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)

Kinesin was extensively purified from bovine brain cytosol by a microtubule-binding step in the presence of 5'-adenylyl imidodiphosphate (AMP-PNP), followed by gel filtration chromatography and sucrose gradient ultracentrifugation. The products consistently contained 124,000 (124K) and 64,000 (64K) dalton polypeptides. These two polypeptides appear to represent heavy and light chains of kinesin, respectively, because they copurified on sucrose gradients to a constant and equimolar stoichiometry and bound stably to microtubules in the presence of AMP-PNP but not ATP. The mobilities of 124K and 64K in sodium dodecyl sulfate-polyacrylamide gels under reducing conditions were the same as under nonreducing conditions. A diffusion coefficient of (2.24 +/- 0.21) X 10(-7) cm2 s-1 and a sedimentation coefficient of (9.56 +/- 0.34) X 10(-13) s were determined for native kinesin by gel filtration and sucrose gradient ultracentrifugation, respectively. These values were used to calculate a native molecular weight of about 379,000 and suggest that kinesin has an axial ratio of approximately 20. Extensively purified kinesin exhibited microtubule-activated ATPase activity, and only the 124K subunit incorporated ATP in photoaffinity labeling experiments using [32P]ATP. Collectively, these data favor the interpretation that bovine brain kinesin is a highly elongated, microtubule-activated ATPase comprising two subunits each of 124,000 and 64,000 daltons, that the subunits are not linked to one another by disulfide bonds, and that the heavy chains are the ATP-binding subunits.
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PMID:Native structure and physical properties of bovine brain kinesin and identification of the ATP-binding subunit polypeptide. 313 48

Interrelationships between the binding by rabbit muscle phosphofructokinase of citrate, ATP, GTP, and adenyl-5'-yl imidodiphosphate (AMP-PNP) were investigated. To allow measurements at 25 degrees C, pyruvate kinase and phosphoenolpyruvate were included in the dialysis media to rephosphorylate ADP formed by the weak ATPase action of phosphofructokinase. Binding of citrate was enhanced by GTP nearly as much as by ATP, although GTP does not inhibit the catalytic activity of the enzyme. The results are consistent with the interpretation that binding of GTP, and, by analogy, ATP, at the catalytic site enhances the binding of citrate. AMP-PNP also enhanced citrate binding. Both ATP and GTP appear to bind at three sites per enzyme subunit, with the apparent third site binding relatively weakly. The estimated dissociation constants for the first two sites, about 33 microM for both for ATP compared with 3 and 280 microM for GTP, are consistent with kinetic results that imply lack of effective competition by GTP for the inhibitory site. When a compound binds at two or more sites on a macromolecule, the position and shape of the binding curve are sensitive to the geometric mean of the binding constants but quite insensitive to the magnitudes of the individual constants; thus, binding affinities cannot be estimated with confidence in such cases.
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PMID:Interactions between citrate and nucleoside triphosphates in binding to phosphofructokinase. 315 47

It was previously reported that 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) (TNP)-nucleotides bind with high affinity to the sarcoplasmic reticulum Ca-ATPase (Dupont, Y., Chapron, Y., and Pougeois, R. (1982) Biochem. Biophys. Res. Commun. 106, 1272-1279 and Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Here we report a study of the Ca-ATPase nucleotide binding sites using TNP-nucleotides. Competition at equilibrium between TNP-nucleotides and ATP was measured in the absence of calcium; it was found that TNP-nucleotides and ATP competitively bind to two classes of sites of equal concentration (3.5 nmol/mg). The ATP dissociation constants for the two classes of sites were found to be sensitive to H+ and Mg2+ concentrations. In the absence of Mg2+ (independently of pH) or at acid pH (independently of Mg2+ concentration), the nucleotide sites behave like one single family of sites of intermediate affinity (Kd = 20 microM). They split into two classes of sites of high (Kd = 2-4 microM) and low (Kd greater than 1 mM) affinity at pH values higher than neutral and in the presence of Mg2+. The calcium-activated ATP hydrolysis is accelerated by TNP-ATP (or TNP-AMP-PNP) binding on the phosphorylated enzyme. It is concluded 1) that the Ca-ATPase enzyme possesses two classes of ATP binding sites, 2) that the affinity of these two sites and the nature of their interaction is modulated by the H+ and Mg2+ concentrations, and 3) that the hydrolytic activity of the high affinity ATP binding site is activated by ATP or TNP-AMP-PNP (or TNP-ATP) binding in a low affinity ATP binding site.
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PMID:Two distinct classes of nucleotide binding sites in sarcoplasmic reticulum Ca-ATPase revealed by 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP. 315 55

