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)

The phospholipid and fatty acid composition and role of phospholipids in enzyme and transport function of gastric (H+ + K+)-ATPase vesicles was studied using phospholipase A2 (bee venom). The composition (%) was phosphatidyl-choline (PC) 33%; sphingomyelin (sph) 25%; phosphatidylethanolamine (PE) 22%; phosphatidylserine (PS) 11%; and phosphatidylinositol (PI) 8%. The fatty acid composition showed a high degree of unsaturation. In both fresh and lyophilized preparations, even with prolonged incubation, only 50% of phospholipids were hydrolyzed, but the amount of PE and PS disappearing was increased following lyophilization. There was a marked decrease in K+-ATPase activity (75%) but essentially no loss of the associated K+ p-nitrophenyl phosphatase was found. ATPase activity could be largely restored by various phospholipids (PE greater than PC greater than PS). There was also an increase in Mg2+-ATPase activity, partially reversed in fresh preparations by the addition of phospholipids (PE greater than PS greater than PC). Proton transport activity of the preparation was rapidly inhibited, initially due to a large increase in the HCl permeability of the preparation. Associated with these enzymatic and functional changes, the ATP-induced conformational changes, as indicated by circular dichroism spectra were inhibited.
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PMID:Effect of phospholipase A2 on purified gastric vesicles. 4 34

The sodium-potassium activated adenosine triphosphatase (NaKATPase) activity of the rat cornea was investigated histochemically using a Pb2+-precipitation technique in which adenosine triphosphate (ATP) is used as substrate and two methods for potassium-dependent para-nitrophenyl-phosphatase (K-NPPase) activity. With all the three techniques used it was demonstrated that the sodium-potassium-activated adenosine triphosphatase (NaK-ATPase) activity is localized in the cell membranes of the endothelium whereas a much weaker activity was observed in the epithelium. When the Pb2+-technique was used, the epithelial cell membranes showed a weaker reaction in the presence of ouabain. This activity was only Mg2+-dependent and was presumably due to an Mg2+-dependent ATPase. The validity of the histochemical techniques for NaK-ATPase activity is discussed. The results emphasize the importance of the endothelium as the main site of Na+ transport in the cornea. Small amounts of the enzyme are also present in the epithelium, which seems to be rich in Mg2+-ATPase. Provided that careful controls are performed, all the methods give consistent results in the cornea.
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PMID:Transport adenosine triphosphatase activity in the rat cornea. 6 3

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.
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PMID:Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+. 13 42

Most biological membranes are functionally asymmetric. To study biochemical control of cardiac transsarcolemmalion fluxes, it would be of obvious advantage to use isolated vesicles of sarcolemma which retains the low passive permeability characteristics of intact sarcolemma because in such vesicles the membrane should exhibit its normal asymmetric character with respect to enzymic activities. The purpose of this investigation was to attempt identify such vesicles in a cardiac microsomal (membrane vesicular) preparation. We studied activation by Na+ and K+ of Na+, K+-ATPase and its associated K+-phosphatase activities, using as substrates ATP or p-nitrophenylphosphate (pNPP) in the presence of Mg2+. Optimal concentrations of K+ alone (10 mM) stimulated p-nitrophenylphosphatase (pNPPase) activity 1.8-fold, and over 80% of the increase could be inhibited by ouabain. Optimal Na+ plus K+ concentrations (100 mM and 10 mM, respectively) stimulated the rate of ATP hydrolysis 2-fold, but only 11 +/- 1.1% of the increased activity was ouabain-sensitive. Optimal pretreatment with sodium dodecyl sulfate (SDS) (0.3 mg/ml) rendered both activities completely sensitive to inhibition by ouabain and reduced the basal Mg2+-ATPase activity by 70-90%. The K+-stimulated pNPPase activity doubled after preincubation in SDS, but the ATPase activity stimulated by Na+ plus K+ fell by 50% under these conditions. A similar pattern of apparent activation was produced by preincubation with deoxycholate (DOC), except that basal Mg2+-dependent activities were resistant to destruction by this detergent. The incremental responses to activation by ions and substrates, and inhibition by oubain, are consistent with the hypothesis that permeability-intact vesicles of sarcolemma are present in the isolated preparation, and that detergent activation renders the vesicles highly permeable to the ions, substrates, and ouabain.
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PMID:Intact vesicles of canine cardiac sarcolemma: evidence from vectorial properties of Na+, K+-ATPase. 18 13

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

Ouabain inhibited 86RbCl uptake by 80% in rabbit gastric superficial epithelial cells (SEC), revealing the presence of a functional Na+,K+-ATPase [(Na+ + K+)-transporting ATPase] pump. Intact SEC were used to study the ouabain-sensitive Na+,K+-ATPase and K+-pNPPase (K+-stimulated p-nitrophenyl phosphatase) activities before and after lysis. Intact SEC showed no Na+,K+-ATPase and insignificant Mg2+-ATPase activity. However, appreciable K+-pNPPase activity sensitive to ouabain inhibition was demonstrated by localizing its activity to the cell-surface exterior. The lysed SEC, on the other hand, demonstrated both ouabain-sensitive Na+,K+-ATPase and K+-pNPPase activities. Thus the ATP-hydrolytic site of Na+,K+-ATPase faces exclusively the cytosol, whereas the associated K+-pNPPase is distributed equally across the plasma membrane. The study suggests that the cell-exterior-located K+-pNPPase can be used as a convenient and reliable 'in situ' marker for the functional Na+,K+-ATPase system of various isolated cells under noninvasive conditions.
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PMID:Half of the (Na+ + K+)-transporting-ATPase-associated K+-stimulated p-nitrophenyl phosphatase activity of gastric epithelial cells is exposed to the surface exterior. 245 13

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations. 255 12

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa. 282 83

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.
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PMID:Subcellular fractionation of pig coronary artery smooth muscle. 299 88

The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain-sensitive, potassium-stimulated p-nitrophenyl phosphatase (K+-pNPPase)], [3H]saxitoxin binding, and 5'-AMPase, was studied in the subcellular fractions prepared from the homogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+-pNPPase activity and [3H]-saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+-pNPPase and [3H]saxitoxin binding, but not 5'-AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+-pNPPase activity and [3H]saxitoxin binding, the 5'-AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on density gradient resulted in copurification of 5'-AMPase but not K+-pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane-bound enzymes, such as high-affinity Ca2+-ATPase, Mg2+-ATPase, and Ca2+-ATPase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5'-AMPase activity is concentrated in the smooth muscle plasma membranes.
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PMID:Subcellular fractionation of the longitudinal smooth muscle/myenteric plexus of dog ileum: dissociation of the distribution of two plasma membrane marker enzymes. 304 Sep 6


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