Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.1.3 (ATPase)
 65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.
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PMID:Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity. 0 69

A prodcedure was developed for the purification of the ATP-dependent deoxyribonuclease of Bacillus subtilis 168. It comprises ammonium sulphate fractionation, Sephadex gel filtration, DEAE-cellulose chromatography and gel electrophoresis on a discontinuous polyacrylamide gradient. The enzyme has been obtained in a homogeneous state. Its molecular weight was estimated to be 270000 by disc electrophoresis. Dodecylsulfate-polyacrylamide gel electrophoresis showed the presence of five nonidentical subunits of the following molecular weights: 81000, 70000, 62000, 52500 and 42500. These values give 308000 as the molecular weight of the native enzyme. The pH optimum of the purified enzyme is 9.6. The optimal concentrations of Mg2+ and ATP for exonuclease activity on native B. subtilis DNA were determined. ATP-requirement for hydrolysis of single-stranded DNA is less strigent. The enzyme also possesses high DNA-dependent ATPase activity. The purification procedure was applied to extracts of a mutant devoid of activity for this enzyme (strain GSY 1290). A protein was isolated which is very similar to the active DNAase as regards electrophoretic mobility, reaction with specific antisera and size of four of the subunits. One subunit is missing (Mr 70000) and is replaced by a smaller polypeptide (Mr 565000). The latter results suggest that the mutant is affected in the genetic locus coding for the 70000-Mr subunit.
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PMID:Isolation, subunit structure and properties of the ATP-dependent deoxyribonuclease of Bacillus subtilis. State of the protein in a mutant devoid of activity. 1 60

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.
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PMID:Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties. 9 4

Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to suing cilution-precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic amino acid, optical and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca-2+ or Mg-2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 times 10-5, which display non-ideality even in solvent buffers of high ionic strength at neurtal pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specif ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.
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PMID:Rabbit cardiac myosin. I. Physical and chemical characterization of the native molecule. 12 22

1. Purification of the coupling factor ATPase from Rhodospirillum rubrum has been achieved by a combination of a previously described procedure with chromatography on DEAE-Sephadex A50. 2. Identification of the coupling factor ATPase during purification, and estimation of the relative amount of the enzyme in each fraction was greatly simplified by utilization of its unusual fluorescence. 3. Preparations of R. rubrum coupling factor ATPase injected into rabbits yielded antisera which were suitable for following the course of purification. 4. Judged by immunoelectrophoretic analysis and polyacrylamide gel electrophoresis the final preparation was pure. Under standardized conditions, apparently pure preparations showed fluorescence ratios at 300/350 nm of 3-6, which are considerably higher than those reported for pure CF1 from chloroplasts. 5. The enzyme lost its activity and changed its immunological identity during prolonged storage and by treatment with urea. Antisera against urea-treated enzyme showed the presence of two distinct antigens in the modified preparations.
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PMID:Immunological and fluorescence studies with the coupling factor ATPase from Rhodospirillum rubrum. 12 80

A low myofibrillar ATPase seems to be established definitely in several experimental models of chronic heart hypertrophy as well as in humans, but the biochemical pathogenesis of this defect is still unclear. Three different preparations of myosin were studied. Their purity was estimated by measuring MgATPase or by polyacrylamide gel electrophoresis. The first preparation was highly contaminated by actin and tropomyosin; the second was rather pure, and the third (chromatography on DEAE-Sephadex) was pure but slightly denaturated. Heart myosin CaATPase (ionic strength 0.6 or 0.06) was decreased in chronic aortic insufficiency in the rabbits (CAI) when all three preparations were tested. Two (molecular weight 18,000 and 26,000), sometimes three light subunits were found in heart myosin. Their charge and molecular weight are normal in CAI. The third subunit (molecular weight 15,500) was found in control as well as in CAI. Search for an inhibitor was unsuccessful since the two myosin ATPases are additive. The nucleoprotein peak separated from myosin during chromatography was identical in control and CAI. Therefore, myosin seems to be abnormal in CAI.
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PMID:Cardiac myosin: preparation, ATPase in chronic heart hypertrophy. 12 56

The three major components of bovine cardiac troponin were separated by successive chromatography on sulfopropyl-Sephadex and DEAE-Sephadex columns in the presence of 6 M urea. All three of the bovine cardiac troponin subunits were necessary to restore full troponin activity in both skeletal and cardiac actomyosin ATPase assay systems. The 38,000-dalton subunit bound tropomyosin, and the 20,000-dalton subunit bound calcium, like skeletal TN-T and TN-C, respectively. The 28,000 component, although presumably analogous to skeletal TN-I, gave very little inhibition of actomyosin ATPase activity. Differences between cardiac and skeletal troponin subunits were also found in the elution patterns from ion exchange columns and in amino acid composition, thus demonstrating a significant muscle-type specificity.
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PMID:Separation and characterization of the troponin components from bovine cardiac muscle. 12 74

Cytoplasmic actin has been isolated from Acanthamoeba castellanii by a new method, employing chromatography on DEAE-cellulose, that improves the yield by more than 20-fold over the previously reported method. This procedure should be particularly useful for isolating actin from cells in which it is present in relatively low concentration because the method does not depend initially on the polymerization of actin or its interaction with myosin. Systematic comparison of the properties of purified Acanthamoeba actin and rabbit skeletal muscle actin shows them to be similar in many ways: viscosity of F-actin, stoichiometry of bound nucleotide, stoichiometry of binding of muscle heavy meromyosin and myosin subfragment 1 in the absence of ATP, and ability to inhibit the KATPase activity of heavy meromyosin. The amino acid compositions of Acanthamoeba and muscle actin are also quite similar, but significant differences, especially the presence of epsilon-N-methyllysines in Acanthamoeba actin, have been confirmed. In addition to this structural difference, we find that Acanthamoeba actin is only one-third as effective as muscle actin as an activator of the MgATPase of muscle heavy meromyosin and subfragment 1. For Acanthamoeba actin, as for muscle actin, this activation exhibits hyperbolic dependence on actin concentration; i.e. the double reciprocal plot of ATPase activation versus actin concentration is linear. From these plots we find that the two actins give the same extrapolated ATPase activity at infinite actin concentration (Vmax) but differ by a factor of three in the concentration of actin needed to produce half-maximal activation (Kapp).
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PMID:Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method. 13 6

An ATPase which strikingly differed from the mitochondrial ATPases of yeast and of animal tissues was obtained when wheat seedling mitochondria, or electron transport particles derived from them, were subjected to ultrasonication and treated with ammonium sulphate. The enzyme which was purified by chromatography on Sephadex G-100 and DEAE-Sephadex (A50) failed to be inactivated as low as 43 000. The enzyme preparation was capable of hydrolysing ADP, in addition to ATP, and several other nucleoside diphosphates and triphosphates. In contrast to the ATPase of animal mitochondria, the activity of the wheat enzyme was almost as insensitive to oligomycin in intact mitochondria as it was after isolation from the organelles.
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PMID:A low-molecular-weight ATPase from wheat-seedling mitochondria. 13 93

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.
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PMID:Myosin and actomyosin from human skeletal muscle. 13 73


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