The effects of adenylylimidodiphosphate (AMP-PNP) and guanylylimidodiphosphate (GMP-PNP) on the kinetics of MgATP, MgITP and MgGTP hydrolysis by mitochondrial ATPase (EC 3.6.1.3) from human placenta were studied. AMP-PNP is a noncompetitive inhibitor of hydrolysis of all substrates used, both in the presence and in the absence of the activating HCO3- anion. At least two binding sites for AMP-PNP are present in the F1. Unlike AMP-PNP, GMP-PNP was shown to be a competitive inhibitor of hydrolysis of all substrates used. The results of the kinetic experiments presented support the alternating three-site mechanism of ATP hydrolysis by mitochondrial ATPase.
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PMID:Mitochondrial adenosine triphosphatase from human placenta--effects of adenylyl and guanylyl imidodiphosphate. 315 4

The binding and cross-linking of the ATP photoaffinity analogue 8-azidoadenosine 5'-triphosphate (azido-ATP) with recA protein have been investigated, and through cross-linking inhibition studies, the binding of other nucleotide cofactors to recA protein has also been studied. The azido-ATP molecule was shown to be a good ATP analogue with regard to recA protein binding and enzymatic function by three criteria: first, the cross-linking follows a simple hyperbolic binding curve with a Kd of 4 microM and a cross-linking efficiency ranging from 10% to 70% depending on conditions; second, ATP, dATP, and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S) specifically inhibit the cross-linking of azido-ATP to recA protein; third, azido-ATP is a substrate for recA protein ATPase activity. Quantitative analysis of the cross-linking inhibition studies using a variety of nucleotide cofactors as competitors has shown that the binding affinity of adenine-containing nucleotides for recA protein decreases in the following order: ATP-gamma-S greater than dATP greater than ATP greater than adenylyl beta,gamma-imidodiphosphate (AMP-PNP) much greater than adenylyl beta,gamma-methylenediphosphate (AMP-PCP) approximately adenine. Similar competition studies also showed that nearly all of the other nucleotide triphosphates also bind to recA protein, with the affinity decreasing in the following order: UTP greater than GTP approximately equal to dCTP greater than dGTP greater than CTP. In addition, studies performed in the presence of single-stranded DNA demonstrated that the affinity of ATP, dATP, ATP-gamma-S, and AMP-PNP for recA protein is significantly increased. These results are discussed in terms of the reciprocal effects that nucleotide cofactors have on the modulation of recA protein--single-stranded DNA binding affinity and vice versa. In addition, it is demonstrated that nucleotide and DNA binding are necessary though not sufficient conditions for ATPase activity. The significance of this result in terms of the possible requirement of critically sized clusters of 15 or more recA protein molecules contiguously bound to DNA for ATPase activity is discussed.
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PMID:Interaction of recA protein with a photoaffinity analogue of ATP, 8-azido-ATP: determination of nucleotide cofactor binding parameters and of the relationship between ATP binding and ATP hydrolysis. 353 81

Axoplasm extruded from the squid giant axon has been used to analyse the molecular mechanisms of intracellular vesicle transport. Using video-enhanced light microscopy, vesicle transport can be observed directly on individual microtubules at the edge of the axoplasm. Here we report that AMP-PNP (adenyl-5'-yl imidodiphosphate), a non-hydrolysable analogue of ATP, reversibly inhibited vesicle transport. Moreover, vesicles in solution attach to the microtubules and form relatively stable complexes. AMP-PNP may produce this effect by binding to an ATP-binding site on the transport machinery, thereby stabilizing the motility complex that is normally formed by a transported vesicle, an ATPase and a microtubule. The effects of AMP-PNP on the vesicle transport system indicate that the enzymatic machinery of this system differs significantly from that of the actomyosin system or the dynein-microtubule system.
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PMID:Attachment of transported vesicles to microtubules in axoplasm is facilitated by AMP-PNP. 403 61

