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

Ca2+ is a powerful inhibitor (Ki is congruent to 16 muM) of basal and prostaglandin E1 (PGE1)-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] activity in membranes obtained from homogenized human platelets. Ca2+ (but not the ionophore A23,187) decreased V(max) of the reaction without an effect on the Ks for ATP. Neither ATP nor PGE1 affected Ki for Ca2+. In intact platelets A23,187 induced Ca2+ influx and markedly inhibited PGE1-stimulated rise in adenosine 3':5'-cyclic monophosphate (cAMP) levels. Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] activity was mainly found in the soluble fraction (greater than 90%). Both soluble and membrane bound enzymes were stimulated by Mn2+ and Ca2+ and inhibited by Zn2+. Adenylate and guanylate cyclase activity were both present in a membrane fraction cyclase activity were both present in a membrane fraction which contained Ca2+ activated ATPase activity, and accumulated Ca2+ from the medium in the presence of ATP and oxalate. Other evidence indicates that these membranes originated in large part from the dense tubular system of the platelets. It is proposed that concurrent inhibition of adenylate cyclase and stimulation of guanylate cyclase facilitates the direct initiating effect of Ca2+ on platelet secretion and aggregation.
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PMID:Interrelationships between Ca2+ and adenylate and guanylate cyclases in the control of platelet secretion and aggregation. 0 60

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.
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PMID:Purification and properties of ATPase inhibitor from rat liver mitochondria. 0 95

The purple membrane of Halobacterium halobium acts as a light-driven proton pump, ejecting protons from the cell interior into the medium and generating electrochemical proton gradient across the cell membrane. However, the type response of cells to light as measured with a pH electrode in the medium consists of an initial net inflow of protons which subsides and is then replaced by a net outflow which exponentially approaches a new lower steady state pH level. When the light turned off a small transient acidification occurs before the pH returns to the original dark level. We present experiments suggesting that the initial inflow of protons is triggered by the beginning ejection of protons through the purple membrane and that the initial inflow rate is larger than the continuing light-driven outflow. When the initial inflow has decreased exponentially to a small value, the outflow dominates and causes the net acidification of the medium. The initial inflow is apparently driven by a pre-existing electrochemical gradient across the membrane, which the cells can maintain for extended times in the absence of light and oxygen. Treatments which collapse this gradient such as addition of small concentrations of uncouplers abolish the initial inflow. The triggered inflow occurs through the ATPase and is accompanied by ATP synthesis. Inhibitors of the ATPase such as N,N'-dicyclohexylcarbodiimide (DCCD) inhibit ATP synthesis and abolish the inflow. They also abolish the transient light-off acidification, which is apparently caused by a short burst of ATP hydrolysis before the enzyme is blocked by its endogenous inhibitor. Similar transient inflows and outflows of protons are also observed when anaerobic cells are exposed to short oxygen pulses.
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PMID:Light-driven proton translocations in Halobacterium halobium. 0 22

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca2+- and Mg2+-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. Km values of Ca2+ and Mg2+ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca2+- or Mg2+-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca2+ or Mg2+ or from two different enzymes are discussed.
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PMID:Membrane-bound ATPase in chloroplasts of Euglena gracilis. 0 24

Reconstituted actomyosin (ATP phosphohydrolase, EC 3.6.1.3) (0.400 mg F-actin/mg myosin) in 10.0 muM ATP loses 96% of its specific ATPase activity when its reaction concentration is decreased from 42.0 mug/ml down to 0.700 mug/ml. The loss of specific activity at the very low enzyme concentrations is prevented by the addition of more F-actin to 17.6 mug/ml. It is concluded that at low actomyosin concentrations the complex dissociates into free myosin with a very low specific ATPase activity and free F-actin with no ATPase. The dissociation of the essential low molecular weight subunits of myosin from the heavy chains at very low actomyosin concentrations may be a contributing factor. Actomyosin has its maximum specific activity at pH 7.8-8.2. The Km for ATP is 9.4 muM, which is at least 20-fold greater than myosin's Km for ATP. The actin-activated ATPase of myosin follows hyperbolic kinetics with varying F-actin concentrations. The Km values for F-actin are 0.110 muM (4.95 mug/ml) at pH 7.4 and 0.241 muM (10.8 mug/ml) at pH 7.8. The actin-activated maximum turnover numbers for myosin are 9.3 s-1 at pH 7.4 and 11.6 s-1 at pH 7.8. The actomyosin ATPase is inhibited by KCl. This KCl inhibition is not competitive with respect to F-actin, and it is not a simple form of non-competitive inhibition.
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PMID:Steady-state studies of the actin-activated adenosine triphosphatase activity of myosin. 0 39

