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1.
The inhibitory effect of ouabain on (Na+ + K+)-activated ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, EC 3.6.1.3) obtained from rat brain microsomal fraction was re-examined using a modified method to estimate the inhibited reaction velocity. This method involves a preincubation of a ouabain-enzyme mixture in the presence of Na+, Mg2+ and ATP to bring the ouabain-enzyme reaction to near equilibrium. The (Na+ + K+)-activated ATPase reaction was subsequently started by the addition of a KCl solution. 相似文献
2.
Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124–132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ + K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ + K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca2+, and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ + K+)-activated ATPase activity averaged 10.07 ± 2.80 μmol Pi/mg protei per h compared to 50.03 ± 11.41 for Mg2+-activated ATPase and 58.66 ± 10.07 for 5′-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ + K+)-activated ATPase without any effect on Mg2+-activated ATPase. Both (Na+ + K+)-activated ATPase and Mg2+-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ + K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ + K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis. 相似文献
3.
A.J. Peacock 《Journal of insect physiology》1976,22(12):1631-1634
The Mg2+ dependent and Na+K+-activated ATPase activities of microsomal preparations from the rectum of Locusta migratoria were both stimulated, to varying extents, by crude extracts of the corpora cardiaca of this species. Mg2+ ATPase activity increased by approximately 549% whereas the hormonal stimulation of Na+K+-activated ATPase depended upon the concentration of sodium and potassium ions. At 100 mM Na+ and 20 mM K+, conditions which approximate to optimum for this enzyme system, Na+K+-activated ATPase activity increased by about 14%. At sub-optimum concentrations of these ions, i.e. 50 and 5 mM Na+ and K+ respectively, the increase in Na+K+-activated ATPase activity was about 205%. Ouabain at a concentration of 10?3 M completely abolished this stimulated activity and was consistently effective in partially reducing the stimulation of Mg2+ ATPase activity by corpora cardiaca extracts. 相似文献
4.
The adenosine triphosphatase (ATPase) system in worker honey-bee brains showed an increased activity of 57 per cent in Na+K+ATPase and 63 per cent in Mg2+ATPase from adult emergence to 7 days post-emergence. Mg2+ATPase activity remained about the same throughout the remainder of adult life, while Na+K+ATPase remained the same until the sixth week, when a decline occurred. The percentage mortality of the bees exceeded 90 per cent at the time of decline of Na+K+ATPase. The in vitro inhibition of Mg2+ATPase and Na+K+ATPase by 10 μM DDT was between 40 and 50 per cent and about 20 per cent, respectively. A somewhat greater sensitivity to DDT was determined in brains of older honey-bees. 相似文献
5.
Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase 总被引:2,自引:2,他引:0
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1. An adenosine triphosphatase membrane system, dependent on Mg2+ and activated further by Na++K+, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na+ in the presence of K++Mg2+, for K+ in the presence of Na++Mg2+ and for Mg2+ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na++K+ were added to preparations from 8°-acclimatized fish the affinity for Mg2+ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg2+-activated adenosine triphosphatase was inhibited by Na+ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na+-inhibited adenosine triphosphatase was separated from the (Na++K+)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg2+-activated and more (Na++K+)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further. 相似文献
6.
The presence of a Na+K+-activated, Mg2+-dependent ATPase (E.C. 3.6.1.3) has been demonstrated in microsomal preparations from the Malpighian tubules of Locusta. The effects of sodium and potassium ions, and different concentrations of ouabain, have been studied in relation to the activity of this enzyme and the ability of in vitro Malpighian tubule preparations to secrete fluid. From these studies it seems highly likely that a Na+K+ activated ATPase ‘pump’ is involved in fluid transport across the walls of the tubules. 相似文献
7.
PbCl2 in micromolar concentrations stimulates phosphorylation of electroplax microsomal protein in the absence of Na+. Other divalent cations showed little or no such effect. The (Mg2+ + Pb2+)- and (Mg2+ + Na+)-dependent membrane-bound protein kinase activities in electroplax particulate preparations exhibit properties in common, including their acid stability, ouabain sensitivity, ATP specificity, and molecular size. It is concluded that the (Mg2+ + Pb2+)-dependent phosphoprotein is part of the Na+-, K+-dependent adenosine triphosphatase [(NaK)ATPase]. The Pb2+-dependent product, in contrast to the Na+-dependent one, is insensitive to K+ and the hydrolysis of ATP is thus inhibited. 相似文献
8.