Five chromatographically distinct DNA-dependent ATPase activities have been identified in high salt-detergent extracts of the Novikoff hepatoma. One of these, ATPase III, has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis and has a specific activity of 12 mumol of ATP hydrolyzed min-1 (mg of protein)-1. The enzyme, a dimer of Mr 65000 subunits, has a sedimentation coefficient of 7.0 S in both high salt and low salt, a Stokes radius of 43 A, and a frictional coefficient of 1.31. In the presence of Mg2+ ion and a polynucleotide effector, the enzyme catalyzes hydrolysis of ATP or dATP to a diphosphate with a Km of 206 microM and 110 microM, respectively, for the two substrates. Although single-stranded effectors are preferred, the enzyme has significant activity with double-stranded effectors. The Km for effector is 0.4 microM (nucleotide). The analogues adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), dideoxyadenosine triphosphate (ddATP), and adenosine 5'-(alpha, beta-methylenetriphosphate) (alpha, beta-Me-ATP) are competitive inhibitors of the enzyme while adenosine tetraphosphate (ATP-P), 8-bromoadenosine 5'-triphosphate (8-Br-ATP), 5'-adenylyl imidodiphosphate (AMP-PNP), and adenosine 5'-(beta, gamma-methylenetriphosphate) (beta, gamma-Me-ATP) do not inhibit. The enzyme is insensitive to nalidixic acid, novobiocin, and berenil but is sensitive to N-ethylmaleimide. ATPase III is capable of stimulating DNA polymerase beta on duplex DNA, but this effect is abolished in the presence of ATP gamma S. Polymerase stimulation is further enhanced in the presence of a single-stranded DNA-binding protein. These data suggest that ATPase III may play a role in DNA repair.
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PMID:Deoxyribonucleic acid dependent adenosinetriphosphatases from the Novikoff hepatoma. Characterization of a homogeneous adenosinetriphosphatase that stimulates DNA polymerase beta. 612 27

In a permeabilized axon model, exogenous ATP can reactivate intraaxonal saltatory organelle movements (microscopically visible manifestations of fast axonal transport). We have studied the dependence of the reactivated movements on the ATP concentration and have also examined the nucleotide specificity of the reactivation. Organelle transport was visualized in isolated lobster giant motor axons using Nomarski optics and video microscopy. The axons were permeabilized with saponin, and movement was reactivated with ATP or other nucleotides. Some slight movement was seen with ATP concentrations as low as 10 microM. The velocity and frequency of the reactivated transport increased with increasing ATP concentrations up to about 5 mM. Movement was also reactivated by deoxyadenosine triphosphate, but not by AMP-PNP (a nonhydrolyzable ATP analogue), ADP, or AMP. Although other nucleotides (CTP, GTP, UTP, ITP) could reactivate transport, movement equivalent to that produced by 0.1 mM ATP was only seen with tenfold or greater concentrations of the other nucleotides. This pattern of specificity is consistent with the hypothesis that a dynein-like ATPase, rather than a myosin, is involved in fast axonal transport.
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PMID:Nucleotide specificity for reactivation of organelle movements in permeabilized axons. 620 49

Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide. 623 23

1. Changes in the intrinsic fluorescence of Na, K-ATPase protein have been used to monitor the interconversion of E(1) (low fluorescence) and E(2) (high fluorescence) forms of the unphosphorylated enzyme.2. In media lacking sodium and nucleotides, 1 mM-potassium was sufficient to convert practically all of the enzyme into the E(2) form. In media containing 1 mM-potassium, 1 mM-EDTA, and no sodium or magnesium, the addition of ATP, or its beta, gamma-imido or methylene analogues, converted the enzyme back into the E(1) form. The relation between nucleotide concentration and the fraction of the enzyme that was in the E(1) form could be described by a rectangular hyperbola, with a K((1/2)) of about 15 muM for ATP, 65 muM for adenylyl-imidodiphosphate (AMP-PNP) and 180 muM for adenylyl (beta, gamma-methylene)-diphosphonate (AMP-PCP). ADP also converted the enzyme back into the E(1) form, with a K((1/2)) of about 25 muM, but the relation between concentration and fraction converted was not well described by a rectangular hyperbola.3. In similar media containing 50 mM-potassium, much higher concentrations of ATP were required to convert the enzyme back into the E(1) form, and the conversion was probably incomplete.4. If we assume that ATP and potassium ions affect each other's binding solely by altering the equilibrium between E(1) and E(2) forms of the enzyme, we are able to conclude (i) that potassium ions bind to the E(1) form with a moderately low affinity, (ii) that, in the absence of nucleotides, the equilibrium between E(1)K and E(2)K is poised strongly in favour of E(2)K, (iii) that the binding of ATP to a low-affinity site alters the equilibrium constant for the interconversion of E(1)K and E(2)K by two to three orders of magnitude, so that, at saturating levels of ATP, the equilibrium is probably slightly in favour of E(1)K, and (iv) that in sodium-free, potassium-containing media, ATP will appear to bind to the enzyme more tightly than would be expected from the dissociation constant of the E(2)K. ATP complex.5. The pattern of the equilibrium constants for the various reactions between E(1), E(2), ATP and potassium is compatible with the hypothesis that the ATP-accelerated conversion of E(2)K into E(1)K, and the subsequent release of potassium ions from low-affinity inward-facing sites, are part of the normal sequence of events during potassium influx in physiological conditions.
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PMID:The equilibrium between different conformations of the unphosphorylated sodium pump: effects of ATP and of potassium ions, and their relevance to potassium transport. 624 81


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