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)

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.
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PMID:ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. 135 66

1. Mercuric chloride inhibited chloride secretion in a dose dependent way in isolated perfused rectal glands. The effect was readily apparent at a concentration of 10(-6) M and profound and irreversible at 10(-4) M. 2. The dithiol dithiothreitol (DTT) 10(-2) M completely prevented the effect of 10(-6) M mercuric chloride, reduced that at 10(-5) M and 10(-4) M, and made the inhibition at the latter concentration reversible. 3. Two organic mercurials, mersalyl and meralluride, that are effective diuretics in the mammalian kidney, and p-chloromercuribenzoyl sulfonic acid (PCMBS), that has no diuretic activity, had no effect on chloride secretion by the rectal gland. 4. The effect of mersalyl was not modified by lowering the pH of the solution perfusing the glands. 5. These results indicate that inorganic mercury and organic mercurials do not share the same mechanism of action. 6. The absence of an effect of organic mercurials on chloride transport in the rectal gland suggests that its effect on another chloride transporting epithelia, the thick ascending limb of the loop of Henle, is not mediated by inhibition of the chloride cotransporter or Na+, K(+)-ATPase, common to both epithelia.
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PMID:The effect of mercury on chloride secretion in the shark (Squalus acanthias) rectal gland. 136 7

Endocytic vesicles that are involved in the vasopressin-stimulated recycling of water channels to and from the apical membrane of kidney collecting duct principal cells were isolated from rat renal papilla by differential and Percoll density gradient centrifugation. Fluorescence quenching measurements showed that the isolated vesicles maintained a high, HgCl2-sensitive water permeability, consistent with the presence of vasopressin-sensitive water channels. They did not, however, exhibit ATP-dependent luminal acidification, nor any N-ethylmaleimide-sensitive ATPase activity, properties that are characteristic of most acidic endosomal compartments. Western blotting with specific antibodies showed that the 31- and 70-kD cytoplasmically oriented subunits of the vacuolar proton pump were not detectable in these apical endosomes from the papilla, whereas they were present in endosomes prepared in parallel from the cortex. In contrast, the 56-kD subunit of the proton pump was abundant in papillary endosomes, and was localized at the apical pole of principal cells by immunocytochemistry. Finally, an antibody that recognizes the 16-kD transmembrane subunit of oat tonoplast ATPase cross-reacted with a distinct 16-kD band in cortical endosomes, but no 16-kD band was detectable in endosomes from the papilla. This antibody also recognized a 16-kD band in affinity-purified H+ ATPase preparations from bovine kidney medulla. Therefore, early endosomes derived from the apical plasma membrane of collecting duct principal cells fail to acidify because they lack functionally important subunits of a vacuolar-type proton pumping ATPase, including the 16-kD transmembrane domain that serves as the proton-conducting channel, and the 70-kD cytoplasmic subunit that contains the ATPase catalytic site. This specialized, non-acidic early endosomal compartment appears to be involved primarily in the hormonally induced recycling of water channels to and from the apical plasma membrane of vasopressin-sensitive cells in the kidney collecting duct.
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PMID:Apical endosomes isolated from kidney collecting duct principal cells lack subunits of the proton pumping ATPase. 138 76

The kinetic properties of type-II ATP diphosphohydrolase are described in this work. The enzyme preparation from the inner layer of the bovine aorta, mostly composed of smooth muscle cells, shows an optimum at pH 7.5. It catalyzes the hydrolysis of tri- and diphosphonucleosides and it requires either Ca2+ or Mg2+ for activity. It is insensitive to ouabain (3 mM), an inhibitor of Na+/K(+)-ATPase, to tetramisole (5 mM), an inhibitor of alkaline phosphatase, and to Ap5A (100 microM), an inhibitor of adenylate kinase. In contrast, sodium azide (10 mM), a known inhibitor for ATPDases and mitochondrial ATPase, is an effective inhibitor. Mercuric chloride (10 microM) and 5'-p-fluorosulfonylbenzoyl adenosine are also powerful inhibitors, both with ATP and ADP as substrates. The inhibition patterns are similar for ATP and DP, thereby, supporting the concept of a common catalytic site for these substrates. Apparent Km and Vmax, obtained with ATP as the substrate, were evaluated at 23 +/- 3 microM and 1.09 mumol Pi/min per mg protein, respectively. The kinetic properties of this enzyme and its localization as an ectoenzyme on bovine aorta smooth muscle cells suggest that it may play a major role in regulating the relative concentrations of extracellular nucleotides in blood vessels.
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PMID:Kinetic properties of type-II ATP diphosphohydrolase from the tunica media of the bovine aorta. 147 95