The specific activity of (Na+ + Mg2+)-dependent ATPase is three times greater in the microsomes of sea-water eels than in freshwater eels; the specific activity is one quarter of that of (Na+ + K+ + Mg2+)-dependent ATPase in both cases.(Na+ + Mg2+)-dependent ATPase is optimally active in a medium containing 8 mM NaCl, 4 mM MgCI2, 4 mM ATP, pH 8.8 and at 30 °C; the enzyme is inhibited by ouabain, by NaCl concentrations > 100 mM and by treatment with urea.It is concluded that the (Na+ + Mg2+)-dependent ATPase activity of gills arises from the presence of a (Na+ + K+ + Mg2+)-dependent ATPase. 相似文献
9.
Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland 总被引:3,自引:3,他引:0
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1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg2+-dependent, (b) Ca2+-dependent and (c) Mg2++Na++K+-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg2++Na++K+-dependent enzyme, whereas the Mg2+- and Ca2+-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg2+- and Ca2+-ATPases; however, the activity of the Mg2++Na++K+-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg2+- and Ca2+-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg2+ or Ca2+ and the other activated only by Ca2+. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity. 相似文献
10.
Ernesto Guiraldes Sanath P. Lamabadusuriya Jose E.J. Oyesiku Anthony E. Whitfield John T. Harries 《生物化学与生物物理学报:生物膜》1975,389(3):495-505
The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques.In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-O-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5±2.9 % per g wet wt of gut) greatly exceeded those of taurocholate (4.3±1.0).In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and Cl−, and inhibited active glucose transport; concomitantly “total” ATPase, (Na+ + K+)-ATPase, and Mg2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg2+-ATPase activity. Taurocholate and cholate at 1 mM had no effect on transport or (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate.These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level. 相似文献
11.
Summary (i) In human red cell membranes the sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is at least ten times larger than the sensitivity to N-ethylmaleimide of (Na++K+)-ATPase and K+-activated phosphatase activities. All activities are partially protected against N-ethylmaleimide by ATP but not by inorganic phosphate or byp-nitrophenylphosphate. (ii) Protection by ATP of (Na++K+)-ATPase is impeded by either Na+ or K+ whereas only K+ impedes protection by ATP of K+-activated phosphatase. On the other hand, Na+ or K+ slightly protects Ca2+-dependent activities against N-ethylmaleimide, this effect being independent of ATP. (iii) The sensitivity to N-ethylmaleimide of Ca2+-dependent ATPase and phosphatase activities is markedly enhanced by low concentrations of Ca2+. This effect is half-maximal at less than 1 m Ca2+ and does not require ATP, which suggests that sites with high affinity for Ca2+ exist in the Ca2+-ATPase in the absence of ATP. (iv) Under all conditions tested the response to N-ethylmaleimide of the ATPase and phosphatase activites stimulated by K+ or Na+ in the presence of Ca2+ parallels that of the Ca2+-dependent activities, suggesting that the Ca2+-ATPase system possesses sites at which monovalent cations bind to increase its activity. 相似文献
12.
External treatment of human erythrocytes with the diazonium salt of sulfanilic acid does not inhibit the Mg2+-dependent ATPase but does markedly inhibit the Ca2+-stimulated ATPase activity. Inhibition of the (Na+ + K+)-dependent activity is dependent upon the concentration of diazonium salt used. Treatment of membrane fragments does not irreversibly inhibit the (Na+ + K+)-dependent ATPase even though the diazonium salt binds covalently to membrane components. However, the Mg2+-dependent and Ca2+-stimulated ATPase activities are irreversibly inhibited. ATP and Mg-ATP will completely protect the (Na+ + K+)-dependent ATPase when present during treatment of membrane fragments with the diazonium salt, but only Mg-ATP will protect the Mg2+-dependent ATPase from inhibition. The Ca2+-stimulated ATPase activity is not protected. 相似文献
13.
《Insect Biochemistry》1991,21(4):399-405
Na+,K+-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na+,K+-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na+,K+-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na+,K+-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg2+, Ca2+ and Na+. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg2+ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca2+-ATPase activity and oligomycin-sensitive Ca2+, Mg2+-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction. 相似文献
14.