The mechanism of biosynthetic, transferase, ATPase, and transphosphorylation reactions catalyzed by unadenylylated glutamine synthetase from E. coli was studied. Activation complex(es) involved in the biosynthetic reaction are produced in the presence of either Mg2+ or Mn2+ ; however, with the Mn2+-enzyme inhibition by the product, ADP, is so great that the overall forward biosynthetic reaction cannot be detected with the known assay methods. Binding studies show that substrates (except for NH3 and NH2OH which are not reported here) can bind to the enzyme in a random manner and that binding of the ATP-glutamate, ADP-Pi or ADP-arsenate pairs is strongly synergistic. Inhibition and binding studies show that the same binding site is utilized for glutamate and glutamine in biosynthetic and transferase reactions, respectively, and that a common nucleotide binding site is used for all reactions studied. Studies of the reverse biosynthetic reaction and results of fluorescent titration experiments suggest that both arsenate and orthophosphate bind at a site which overlaps the gamma-phosphate site of nucleoside triphosphate. In the reverse biosynthetic and transferase reactions, ATP serves as a substrate for the Mn2+-enzyme but not for the Mg2+-enzyme. The ATP supported transferase activity of Mn2+-enzyme is probably facilitated by the generation of ADP through ATP hydrolysis. When AMP was the only nucleotide substrate added, it was converted to ATP with concomitant formation of two equivalents of glutamate, under the reverse biosynthetic reaction conditions, and no ADP was detected. The reversibility of 180 transfer between orthophosphate and gamma-acyl group of glutamate was confirmed. ATPase activity of Mg2+ and Mn2+ unadenylylated enzymes is about the same. Both enzymes forms catalyze transphosphorylation reactions between various purine nucleoside triphosphates and nucleoside diphosphates under biosynthetic reaction conditions. The data are consistent with the hypothesis that a single active center is utilized for all reactions studied. Two stepwise mecanisms that could explain the results are discussed.
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PMID:Mechanistic studies of glutamine synthetase from Escherichia coli. An integrated mechanism for biosynthesis, transferase, ATPase reaction. 0 53

The kinetic study of the C2+ ATPase activity of lymphocyte plasma memebranes allowed some properties of this enzyme to be evidenced. The Ca2+-activated hydrolysis of ATP is independent of a non-specific alkaline phosphatase. The substrate of the ATPase activity is the chelate Ca2+- ATP. Mg2+ may substitute for Ca2+ both as chelating ion and as activating ion. Several results suggest that we have only one ATPase, activated either by Ca2+-, or by Mg2+ with less efficiency; both chelates hve the same Km; pH values for maximum activity and transition temperatures are identical; the effects of free ions are also the same, activation at low concentration and inhibition at high concentration.
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PMID:[Kinetics of Ca 2+ or Mg 2+ activated ATPase from lymphocyte plasma membranes]. 0 56

Cardiac myosin from thyrotoxic animals (myosin-T) exhibits elevated Ca2+ -ATPase activity which is resistant to further stimulation by sulfhydryl modification. In the present study, we have compared the enzymatic properties of myosin-T with those of myosin from euthyroid rabbits (myosin-N) and the derivatives of myosin-T and myosin-N formed by blocking the most rapidly reacting class of thiols (SH1) with N-ethylmaleimide (NEM). Vmax for Ca2+ -ATPase of myosin-T was about 250% greater than myosin-N and was nearly the same as NEM-modified myosin-N. Values for the apparent Km of myosin-T and NEM-modified myosin-N were 200% greater than the value for unmodified myosin-N. Vmax and Km for K+ (EDTA)-ATPase activity of NEM-modified myosin-T and myosin-N were identical. The Ca2+ saturation, pH, and salt-dependency curves for the ATPase activity of myosin-T were parallel to the curves for myosin-N and differed from those for the NEM-modified myosins. Myosin-T exhibited an increased rate of hydrolysis of ATP, CTP, and UTP in both low (0.05m) and high (0.5m) KCl medium. NEM-modified myosin-N showed increased hydrolysis of ATP and CTP in low KCl medium and increased hydrolysis of ATP, CTP, and UTP in high KCl medium. These results support the hypothesis that the enzymatic behavior of myosin-T may be caused by an alteration in the active site near the SH, thiols. The unique enzymatic properties of myosin-T did not seem to be the result of a major change in structure. The electrophoretic pattern of light chains from myosin-T and myosin-N was the same in polyacrylamide gels containing either 8 M urea at pH 8.6 or sodium dodecyl sulfate. Also, myosin-T had a normal amino acid composition and lacked 3-methyl-histidine and hot acid-stable phosphate.
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PMID:Enzymatic properties of native and N-ethylmaleimide-modified cardiac myosin from normal and thyrotoxic rabbits. 0 19

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.
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PMID:Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium. 0 39


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