Intraperitoneal administration of a single dose (6 mg/kg body wt.) of mercuric chloride led to a rapid and irreversible inhibition of Na+/K(+)-ATPase activity in rat cerebral capillaries. The activity measured at 1 h, 18 h and 5 days after injection was, respectively, 53, 44 and 26% of the control. By contrast, Mg(2+)-ATPase activity in the capillaries remained uninhibited throughout the observation period. Mercuric chloride administration did not affect either of the two enzyme activities in nerve endings, which is consistent with the inability of the compound to penetrate the blood-brain barrier. The mercuric-chloride-induced impairment of the capillary sodium pump may contribute to disturbances of ion homeostasis in the brain and thus to the neurophysiological abnormalities accompanying this exposure. Direct treatment of the isolated cerebral capillary preparations with mercuric chloride evoked a stronger inhibitory effect on Mg(2+)-ATPase (IC50 = 0.25 microM) than on Na+/K(+)-ATPase (IC50 = 5.0 microM). This result indicates that the effect in vivo may not have resulted from direct interaction of the compound with the latter enzyme.
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PMID:Durable inhibition of rat cerebral capillary Na+/K(+)-ATPase after in vivo administration of mercuric chloride. 166 49

Mercuric chloride (Hg) in micromolar concentrations inhibited Mg(++)-dependent ATPase activity in rat brain microsomes. Inhibition was higher in oligomycin-sensitive (O.S.) than oligomycin-insensitive (O.I.) Mg(++)-ATPase. Hydrolysis of ATP with 15 and 50 micrograms of microsomal protein for 45 min without and with (2.10(-7M) Hg showed linear rates for 15-20 min. Altered pH vs activity demonstrated comparable inhibitions by Hg in buffered (neutral greater than acidic greater than basic) pH ranges. Inhibition of enzyme activity by Hg was found to be greater at 37 degrees C than at lower temperatures suggesting positive correlation trend. An uncompetitive inhibition with respect to the activation of Mg(++)-ATPase, O.S. Mg(++)-ATPase and O.I. Mg++ ATPase by ATP was indicated by a decrease in apparent Vmax and Km. Mg(++)-activation kinetic studies indicated that Hg causes uncompetitive inhibition of Mg(++)-ATPase and O.I. Mg(++)-ATPase and mixed inhibition of O.S. Mg(++)-ATPase. Inhibition was partially restored by repeated washings. These results indicate that the inhibition of microsomal Mg(++)-ATPase by Hg was pH, temperature, enzyme and Mg++ concentration dependent. Additionally, the data also suggest that O.S. compared to O.I. Mg(++)-ATPase is more sensitive to Hg toxicity.
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PMID:Effects in vitro of mercury on rat brain Mg(++)-ATPase. 170 94

Information regarding the cytopathologic mechanism of action of the retinoids [isotretinoin (IR) and 4-oxo-isotretinoin (4-OIR)] on neural crest cells (NCCs) in culture was sought. Those pathophysiologic alterations in cell metabolism studied were: cell blebbing (xieosis), free radical formation, cell viability, and cellular calcium homeostasis. Cells were treated with IR or 4-OIR in the presence of high (1.4 mM) and low (5.0 microM) levels of extracellular calcium ions. Recently developed techniques utilizing fluorescent molecular probes for calcium analyses, i.e., Fura 2AM, were used to study the effects of these drugs on the cytosolic calcium concentration of NCCs. The effects of IR and 4-OIR on NCC viability, [Ca++]int, were contrasted with the effects of certain sulfhydryl drugs (HgCl2, NEM, PCMBS) and calcium ionophores (ionomycin, A23187), agents known to perturb cell membranes, increase cytosolic calcium loads, and induce cell injury and subsequent cell death. Both retinoids were shown to induce an increase in the generation of superoxide radicals (SO) and increase the influx of calcium ions by the NCCs, thus increasing [Ca++]int by several hundred percent within 5 to 10 min. The liberation of SO was calcium dependent. These early effects were accompanied by an increase in cell blebbing activity. Also, a significant decrease in NCC viability was seen as early as 10 min after the addition of IR or 4-OIR to the incubation medium. 4-OIR proved to be the more potent of the two retinoids tested. The severity of these effects on NCC metabolism was dependent on medium calcium concentration with all changes being increased in the presence of the higher extracellular calcium levels. From the data presented it appears as though the retinoids cause a rapid elevation in cytosolic [Ca++]int possibly by purturbing the integrity of the cell membrane, denaturing membrane Ca-ATPase activity, or both. Retinoid-induced changes in membrane activity are evidenced by increased surface blebbing and superoxide formation. The prolonged elevation of intracellular [Ca++] may be directly related to depressed NCC viability and thus explain the known teratogenic effects of these drugs and their relationship to ectomesenchymal cell hypoplasia and craniofacial dysmorphogenesis.
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PMID:Changes in cytosolic calcium, bleb formation, and cell death in neural crest cells treated with isotretinoin and 4-oxo-isotretinoin. 186 15

