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)

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.
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PMID:The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. 12 31

When stoichiometric amounts of tropomyosin (TM) are bound to F-actin in the presence of 2 mM ATP, the MG2+-activated acto-heavy meromyosin (HMM) ATPase is inhibited by about 60% in 5 mM MgCl2-30 mM KCl. If the concentration of MgCl2 is reduced to 1 mM, the inhibition disappears because TM no longer binds to F-actin. Increasing the concentration of KCl to 100 mM restores both the binding and the inhibition. Thus, the binding of TM alone to F-actin causes significant inhibition of the ATPase provided that the HMM is saturated with ATP. (When the HMM is not saturated, TM activates the ATPase). When TM alone can bind stoichiometrically to F-actin, addition of troponin I (TN-I) increases the inhibition from 60% to about 85%, but the TM binding to F-actin is not affected. Under conditions such that TM alone neither inhibits the acto-HMM ATPase nor binds to F-actin, the inhibition caused by TN-I plus TM still approaches 100%. Direct binding studies under these conditions show that TN-I induces binding between TM and F-actin. A dual role for TN-I is proposed: first, TN-I can induce TM to bind to F-actin, causing inhibition of the ATPase; and second, TN-I can itself enhance the inhibition of the ATPase in a cooperative manner. The addition of TN-C in the absence of CA2+ has only a limited effect on the first role, but seems to be able to block completely the cooperative inhibition caused by TN-I such that the residual inhibition is a function only of the TM which remains bound.
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PMID:Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C. 12

1. Conditions for binding of [gamma-32P]ATP to bovine brain Na+,K+-stimulated ATPase were investigated by the indirect technique of measuring the initial rate of 32P-labelling of the active site of the enzyme. 2. At 100 muM [gamma-32P]ATP in the presence of 3 mM MgCl2, approximately the same very high rate of formation of [32P]phosphoenzyme was obtained irrespective of whether [gamma-32P]ATP was added to the enzyme simultaneously with, or 70 ms in advance of the addition of NaCl. A comparatively slow rate of phosphorylation was obtained at 5 muM[gamma-32P]ATP without preincubation. However, on preincubation of the enzyme with 5 muM[gamma-32P]ATP a rate of formation of [32P]phosphoenzyme almost as rapid as at 100 muM[gamma-32P]ATP was observed. 3. A transient [32P]phosphoenzyme was discovered. It appeared in the presence of K+, under conditions which allowed extensive binding of [gamma-32P]-ATP. The amount of [gamma-32P]ATP that could be bound to the enzyme seemed to equal the amount of [32P] phosphorylatable sites. 4. The formation of the transient [32P] phosphoenzyme was inhibited by ADP. The transient [32P] phosphoenzyme was concluded mainly to represent the K+-insensitive and ADP-sensitive E1-32P. 5. When KCl was present in the enzyme solution before the addition of NaCl only a comparatively slow rate of phosphorylation was observed. On preincubation of the enzyme with [gamma-32]ATP an increase in the rate of formation of [32P] phosphoenzyme was obtained, but there was no transient [32P]-phosphoenzyme. The transient [32P]phosphoenzyme was, however, detected when the enzyme solution contained NaCl in addition to KCl and the phosphorylation was started by the addition of [gamma-32P]ATP.
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PMID:Bovine brain Na+, K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Detection of a transient [32P]phosphoenzyme formed in the presence of potassium ions. 12 4

Two reaction intermediates of H-meromyosin (HMM) ATPase [EC 3.6.1.3], E2AT32P, and (see article), were formed by mixing excess HMM with AT32P. Then a large excess of unlabelled ATP was added, and the amount of AT32P liberated from E2AT32P was measured as the difference between the total amount of AT32P in the reaction mixture and the amount of AT32P bound to HMM, obtained by filtering the mixture after adding charcoal to adsorb nucleotides (charcoal-filtration method). The amount of free AT32P was also measured as the amount of glucose-6-32P formed within 15 sec after adding large excesses of hexokinase [EC 2.7.1.1] and glucose to the reaction mixture. The rate constant, k-2, for the step E2ATP yields E plus ATP was calculated at various KCl concentrations from the time-course of liberation of AT32P. The intermediate, (see article), was formed by mixing HMM with AT32P in a molar ratio of 1:2, and the rate constant, k-6, for the step (see article) was also determined by the same procedures used for k-2. In 0.5 M KCl and 2 mM MgCl2 at pH 7.8 and 0 degrees, k-2 and k-6 were 0.002 sec-1 and 0.1 sec-1 or more, respectively. From the rate constants determined in this work and the rate and equilibrium constants which we reported previously, the standard free energy changes (kcal/mole) for formation of various reaction intermediates in the reaction of HMM ATPase in 0.5 M KCl and 2 mM MgCl2 at pH 7.8 and 0 degrees were calculated to be as follows: (see article).
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PMID:Standard free energy changes for formation of various intermediates in the reaction of H-meromyosin ATPase. 12 76

