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:3.6.3.1 (Mg2+-ATPase)
1,484 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenosine 5'-triphosphate (ATP) synthesis driven by an artificially imposed membrane potential in right-side-out membrane vesicles of Escherichia coli was investigated. Membrane vesicles prepared in the presence of adenosine diphosphate were loaded with K+ by incubation with 0.5 M potassium phosphate. Addition of valinomycin resulted in the synthesis of 0.2 to 0.3 nmol of ATP/mg of membrane protein, whereas no synthesis was observed after addition of nigericin. Addition of K+, dicyclohexylcarbodiimide, carbonylcyanide p-trifluoromethoxyphenylhydrazone, or azide to the assay buffer inhibited ATP synthesis. Adenosine diphosphate and Mg2+ were found to be required. Ca2+, which can replace Mg2+ for the hydrolytic activity of the Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3), could not replace Mg2+ in the synthetic reaction and, in fact, inhibited ATP synthesis even in the presence of Mg2+. Strain NR-70, a mutant lacking the Mg2+-ATPase, was unable to synthesize ATP using an artificially imposed membrane potential. Additionally, the Mg2+-ATPase was found to contain tightly bound ATP.
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PMID:Adenosine 5'-triphosphate synthesis energized by an artificially imposed membrane potential in membrane vesicles of Escherichia coli. 0 30

A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions.
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PMID:Isolation of the basal and lateral plasma membranes of rat kidney tubule cells. 13 91

Membrane vesicles of Escherichia coli prepared by osmotic lysis of lysozyme ethylenediaminetetracetate (EDTA) spheroplasts have approximately 60% of the total membrane-bound reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase (ED 1.6.99.3) and Mg2+-adenosine triphosphatase (ATPase) (EC 3.6.1.3) activities exposed on the outer surface of the inner membrane. Absorption of these vesicles with antiserum prepared against the purified soluble Mg2+-ATPase resulted in agglutination of approximately 95% of the inner membrane vesicles, as determined by dehydrogenase activity, and about 50% of the total membrane protein. The unagglutinated vesicles lacked all dehydrogenase activity and may consist of outer membrane. Lysozyme-EDTA vesicles actively transported calcium ion, using either NADH or adenosine 5'-triphosphate (ATP) as energy source. However, neither D-lactate nor reduced phenazine methosulfate energized calcium uptake, suggesting that the observed calcium uptake was not due to a small population of everted vesicles. Transport of calcium driven by either NADH or ATP was inhibited by simultaneous addition of D-lactate or reduced phenazine methosulfate. Proline transport driven by D-lactate oxidation was inhibited by either NADH oxidation or ATP hydrolysis. These results suggest that the portion of the total population of vesicles capable of active transport, i.e., the inner membrane vesicles, are functionally a homogeneous population but cannot be categorized as either right-side-out or everted, since activities normally associated with only one side of the inner membrane can be found on both sides of the membrane of these vesicles. Moreover, the data indicate that oxidation of NADH or hydrolysis of ATP by externally localized NADH dehydrogenase or Mg2+-ATPase establishes a protonmotive force of the opposite polarity from that established through D-lactate oxidation.
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PMID:Functional mosaicism of membrane proteins in vesicles of Escherichia coli. 19 Feb 12

Insulin receptor characteristics were examined in purified brush border membrane from the syncytiotrophoblast of the normal human placenta and quantified during membrane preparation. Insulin receptor concentration was enriched 10- to 15-fold in this preparation, and insulin receptor specific activity followed closely the enrichment values for microvillus plasma membrane markers, alkaline phosphatase, Ca2+- and Mg2+-ATPase, and 5'-nucleotidase during cell fractionation. Insulin receptor concentrations and marker enzyme analyses were compared in whole homogenate, mitochondrial, microsomal, and microvillus fractions, and these fractions were characterized by SDS-gel electrophoresis. Microvillus insulin receptor interactions were dependent on time, [125I]iodoinsulin concentration, protein, and unlabeled hormone concentrations. Competition studies with porcine insulin and [125I]iodoinsulin for this receptor revealed a curvilinear Scatchard plot. Insulinase was demonstrated at 37 C but was minimal at 24 C in the microvillus fraction. Electron microscopy of the microvillus membrane preparation revealed its composition to be mainly spherical closed membrane vesicles and brush border fragments. Sodium dodecyl sulfate polyacrylamide and isoelectric focusing gels of membrane fractions were compared. Actin was tentatively identified as a major microvillus membrane protein and was further fractionated: beta-Actin and gamma-actin were present in approximately equal concentrations. The localization of the insulin receptor in the microvillus brush border of the human placenta suggests that this receptor interacts with maternal, rather than fetal insulin.
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PMID:Characteristics of the microvillus brush border of human placenta: insulin receptor localization in brush border membranes. 75 22

The phenomenon of multidrug resistance is correlated with the presence of a membrane protein, P-glycoprotein, which pumps a wide variety of drugs out of cells thus reducing their toxicity. However, the mechanism of this pumping action remains unclear. In this article, we suggest that several properties of the multidrug transporter may be explained if it acts as a 'flippase' to transport drugs from the inner leaflet of the lipid bilayer to the outer or to the external medium.
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PMID:Is the multidrug transporter a flippase? 137 41