—In the presence of synaptosomes prepared from rat brain, only ATP, dATP and ADP but not dADP were active as substrates of phosphatase (ATP phosphohydrolase; EC 3.6.1 4) in the presence of 150mm-Na+ and 20mm-K+. An active adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3.) was demonstrated in the synaptosomal fractions by means of paper chromatography, paper electrophoresis and enzymic reactions, so that the high activity with ADP as substrate could represent an activity of an ATPase. Apparently dADP was not a substrate for the kinase; no dATP was formed when dADP was incubated with the synaptosomal fraction in the presence of Na+, K+ and Mg2+. Small amounts of P1 were liberated with dADP, IDP, GDP or CDP, but not UDP, as substrates, but none was produced in the presence of mononucleotides. The adenine-deoxyribose bond, but not the adenine-ribose bond, was hydrolysed upon the addition of 5% (w/v) TCA to the reaction mixture. The KM for the hydrolysis of ATP but not ITP, in the presence of Mg2+, or of Na+, K+ and Mg2+, was lower for the synaptosomal ATPase than for the microsomal ATPase, and the values for Vmax for synaptosomal ATPase were higher. The activation increment was generally higher for the synaptosomal ATPase and no distinct differences in the properties of the enzyme from either particulate fractions were observed. Mg2+ could be partially replaced by Mn2+ in the synaptosomal ATPase system, but there was little Na+-K+-activation observed in the presence of the latter. The effects of ouabain and of homogenization under various conditions suggested localization of the K+-sensitive site of the ATPase on the surface of the synaptosomal membrane. Activity of the Na+-K+-Mg2+ ATPase increased after freezing and thawing of the sonicated, sucrose or tris-treated preparations but decreased considerably in the synaptosomes treated with 001 m-deoxycholate. Activity of the Mg2+ ATPase in the latter preparation showed little change. 相似文献
15.
Sugar beet leaf homogenate contains Mg2+-stimulated ATPase activity with the highest specific activity in the 25,000–30,000 ×g-fraction. This fraction also has (Na++ K+)-activated ATPase activity. Both activities have two pH optima, one stable at pH 7.9 and one variable at lower pH. When optimal conditions of Na+ and K+ were tested with 64 combinations of these ions, at least two mountains of activity were revealed. The (Na++ K+)-ATPase had a high specificity for ATP. It had lost about 50% of its original activity after 56 days of storage at ?85°C. The activity drop was most pronounced at high ionic concentrations in the test medium. The (Na++ K+)-ATPase shows four peaks of activity when tested at constant ionic strength. The idea is put forward that the four peaks reflect two ATPases, one in the tonoplast and one in the plasmalemma, which undergo conformational changes in relation to the ionic milieu. 相似文献
16.
Variable ATPase composition of human tumor plasma membranes 总被引:2,自引:0,他引:2
Purified plasma membranes from several transplantable human tumors exhibit very high Mg2+-dependent ATPase activities. Three types of Mg2+-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na+, K+-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg2+-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na+ (or K+) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg2+-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed. 相似文献
17.
5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na+ + K+)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K+-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na+, K+, Mg2+, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na+, and Mg2+, and decreased by K+. These fluorescence changes likely reflect ligand-induced stabilization of the E1 or E2 states of the enzyme. 相似文献
18.
JAN KARLSSON 《Physiologia plantarum》1975,35(2):77-79
Electrophoretic measurements on membrane coated particles were performed with a Zytopherometer. Tris-HCl buffer 0.2 M pH 7.0 at 37°C with addition of different combinations of Na+, K+, Mg2+ and ATP was used as test medium. The membranes were of two types, an untreated preparation with low NaK ATPase activity and a deoxycholate treated preparation with high NaK ATPase activity. There was no marked difference in reaction between the two types of membranes. To both types of membranes Mg2+ gave a strong positive and ATP a slight negative addition to the membrane charge. In the presence of ATP Na+ gave a higher charge contribution than did K+ or a combination of Na+ and K+. This implies that K+ gives a higher affinity for ATP than Na+ does and or that ATP mediates a higher affinity for Na+ than for K+. 相似文献
19.
20.
[14C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na+, but decreased by K+, Mg2+ and Ca2+. Na+ abolished the effect of K+ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg2+ and Ca2+. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na+ + K+)-activated ATPase. 相似文献