The action of EGTA on the catecholamines stimulation of rat brain Na-K-ATPase (original) (raw)
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Advances in Enzyme Research, 2014
The work is a study of the influence of Ca 2+ (0.01-1 mM) on neuronal Cl − , 3 HCO −-ATPase complex: an enzyme that is a Cl −-pump which is functionally and structurally coupled to GABAA-receptors. It is found that influence of Ca 2+ on the multifunctional complex starts at concentration of 50 µM and at concentration of 0.1 mM, it reduces the "basal" one and increases the Cl − , 3 HCO −-stimulated Mg 2+-ATPase activities. GABA (0.1-100 µM) activates the "basal" Mg 2+-ATPase activity in the absence of calcium. The effect of GABA on the enzyme in the presence of 0.01 µM Ca 2+ does not change. At the same time, 1 mM Ca 2+ eliminates the GABA effect on the "basal" Mg 2+-ATPase activity. Competitive blocker of GABAA-receptors bicuculline (5-20 µM) in the absence of Ca 2+ ions eliminates the stimulation of the "basal" Mg 2+-ATPase by anions. When 0.25 mM Ca 2+ is added to the incubation medium the inhibitory bicuculline effect on the enzyme does not appear. We found that 0.1 mM o-vanadate (protein tyrosine phosphatase blocker) reduces the GABA-activated ATPase activity. At the same time, 0.1 mM genistein (a protein tyrosine kinase blocker) has no effect on enzyme activity. In the presence of Ca 2+ (0.25 mM), the effect of o-vanadate on the "basal" and Cl − , 3 HCO −-ATPase activities does not appear. It is shown for the first time that high concentrations of Ca 2+ prevent the action of GABAA-ergic ligands on the study ATPase. It is assumed that there is the involvement of protein kinases and protein phosphatases in the modulation of the enzyme activity by calcium. The observed effect of calcium on the ATPase may play an important role in the study of the mechanisms of epileptogenesis and seizure activity.
Biochimica Et Biophysica Acta - Biomembranes, 1978
Effects of commonly used purification procedures on the yield and specific activity of (Na ÷ + K*)-ATPase (Mg2÷-dependent, Na* + K÷-activated ATP phosphohydrolase, EC 3.6.1.3), the turnover number of the enzyme, and the kinetic parameters for the ATP<tependent ouabain-enzyme interaction were compared in canine brain, heart and kidney. Kinetic parameters were estimated using a graphical analysis of non-steady state kinetics. The protein recovery and the degree of increase in specific activity of (Na*+ K*)-ATPase and the ratio between (Na*+ K*)-ATPase and Mg2÷-ATPase activities during the successive treatments with deoxycholate, sodium iodide and glycerol were dependent on the source of the enzyme. A method which yields highly active (Na'+ K*)-ATPase preparations from the cardiac tissue was not suitable for obtaining highly active enzyme preparations from other tissues. Apparent turnover numbers of the brain (Na*+ K*)-ATPase preparations were not significantly affected by the sodium iodide treatment, but markedly decreased by deoxycholate or glycerol treatments. Similar glycerol treatment, however, failed to affect the apparent turnover number of cardiac enzyme preparations. Cerebral and cardiac enzyme preparations obtained by deoxycholate, sodium iodide and glycerol treatments had lower affinity for ouabain than renal enzyme preparations, primarily due to higher dissociation rate constants for the ouabain • enzyme complex. This tissue-dependent difference in ouabain sensitivity seems to be an artifact of the purification procedure, since less purified cerebral or cardiac preparations had lower dissociation rate constants. Changes in apparent association rate constants were minimal during the purification procedure. These results indicate that the presently used purification procedures may alter * To whom reprint requests should be sent. the properties of membrane (Na*+ K÷)-ATPase and affect the interaction between cardiac glycosides and the enzyme. The effect of a given treatment depends on the source of the enzyme. For the in vitro studies involving purified (Na ÷ + K÷)-ATPase preparations, the influence of the methods used to obtain the enzyme preparation should be carefully evaluated.