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver. 240 82

Isothiocyanates are potent modifiers of thiol groups, and they have been successfully applied in studying the active site structure of renal (Na+ + K+)-ATPase. However, very little has been known on interactions of isothiocyanates with myocardial sarcolemmal ATPases. In the present study the mode of interaction and inhibitory effect of p-bromophenyl isothiocyanate (BPITC) on isolated rat heart sarcolemmal preparation ATPase activities not exhibiting (Mg-Ca)-ATPase activity was investigated. BPITC in concentrations of 10(-7)-10(-4) mol . l-1 inhibited selectively and non-competitively the (Na+ + K+)-ATPase activity in the sarcolemma with an ID50 around 2.10(-7) mol . l-1. The non-specific interaction of BPITC with bivalent cations, namely with Mg2+ and Ca2+, in the reaction system was eliminated by preincubation of membranes with BPITC keeping the ratio of inhibitor to membrane protein concentration constant. Under these conditions no considerable inhibitory effects were observed on Mg2+-ATPase or the low-affinity Ca2+-ATPase of sarcolemma. Preincubation of membranes with 2 mmol . l-1 ATP protected (Na+ + K+)-ATPase activity against inhibition by BPITC. The interaction of BIPTC with the sarcolemma proved to be reversible in the presence of beta-mercaptoethanol or dithiothreitol.
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PMID:Selective and reversible inhibition of heart sarcolemmal (Na+ + K+)-ATPase by p-bromophenyl isothiocyanate. Evidence for a sulfhydryl group in the ATP-binding site of the enzyme. 608 62

Because it has been suggested that decreased activity at the erythrocyte sodium pump might be the cause of age-related decreases in basal oxygen consumption, we have examined age-associated changes in Na+,K+-ATPase activity in red cell membranes. The initial portion of this study was directed toward elucidating possible methodological pitfalls in membrane preparation which might account for some of the variable results reported in prior erythrocyte Na+,K+-ATPase studies. We found that two of four red cell membrane fractions have substantial Mg2+-ATPase activity and contribute a significant portion of total membrane protein. As these two fractions contain little Na+,K+-ATPase activity their contamination of the other two fractions could cause significant variation in measured Na+,K+-ATPase activity. Additionally, we found that meal feeding raised Na+,K+-ATPase activity necessitating that measurements be made in the fasting state. With these methodological variables controlled, we found only a 10.8% coefficient of variation between fasting samples obtained on separate days in eight subjects. Using this methodology, we observed no correlation of Na+,K+-ATPase specific activity with age in males, and only a weak correlation in females who showed decreasing Na+,K+-ATPase specific activity occurring with advancing age. These observations suggest that changes in erythrocyte Na+,K+-ATPase activity do not cause the age-related fall in basal oxygen consumption.
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PMID:Variables affecting measurement of human red cell Na+,K+ATPase activity: technical factors, feeding, aging. 614 85

P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is an ATPase thought to actively export cytotoxic drugs. It has been proposed that Pgp transports drugs directly from the lipid bilayer to the external medium ("vacuum cleaner" hypothesis). A possible mechanism for this model is that the Pgp is a flippase--i.e., it catalyzes the translocation of hydrophobic substrates from the inner to the outer leaflet of the cell membrane. Two immediate predictions of the vacuum cleaner and flippase hypotheses are that the apparent unidirectional influx of substrate should be less in Pgp-expressing than in Pgp-lacking cells and that this difference should be abolished by inhibition of the Pgp. We used Chinese hamster fibroblasts with different levels of Pgp expression to measure true unidirectional fluxes of rhodamine 123 (R123), a Pgp-transported fluorescent dye that accumulates in mitochondria (hence, its cytosolic concentration remains low at short times after external addition). The unidirectional efflux of R123 was proportional to the level of Pgp expression and was reduced by Pgp inhibitors. The unidirectional influx of R123 was the same in sensitive and resistant cells--i.e., independent of the level of Pgp expression and insensitive to inhibitors of R123 efflux. From these results, we rule out the vacuum cleaner and flippase hypotheses and conclude that Pgp extracts the actively transported substrates from the cytosol and not from the plasma membrane.
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PMID:Unidirectional fluxes of rhodamine 123 in multidrug-resistant cells: evidence against direct drug extrusion from the plasma membrane. 791 Sep 61

Vectorial sorting of plasma membrane protein-containing vesicles is essential for the establishment and maintenance of cell polarity. In the present study, the involvement of altered vesicle transport in the redistribution of membrane-bound Ca2+, Mg2+-ATPase resulting from cholestasis was investigated in hepatocytes. Cholestasis was induced in rat liver by common bile duct ligation. Ca2+, Mg2+-ATPase activity was demonstrated histochemically at the light and electron microscopical levels. Microtubules, an important factor for transcellular transport of vesicles, were studied in situ by immunofluorescence microscopy and electron microscopy in detergent-extracted preparations. The results showed that microtubules underwent significant changes after common bile duct ligation. The most pronounced alteration was focal accumulation of beta-tubulin in the cytoplasm of hepatocytes after 7 days of common bile duct ligation. At the electron microscopical level, the number of microtubules was increased considerably. In control livers, the activity of Ca2+, Mg2+-ATPase was localized only at the apical plasma membrane of hepatocytes, but it was also present at the basolateral plasma membrane after common bile duct ligation. The number of intracellular vesicles containing Ca2+, Mg2+-ATPase activity was increased strikingly, and some of them were associated with lateral membrane domains in which Ca2+, Mg2+-ATPase activity was found. It is concluded that common bile duct ligation induces the rearrangement of microtubules, which may disturb vectorial transport of Ca2+, Mg2+-ATPase-containing vesicles in hepatocytes, leading to the redistribution of Ca2+, Mg2+-ATPase.
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PMID:The involvement of altered vesicle transport in redistribution of Ca2+, Mg2+-ATPase in cholestatic rat liver. 1010 Jul 33


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