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The effect of insulin on total and ouabain-inhibited membrane-bound adenosine triphosphatase (ATPase) activity in renal glomeruli isolated from adult white rats was examined. In concentrations of 1-10 micrograms/ml, insulin significantly stimulated the ouabain-inhibited (Na+ + K+)-ATPase activity, without affecting total (composite) ATPase activity. These results, coupled with previous findings demonstrating that glomerular (Na+ + K+)-ATPase activity is reduced in acute streptozotocin diabetes, suggest that the renal glomerulus is a target tissue with respect to this biologic effect of insulin. 相似文献
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The effects of alloxan-diabetes on the partial reaction of (Na++K+)-ATPase, K+-activated para-nitrophenylphosphatase, and on ouabain binding were studied in isolated adult dog heart myocytes. The Km of K+-activated para-nitrophenylphosphatase for K+ activation was increased from 2.5 to 7.7 mM with no change in Vmax. The Scatchard plots for ouabain binding between control and diabetic animals were indistinguishable. These results indicate that in acute diabetes induced by alloxan, the number of Na+-K+ pumping sites in the heart is not altered but the affinity of the system for K+ is decreased. It is suggested that the decrease in K+ affinity of the (Na++K+)-ATPase enzyme system is at least in part responsible for the altered K+ homeostasis in the diabetic state. 相似文献
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We have described a simple, rapid, reliable, and sensitive method for measuring the covalent phosphoenzyme intermediates that form in the reaction of ATP and sodium-potassium sensitive adenosine triphosphatase. Results are reproducible even when microgram amounts of microsomal protein are used. The method involves recovery of 32P-labeled phosphoenzyme intermediates on a Millipore filter. 相似文献
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Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis. 总被引:2,自引:0,他引:2
The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments. 相似文献
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Releases of unchanged epinephrine from the extraneuronal site of the isolated guinea-pig vas deferens were fluorimetrically determined by direct trihydroxyindole reaction at two different pH. The spontaneous release of the amine from the normal preparation preloaded with 3 x 10?5 M epinephrine was significantly enhanced by inhibitions of Na-K ATPase or by Ca-removal. After epinephrine loading, the release of the amine from the preparation pretreated with pargyline and pyrogallol or from the denervated preparation was also enhanced by the both procedures. These enhanced releases of the amine were prevented by pretreatment of clonidine, Uptake 2 inhibitor, preceded the epinephrine loading. From these results, it was suspected that the retention or the release of the extraneuronal catecholamine in the vas deferens might be partly modified through the alteration of the Na-K ATPase activity and Ca concentration of the cell membrane. 相似文献
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Electron transport dependent adenosine triphosphatase activity in castor bean endosperm mitochondria
The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg++, but not by Ca++. EDTA, succinate, NADH2or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; ) 相似文献
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Summary The ultrastructural localization of Ca2+, Mg2+-activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3mm ATP, 5mm CaCl2 and 2.4mm Pb(NO3)2 in 0.1m tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized i the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes.The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca2+ or Mg2+ and was greatly decreased in the absence of these cations or in the presence ofp-chloromercuribenzoate orN-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium -glycophosphate as substrate. ATP was, however, the preferential substate. 相似文献
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—10 μm 5-Phenyloxazolidinedione (PKO) stimulated Na–K–Mg-ATPase activity in mouse brain homogenates by 45%. This activation effect was also observed for trimethadione and dimethadione. Inhibition of Na–K–Mg-ATPase by 0.1 mm ouabain was reduced in the presence of PKO. When PKO (4 mg/kg) was administered to mice intravenously, maximum levels of 2 μg (10 nmol)/g were attained in the brain within 15 min. PKO had an ld 50 of 872 mg/kg and an ed 50 of 375 mg/kg for protection against metrazol-induced seizures in mice. 相似文献
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Abstract— The activity of Na-K ATPase was determined in twenty-one regions of the Rhesus monkey brain. White matter contained the lowest activity. The highest activities were found in the cerebral cortex, cerebellar cortex, thalamus, and colliculi. The activities of the enzyme in gray matter were fairly regular in distribution with only a twofold difference between highest (occipital cortex) and lowest (globus pallidus). The possibility that the regional variation in the activity of the enzyme reflects differences in electrical activity and (or) the concentration of dendritic processes was discussed. 相似文献
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An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit. 相似文献
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MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.
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1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined. 相似文献