Properties of a Ca2+ and Mg2+ stimulated ATPase in the rat caudate nucleus
Biochemical Pharmacology, 1985
Adenosine triphosphatase (ATPase) activity which could be stimulated maximally by either Ca 2÷ or Mg 2÷ was identified in a synaptosomal fraction from rat brain caudate nucleus. The thermodynamic properties of the Ca 2÷ and Mg 2÷ stimulated enzymes were similar to each other. Oligomycin, sodium azide and dinitrophenol had no significant inhibitory effects on stimulation by either cation. In vitro incubation of the ATPase with c/s-or trans-flupenthixol, chlorpromazine or trifluoperazine, but not with haloperidol, significantly inhibited stimulation by either cation. The DA receptor agonist 2amino-6,7-dihydroxy-l,2,3,4-tetrahydronaphthalene (ADTN) inhibited stimulation of the enzyme by either cation, while d-amphetamine, SKF 38393, pergolide and LY-171555 had no significant effects. Nomifensine at 10-3 M inhibited the cation stimulation by about 33%. In vivo administration of dopamine (DA) receptor antagonists (haloperidol, c/s-and trans-flupenthixol, spiperone, chlorpromazine and trifluoperazine) and the agonist apomorphine neither inhibited nor stimulated ATPase activity. It appears from these data that the ATPase activity is not under DA receptor modulation. In addition, our tentative conclusion is that one enzyme is involved, because both Ca 2÷ and Mg 2÷ produced similar maximal stimulations, the activities as a function of temperature were similar, the enzyme could not be further stimulated with Ca 2÷ after maximal stimulation by Mg 2~ (and vice versa), and the behaviour of the ATPase activity to all drugs tested was similar.
A simple method for the purification of rat brain Na+,K+-adenosine triphosphatase (ATPase)
Journal of pharmacological methods, 1982
Several methods of purification of Na+,K+-adenosine triphosphatase (ATPase) have been previously described for a wide variety of tissues. In general, highest activity preparations have necessitated large amounts of tissue and many purification steps. This article describes a technique that allows partial purification of Na+,K+-ATPase from as few as 15 rat brains and should be of interest to investigators of the pharmacology of this particular enzyme system. In this modified version of the Jorgensen procedure (Biochim Biophys Acta 356:36--52, 1974) we purified the Na+,K+-ATPase from 15--90 rat brains, and obtained enzyme preparations with a mean specific activity of 552 +/- 37.6 mumol Pi/mg of protein/hr (95.5% ouabain sensitive). This "purified" enzyme had an activity ratio (Mg2+ + Na+ + K+)/(Mg2+ + Na+) of 47.4 +/- 12.3 SEM, compared to 3.29 +/- 0.17 SEM for the untreated microsomes. Ouabain inhibited the "purified" enzyme with an I50 of 6 X 10(-9) M. Ouabain bi...
Some properties of the Ca2+-stimulated ATPase of a rat liver microsomal fraction
The Biochemical journal, 1983
1. The heavy microsomal fraction from rat liver apparently has very little Ca2+-stimulated ATPase activity, although it has an active, ATP-driven Ca2+ accumulation system. 2. The addition of ionophore A23187 to the ATPase assay, to allow continuous Ca2+ recycling during the assay time, reveals the presence of a substantial Ca2+-stimulated ATPase with Vmax. 160 nmol of Pi/10 min per mg of protein and Km for Ca2+ 0.19 microM. 3. The Ca2+-stimulated ATPase, but not the basal Mg2+-stimulated ATPase, is potently inhibited by orthovanadate. Both the Ca2+-stimulated ATPase and the vanadate inhibition are enhanced by the presence of Mg2+. 4. Ca2+-stimulated ATPase activity is not responsive to calmodulin or the calmodulin antagonist trifluoperazine.
Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8
Brazilian Journal of Medical and Biological Research, 2002
SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,K-ATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8-solubilized and purified Na,K-ATPase by 99, 81 and 98.5%, respectively. We have shown that Na,K-ATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.