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)

1. The influence of KCl and CaCl2 on ATPase activity of ventricular myosin of the mouse, rat, rabbit and cow, the temperature dependence of ATPase and the effect of pCMB treatment and tryptic digestion on ATPase activity of these myosins were studied. 2. Ca2+ - and K+ -ATPase activities of myosins were inversely related to body size of the animal species; when K+ -ATPase activities were measured in the absence of EDTA, the body size/ATPase dependence was only slightly apparent. 3. The influence of temperature, the effect of pCMB and the influence of tryptic digestion on Ca2+ - ATPase activity distinguished the compared myosins. 4. There was a marked alteration of the effect of myosin treatment with pCMB or trypsin on K+ -ATPase activity of these myosins and in this case differences in K+ -ATPase activities were less pronounced.
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PMID:The relation between myosin Ca2+ - and K+ -adenosine triphosphatase activity of heart muscles in mammals of different size. 612 14

Sarcoplasmic reticulum (SR) isolated from rabbit skeletal muscle was solubilized with a nonionic detergent, dodecyl octaethyleneglycol monoether (C12E8), at a weight ratio of detergent to protein of greater than 10, so that the Ca2+, Mg2+ dependent ATPase existed mainly in a monomeric form (7). The solubilized ATPase was reacted with 10 microM N-1-P or 5 microM DACM in the presence of 5 mM CaCl2, 0.4 M KCl, 20% glycerol and 50 mM TES at pH 7.5 and 20 degrees C. Under these conditions, about 1 mol of N-1-P was incorporated into 10(5) g SR protein on 10 min incubation and 1 mol of DACM was incorporated into the same amount of SR on 5 min incubation. Analysis of the tryptic digest of the N-1-P- or DACM-labeled. ATPase on SDS polyacrylamide gel revealed that almost all the fluorescence was associated with the 30K m.w. subfragment of the ATPase protein. Even when the amount of the probe incorporated into SR-ATPase was increased from 1 to 3 mol per 10(5) g SR protein, all was incorporated into the 30K subfragment. Both the activities of formation and decomposition of the phosphorylated intermediate (EP) were unaffected by these modifications. When the separately labeled ATPases were mixed together in the presence of C12E8 and the detergent was removed by incubation with Bio-Beads SM-2, a significant amount of fluorescence energy transfer was observed between N-1-P and DACM. However, energy transfer did not occur when the labeled ATPases were mixed after removal of C12E8.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Energy transfer between fluorescent dyes attached to Ca2+,Mg2+-ATPase in the sarcoplasmic reticulum. 614 Feb 62

The ATPase activity of rabbit-kidney brush border can be activated almost equally well by Ca2+ and Mg2+ and, therefore, should be called (Ca2+ or Mg2+)-ATPase. This enzyme was solubilized and enriched 14-fold by the following steps: pretreatment with papain removed 69% of alkaline phosphatase without attacking a significant portion of the ATPase activity. Addition of 1% cholate removed 65% of the protein but no ATPase activity. The combination of cholate (0.5%) and deoxycholate (0.4%) solubilized most of the ATPase activity and most of the remaining protein. A column chromatography of the extract on Sepharose CL-2B resulted in an 6.5-fold increase of specific ATPase activity. A precipitation by ammonium sulfate (40% saturation) produced an additional 1.9-fold increase. The yield of this partial purification was 16%. Towards the nucleotides UTP and GTP the enzyme showed an activity slightly higher, and towards ITP and CTP an activity slightly lower than that with ATP. ADP was split about half as fast as ATP. AMP was not accepted by the enzyme. Replacing MgCl2 by CaCl2 resulted in an ATPase activity of 92% of that with MgCl2. Using calcium- and magnesium-ATP as substrates, apparent Km values of 0.22 and 0.33 mM, respectively, were obtained. The gel electrophoresis revealed the enrichment of a protein with an apparent Mr of 95000 and also that of microvillus actin.
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PMID:Partial purification and characterization of rabbit-kidney brush-border (Ca2+ or Mg2+)-dependent adenosine triphosphatase. 614 3