In mammalian hepatocytes the L-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate (Raja erinacea), HgCl2 inhibits Na(+)-dependent alanine uptake and Na+/K(+)-ATPase and increase K+ permeability. To distinguish between direct effects of HgCl2 on the Na(+)-alanine cotransporter and indirect effects on membrane permeability, [3H]alanine transport was studied in plasma membrane vesicles. [3H]Alanine uptake was stimulated by an "out-to-in" Na+ but not K+ gradient and was saturable confirming the presence of Na(+)-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl2 for 5 min reduced initial rates of Na(+)-dependent but not Na(+)-independent alanine uptake in a dose-dependent manner (10-200 microM). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 microM HgCl2 directly inhibited sodium-dependent alanine-[3H]alanine exchange, demonstrating that HgCl2 directly affected the alanine cotransporter. Inhibition of Na(+)-dependent alanine uptake by 30 microM HgCl2 was reversed by dithiothreitol (1 mM). HgCl2 (10-30 microM) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl2 concentrations greater than 50 microM. Higher concentrations of HgCl2 (100-200 microM) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl2 inhibits Na(+)-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na+ gradient), and loss of membrane integrity. Inactivation of the Na(+)-coupled alanine carrier by mercury in hepatocytes of this evolutionarily primitive vertebrate, as in mammals, suggests that the sulfhydryl groups on this transport protein are highly conserved.
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PMID:Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea. 199 17

We have shown previously that mercuric chloride (HgCl2) inhibits in vitro vasopressin release from the isolated rat neurohypophysis with maximum inhibition occurring with 0.5 mM HgCl2. Associated with the inhibition of hormone release is an increase in 45Ca+2 uptake, an increase in cytosolic 45Ca+2, and a reduction of 45Ca+2 accumulation by mitochondria in the intact gland. In the present series of studies, the effect of HgCl2 on calmodulin (CM) function in neural tissue preparations is reported. Mercuric chloride (0.5 mM) reduced 45Ca+2 binding to CM purified from bovine neurohypophyses by 20% and inhibited endogenous CM-stimulated Ca,Mg-ATPase activity from rat brain mitochondria in a dose-dependent fashion. Ca,Mg-ATPase activity was inhibited by 50 and 80% with 0.5 and 5.0 mM HgCl2, respectively. CM-stimulation of Ca,Mg-ATPase activity was inhibited by calmidazolium (CMZ) with maximal inhibition seen with 0.1 mM CMZ. Reversibility of the HgCl2 interaction with CM was demonstrated using CM-stimulated phosphodiesterase (PDEase) activity from rat brain. HgCl2 inhibited both basal and CM-stimulated PDEase activity in a dose-dependent manner with maximum inhibition occurring with 1.0 mM HgCl2. Preexposure of CM to an inhibitory concentration (1.0 mM) of HgCl2 resulted in no loss of stimulatory PDEase enzyme activity. From these results, we conclude that HgCl2 reversibly interferes with 45Ca+2 binding to CM and also inhibits CM-regulated Ca+2 pumping enzyme systems in the neurohypophysis. The inhibition of vasopressin release from the intact gland in the presence of HgCl2 thus, may be associated with a disruption of calcium in the neurohypophysis.
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PMID:The effects of mercuric chloride on calmodulin-mediated Ca2+ transport in rat brain. 215 38

This study concerned the effects of mercuric chloride on Na+/K(+)-ATPase and [3H]ouabain binding in rat brain microsomes in vitro. The data showed that HgCl2 inhibited Na+/K(+)-ATPase effectively at micromolar concentrations. The degree of inhibition was decreased with increases in enzyme concentration and incubation time. Variations in the ionic strength of Na+ and K+ did not alter the percent inhibition of Na+/K(+)-ATPase activity by HgCl2. Repeated washings partially restored enzyme activity. The binding of [3H]ouabain to microsomal membranes was inhibited by HgCl2 in a concentration-dependent manner. Cumulative inhibition studies with HgCl2 and ouabain indicated that these inhibitors did not act concurrently and independently on Na+/K(+)-ATPase.
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PMID:Inhibition of rat brain microsomal Na+/K(+)-ATPase and ouabain binding by mercuric chloride. 215 58


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