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.
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PMID:A study of the binding of adenosine diphosphate to myosin subfragment-1. 12 50

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.
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PMID:The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction. 12 89

A comparative study was carried out in the properties of ATPase system of the skeletal muscle nuclei in the rabbits in norm and with experimental muscular dystrophy conditioned by E-avitaminosis. It is shown that in the system, containing 1.5 mM of MgCl2, ATPase system of the nuclei is activated by sodium and potassium ions. In norm maximum activation is observed with their presence in the medium, the concentration being 80 and 70 mM, respectively. With experimental muscular dystrophy maximum activating concentrations decrease and are equal for both cations - 30 mM. Activation of the enzymatic system by these ions is specific because the introduction of equimolar quantities of cholin-chloride or lithium, cesium ions instead of sodium ions into the incubation medium evokes no activation of the ATPase system of the rabbit skeletal muscles both in norm and with experimental muscular dystrophy. A simultaneous presence of sodium and potassium ions in optimum concentrations in the incubation medium makes for an increase of ATPase activity to the same extent as the presence of one of these cations. Oubain, a specific inhibitor of Mg2+, Na+, K+- ATPase, taken in the concentrations of 10(-4) and 10(-3) M did not decrease the intensity of ATP hydrolysis and its activation conditioned by the presence of sodium or potassium. A conclusion is made that Mg2+, Na+, K+-ATPase taking part in the work of "sodium pump" is absent in the nuclei of skeletal muscles.
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PMID:[Availability of Mg2+, Na+, and K+-ATPase in the nuclei of the skeletal muscles of rabbits normally and during experimental muscular dystrophy]. 12 69

Using a rapid method of preparation, spectrin has been isolated from human erythrocytes and its ATPase activity investigated. The ATPase activity with calcium has two distinct components, one with optimal activity when calcium and ATP are of equal concentration (low-Ca-ATPase) and another which is activated above 1 mM CaCl2 and is maximal at 100 mM CaCl2. There is also a Mg-ATPase with maximal activity at 10 mM MgCl2. The high-Ca-ATPase of spectrin, but not the low-Ca-ATPase, is inhibited by magnesium, while the Mg-ATPase is inhibited by Ca in excess of ATP. None of these activities exhibits the calcium-stimulated magnesium-dependent activity characteristic of the red cell calcium pump.
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PMID:Calcium and magnesium ATPases of the spectrin fraction of human erythrocytes. 12 59

Na+-K+-Dependent ATPase [EC 3.6.1.3] was preincubated with ATP in the presence of a high concentration of MgCl2, and the phosphorylated intermediate, EP, was formed by adding a high concentration of NaCl. The following results showed that EP was converted from an ADP-sensitive to an ADP-insensitive form by a single turnover of the ATPase reaction. 1. After initiating the reaction by adding NaCl, almost all the EP was at first sensitive to added ADP, but its sensitivity to ADP decreased with increase in the time interval between the additions of NaCl and of ADP. 2. Both in the presence and absence of KCl, the time course of the replacement of ADP-sensitive EP by ADP-insensitive EP coincided with the time course of the decomposition of EP after addition of EDTA.
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PMID:The pre-steady state of Na+-K+-dependent ATPase after addition of Na+ ions. Transition of the phosphorylated intermediate from an ADP-sensitive to an ADP-insensitive form. 12 69

Cytochalasin B stimulated polymerization and decreased the concentration of G-actin remaining in equilibrium with F-actin filaments. Polymerization in the presence of cytochalasin B gave rise to a smaller increase of viscosity but to the same increase in light scattering, compared to polymerization in the absence of cytochalasin B. Cytochalasin B reduced the viscosity of F-actin and caused the appearance of ATP hydrolysis by F-actin. The cytochalasin B-induced ATPase activity was inhibited by concentrations of KCl higher than 50 mM. The cytochalasin B-induced ATPase activity was enhanced by ethyleneglycol bis(alpha-aminoethyl ether)-N,N'-tetraacetic acid and reduced by MgCl2 at concentrations higher than 0.75 mM. The findings suggest that the stability of actin filaments is reduced by cytochalasin B.
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PMID:Effect of cytochalasin B on formation and properties of muscle F-actin. 13 90


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