The amount of phosphoric acid liberated from ATP by Ca2+ (Mg2+)-ATPase in microsomal fraction of guinea-pig thoracic aorta decreased with decreasing concentrations of calcium ions from 20.0 to 2.5 mM in the mixture of the enzyme and substrate. When CaCl2 (2.5 mM) and MgCl2 (5.0 mM) were present in the substrate, both nitroglycerin (0.1 to 1.0 mM) and SIN-1 A (a molsidomine derivative, 0.05 to 1.0 mM) increased the liberated phosphoric acid in a concentration-dependent manner. The contractile tension of smooth muscle prepared from guinea-pig thoracic aorta, which was previously increased by the pretreatment with prostaglandin F2 alpha (5.0 microM), was relaxed by both nitroglycerin and SIN-1 A (0.01 to 100 microM each) in a concentration-dependent manner. From the results, it is assumed that the stimulation of Ca2+ (Mg2+)-ATPase [Ca2+-pump ATPase] activity induced by nitroglycerin and SIN-1 A in the microsome of thoracic aorta takes part in the relaxation of contractile tension in the tissue.
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PMID:Increase in Ca2+ (Mg2+)-ATPase activity induced by a molsidomine derivative (SIN-1 A) and nitroglycerin in microsomal fraction of guinea-pig thoracic aorta. 614 24

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 3 mM ATP, 5 mM CaCl2 and 2.4 mM Pb(NO3)2 in 0.1 M tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized in 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 of p-chloromercuribenzoate or N-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 beta-glycophosphate as substrate. ATP was, however, the preferential substrate.
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PMID:Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin. 618 Oct 20

pH changes of the reaction medium (pH 6.35) following addition of MgATP were determined at 4 degrees C with sarcoplasmic reticulum vesicles in the presence of 0.1 mM CaCl2 and 5 mM MgCl2. With intact vesicles, a pronounced acidification following a slight alkalinization was induced by MgATP, indicating H+ ejection from vesicles. This agrees with previous observations by several investigators that suggested H+ counter-transport coupled to Ca2+ uptake. When vesicles were made leaky, a markedly enhanced alkalinization occurred after addition of MgATP, and then the pH returned to the level before the MgATP addition. The decay of this alkalinization coincided with the disappearance of phosphoenzyme (EP) which was formed from MgATP and the Ca-ATPase of sarcoplasmic reticulum. The CaCl2 concentration dependence of the alkalinization agreed well with that of EP formation. Furthermore, the alkalinization was prevented by modification of a specific SH group of the enzyme essential for EP decomposition. These findings give evidence that the alkalinization with leaky vesicles represents protonation of the enzyme during ATP hydrolysis. The enhanced alkalinization with leaky vesicles suggests protonation occurring inside vesicles. When CaATP in place of MgATP was added in the absence of MgCl2, neither the H+ ejection from intact vesicles nor the enhanced alkalinization with leaky vesicles occurred. Under these conditions, CaATP did not efficiently support active Ca2+ uptake. These results are compatible with the view that the observed protonation could represent an initial event occurring inside vesicles in the H+ countertransport coupled to Ca2+ uptake.
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PMID:Protonation of the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis. 620 83

The ATP-dependent phosphoenzyme formation and its reversal were studied at 0 degrees C and pH 7.0 in the ATPase of sarcoplasmic reticulum. Addition of KCl or several other salts (approximately 100 mM) decreased the maximum rate of ADP-induced dephosphorylation of phosphoenzyme as well as the apparent affinity of the phosphoenzyme toward ADP. High ATP had a similar effect on the latter, whereas it had little effect on the former. In contrast, high KCl or a considerable change in the ionic strength had little effect on the initial rate of phosphoenzyme formation at saturating ATP concentrations. During steady state phosphorylation at 1.0 mM MgCl2 and 5.0 mM CaCl2 in the absence of added KCl, a significant amount of [gamma-32P]ATP remained bound to the enzyme even when the enzyme concentration was much in excess over that of [gamma-32P]ATP. Evidence is presented that this enzyme-ATP complex represents a precursor to the phosphoenzyme. ATP dissociated slowly (0.20 s-1) from this enzyme-ATP complex and addition of high KCl or other salts accelerated its dissociation. In contrast, when the enzyme was complexed with adenyl-5'-yl (beta, gamma-methylene)diphosphonate in the absence of added KCl under these conditions, dissociation of the nucleotide from the complex as estimated in the displacement experiment with [gamma-32P]ATP, was found to be much faster than that of ATP.
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PMID:Phosphoenzyme formation from ATP in the ATPase of sarcoplasmic reticulum. Effect of KCl or ATP and slow dissociation of ATP from precursor enzyme-ATP complex. 621 44

