activity of the (Ca++-Mg++)ATPase and its stimulation by calmodulin were decreased by all four drugs, but the anion transport kinetics were unaltered. No lipid peroxidation could be detected, as estimated by the production of malonyldialdehyde. The red cell GSH was depleted by 4.5-NO2-METRO, probably due to the formation of a complex between GSH and the drugs [Varghese 1983]. The mechanism of the inhibition of the ATPases is not yet clearly apparent; it is presently sought in a direct interaction of the drugs with some thiol reactive groups of the ATPases.
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PMID:The influence of nitroheterocyclic radiosensitizers on the membrane of red blood cells. 243 Sep

The effect of ultraviolet light-B (UVB) irradiation on the activity of prostaglandin (PG) D synthetase was investigated in adult rat skin. Rats were irradiated with 500 mJ/cm2 of UVB, and PGD synthetase activity was determined in 100,000 g supernatant of the homogenate of rat skin in the presence of glutathione (GSH) before and 3, 6, 12, and 24 h after irradiation. The PGD synthetase activity was decreased time dependently, and within 24 h after UVB irradiation it had dropped to 50% of the control level before irradiation. In contrast, the synthesizing activities of PGE2 and PGF2 alpha were unaffected by UVB irradiation. The reduction of PGD synthetase activity after UVB irradiation was much more prominent in the epidermis than in the dermis, which was separated by heat treatment (55 degrees C, 30 sec). Immunohistochemical studies, using anti-(rat spleen PGD synthetase) antibody, revealed that the number of immunopositive cells, which were identified as Langerhans cells, decreased in the basal layer of the epidermis 24 h after UVB irradiation. These results, together with the reduction of ATPase positive cells in the epidermis after UVB irradiation, suggest that the decrease of PGD synthetase activity in rat skin by UVB irradiation is, at least in part, due to the reduced Langerhans cell population in the basal layer of the epidermis.
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PMID:Effect of ultraviolet irradiation on the activity of rat skin prostaglandin D synthetase. 252 10


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