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)

Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.
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PMID:Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity. 21 24

2-Mercaptopropionylglycine was used for the treatment of chronic hepatitis. The drugs showed very good clinical results. In biochemical studies it was found that there was no effect on phosphate transport. Instead, there was an increase of reactive SH-groups sensitive to oligomycin in the membrane of rat liver mitochondria. The ATPase activity in mitochondria was diminished, while ATP-contents of the mitochondrial suspensions were increased by the drug. In the presence of ATP-MG++ there was an increase of oligomycin-sensitive contraction of the mitochondria. In aged mitochondria, the P/O ratios were improved. Reversal of electron transport with formation of NADH by succinate in the presence of rotenone and ATP was, concentration dependently, improved by MPG. Thus, MGP obviously acts at some specific sulfhydryl groups of the mitochondrion. It is concluded that the action of the drug in chronic hepatitis may be interpreted via an improvement of mitochondrial function.
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PMID:[Biochemical and clinical studies on the influence of 2-mercaptopropionylglycine on liver cell function (author's transl)]. 21 69

The effects of vitamin E deficiency on membrane integrity were studied by examining the temperature dependence of membrane-bound enzyme activities in liver mitochondria and microsome and in muscle sarcoplasmic reticulum. In vitamin E-deficient rabbits, the specific activities at 37 degrees of mitochondrial oligomycin-sensitive ATPase (EC 3.6.1.3), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30), and microsomal glucose-6-phosphatase (EC 3.1.3.9) were increased, whereas those of microsomal NADH cytochrome C reductase (EC 1.6.99.3) and sarcoplasmic reticulum Ca-ATPase were reduced in comparison to control rabbits. Arrhenius plots of activity against temperature yielded a linear plot over the range 10 to 40 degrees in the case of beta-hydroxybutyrate dehydrogenase, NADH cytochrome C reductase and Ca-ATPase, and multiple discontinuities for glucose-6-phosphatase and oligomycin-sensitive ATPase. In control rabbits, all five enzymes showed a single discontinuity in the Arrhenius plot over the range 16 to 19 degrees. These results reflect changes in the microenvironment of membrane-bound enzymes as a consequence of vitamin E depletion.
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PMID:Effects of vitamin E deficiency on the activities of lipid-requiring enzymes in rabbit liver and muscle. 22 Mar 97

A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.
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PMID:The microbial metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1. 22 Sep 60

The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Normarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with 125I. The basal-lateral components yielded a hetero-disperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochrome c reductase activities, were separated from the radio-iodine labeled by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 x g x 1 hr after removal of the mitochrondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.
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PMID:Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium. 22 11

The distribution of simian virus 40 large tumor antigen in subcellular fractions from simian virus 40-transformed hamster (H-50) and mouse (VLM) cells and from simian virus 40-infected monkey cells was determined. Solubilized [(35)S]-methionine- or (32)P(i)-labeled surface membrane and nuclear fractions were prepared, immunoprecipitated with hamster anti-T serum, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor antigen with an apparent molecular weight of approximately 96,000 was detected in both subcellular fractions. Minor components of approximately 68,000 and approximately 56,000 with anti-T reactivity which labeled with [(35)S]methionine were also detected in both fractions from H-50 cells, as were components of approximately 140,000 and approximately 56,000 from VLM cells. The 56,000 component appeared to be greatly reduced in (32)P(i)-labeled surface membrane fractions. Normal cells or cells transformed with a heterologous agent, such as polyoma virus or a chemical carcinogen, lacked immunoprecipitable tumor antigen. Cell fractionation was monitored by [(3)H]thymidine labeling, NADH-diaphorase activity, and Na(+)-K(+)-dependent ATPase activity. These analyses revealed only trace contamination of surface membranes by nuclei, extremely low levels of nuclear rupture during homogenization, and an approximate 10-fold enrichment of surface membrane. Reconstruction experiments demonstrated that soluble tumor antigen failed to associate or copurify with surface membranes during fractionation procedures. These results indicate the presence of a protein in the plasma membrane of cells transformed or infected by simian virus 40 that is immunologically indistinguishable from nuclear tumor antigen.
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PMID:Subcellular Localization of simian virus 40 large tumor antigen. 22 15

A procedure was developed for the isolation of cardiac sarcolemmal vesicles. These vesicles are enriched about ten-fold (with respect to the tissue homogenate) in K+-stimulated p-nitrophenylphosphatase, (Na+ + K+)-ATPase, 5'-nucleotidase activities and sialic acid content, all of which are believed to be components of the sarcolemma. The sarcolemma of tissue culture cardiac cells were radioiodinated and the distribution of this radioiodine paralleled the distribution of the other membrane markers above. There was very little contamination of the sarcolemmal fraction by sarcoplasmic reticulum (as judged by Ca2+-ATPase and glucose-6-phosphatase activities) or inner mitochondrial membranes (as judged by succinate dehydrogenase activity). There may, however, be some contamination by outer mitochondrial membranes (as judged by monoamine oxidase and rotenone-insensitive NADH cytochrome c reductase activities) which have rarely been monitored in cardiac sarcolemmal preparations. The purity of this preparation is good when compared with other cardiac sarcolemmal preparations. This preparation should be very useful in studying the roles of the cardiac sarcolemma (e.g. in excitation contraction coupling and Ca2+ binding).
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PMID:Isolation and characterization of cardiac sarcolemma. 22 23

1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.
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PMID:The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles. 22 34

Sera containing antimitochondrial antibodies (MTA) were tested for binding to intact mitochondria, sonic fragments (SMP), Complex I + III and to oligomycin sensitive ATPase (OS-ATPase) from bovine heart by indirect immunofluorescence. Antigens capable of binding to MTA were present in mitochondria and its fragments tested. Maximum binding was observed with SMP. It appears that one or more antigen binding sites are present on the matrix side of the inner mitochondrial membrane or some location exterior to the inner membrane. Normal human serum or sera containing MTA did not effect the respiration of intact mitochondria or sonic particles. However, NADH-cytochrome c reductase activity of complex I + III was enhanced by 10-60% by sera containing MTA antibodies.
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PMID:Mitochondrial antibodies--heterogeneity and effects on mitochondrial respiration. 23 57

By means of a preparation technique based on the discontinuous sucrose density gradient, subcellular fractions were isolated from guinea pig intestinal smooth muscle cells. A fraction which distributed to a 33% sucrose layer showed relatively high activities of 5'-nucleotidase, Na+ . K+-ATPase and ouabain sensitive Na+ . K+-ATPase. The fraction had a low NaN3 sensitive Mg2+-ATPase activity. On the other hand, the high activity of glucose-6-phosphatase showed a broad distribution. Though the sucrose density gradient proceeded over a series of the fine layers, cross-contamination of microsome into the 33% sucrose fraction was not reduced. To reduce microsomal cross-contamination, another procedure was employed. The homogenization time of 77000 xg sediment to be layered on the top of the sucrose density gradients was prolonged. This procedure did not change the distribution of K+ activated p-nitrophenylphosphatase, K+ activated ouabain sensitive p-nitrophenylphosphatase and ouabain sensitive Na+ . K+-ATPase activities. The peak of NADH cytochrome c reductase activity was shifted to a 38% sucrose fraction from a 33% sucrose fraction and the activity of this marker enzyme in the 33% sucrose fraction decreased to 60% of that of the prior procedure.
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PMID:[Examination of plasma membrane-enriched fraction from guinea pig intestinal smooth muscle by means of some marker enzymes (author's transl)]. 23 74


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