We recently demonstrated that elevated concentrations (greater than 20 microM) of the dynein substrate MgATP2- inhibit the spontaneous ATP-induced sliding disintegration of isolated, Triton-demembranated Tetrahymena cilia. We have used a turbidimetric assay (delta A350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+ are each capable of activating sliding, but only with Mg2+ and Mn2+ is disintegration inhibited by elevated ATP concentrations (greater than or equal to 1 mM). The two major ATPase activities obtained by KCl extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+ and Ca2+ activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by approximately 50% when CaATP2- replaces MgATP2- as substrate. With 1 mM MgATP2- as substrate, 10(-7) to 10(-2) M added CaCl2 alleviates the ATP-dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+ has no effect on either the disintegration response or Mg-ATPase activity. In contrast to Triton-treated cilia, glycerinated cilia, which beat in 1 mM MgATP2-, are inhibited from beating by high CaATP2- concentrations. These substrate specificities suggest that concentration-dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2 probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2- competing for MgATP2- binding sites on the 13S ATPase, thereby blocking expression of the 13S ATPase.
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PMID:Evidence for a role of 13S axonemal ATPase in modulation of ciliary microtubule sliding. 622 Aug 5

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.
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PMID:Purification and characterization of myosin from calf brain. 622 62

(1) Intestinal absorption is altered under a variety of circumstances in health and disease and to determine a possible relationship between intestinal absorptive function and intestinal brush border membrane composition, we undertook the isolation and purification of rabbit jejunal and ileal brush borders, to allow further studies of their lipid composition under varied experimental conditions. (2) A modification of an established method (Schmitz, J., Preiser, H., Maestracci, D., Ghosh, B.K., Cerda, J.J. and Crane, R.K. (1973) Biochim. Biophys. Acta 323, 98-112) utilized CaCl2 aggregation and sequential centrifugation followed by purification of the brush border pellet (P2) at 27,000 X g on a PercollTM (Pharmacia) self-forming gradient. The PercollTM was removed by ultracentrifugation for 30 min at 100 000 X g, utilizing a batch rotor in the Beckman airfugeTM. (3) Pure brush border membrane vesicles were obtained and characterized by specific marker analysis and electron microscopy. Comparative marker analyses performed on P2 and final PercollTM preparations from animals showed that the purification achieved was 8-11-fold greater when compared to the original homogenates. Verification of purity was also demonstrated by the absence of DNA and very low levels of Beta-gluconridase and (Na+ + K+)-ATPase in the PercollTM preparations. (4) Comparative lipid analyses of P2 and final PercollTM preparations showed that levels of total phospholipid and free fatty acids were several-fold higher in the PercollTM preparations on a per mg protein basis. (5) A comparison of the activity of enzyme markers and the levels of total free fatty acids in P2 pellets obtained after Cacl2 and MgCl2 aggregation showed that CaCl2 aggregation gave the more consistently reproducible results. (6) Although standard procedures of membrane preparations not involving density gradient separation provide membranes of reasonable purity for the estimation of lipid components, we consider the final purification step of density gradient separation using PercollTM is essential for determining small quantitative changes which might occur in the membrane lipid composition under experimental conditions were intestinal absorptive function is altered.
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PMID:Use of percollTM in the isolation and purification of rabbit small intestinal brush border membranes. 628 96


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