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)

The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the "light" 30S dynein ATPase fractions are more activated than the "heavy" 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.
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PMID:Specific anion effects on ATPase activity, calmodulin sensitivity, and solubilization of dynein ATPases. 623 71

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10-fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++-dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++-dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++-dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.
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PMID:Calmodulin confers calcium sensitivity on ciliary dynein ATPase. 644 62

Protein carboxylmethylase (S-adenosyl-L-methionine:protein O-methyltransferase, EC 2.1.1.24.) is believed to be involved in the regulation of sperm motility. To test this hypothesis, we investigated the effects of erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA) which, in combination with adenosine and homocysteine thiolactone, inhibits protein carboxylmethylase activity in monocytes. This group of compounds inhibited sea urchin sperm motility. Unexpectedly, EHNA alone inhibited the motility., This observation was confirmed in intact spermatozoa from rats, rabbits, and humans. EHNA also inhibited the motility of demembranated, reactivated sea urchin and rat spermatozoa from which protein carboxylmethylase had been extracted. In these preparations, motility was restored by ATP. These observations suggested that EHNA arrests sperm motility by inhibiting the axonemal dynein ATPase on which motility depends. Kinetic analysis demonstrated that EHNA produced mixed inhibition of both the axonemal ATPase and the partially purified dynein 1 from sea urchin sperm tails, as well as the axonemal ATPase of rat sperm tails. These observations also provide evidence for the similarity of the active site of the dynein ATPase in sea urchin and rat spermatozoa.
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PMID:erythro-9-[3-(2-Hydroxynonyl)]adenine is an inhibitor of sperm motility that blocks dynein ATPase and protein carboxylmethylase activities. 645 42

The action of carnitine in regulating fowl sperm motility was investigated. As the concentration of L-carnitine was increased (0-20 mM), the motility of intact and demembranated fowl spermatozoa was reduced at 30 degrees C. Even the presence of 1 mM CaCl2 before the addition of 10 mM carnitine could not prevent the inhibition of motility at 30 degrees C and 40 degrees C. However, motility was restored by reducing the concentrations of carnitine. Carnitine also inhibited the oxygen consumption and ATP concentrations of intact spermatozoa, and caused a reduction in intracellular free Ca2+ concentrations. Phosphorylation of a 50 kDa protein and dephosphorylation of 24 kDa and 30 kDa proteins of demembranated spermatozoa were observed after the addition of carnitine. In contrast, the flagellar ATPase activity of crude dynein extract was not affected by the addition of carnitine. These results suggest that inhibitory effect of carnitine for motility may be directly on the axonemal phosphoproteins, but not directly on the dynein ATPase activity. The physiological role of carnitine for fowl spermatozoa in the ductus deferens is discussed.
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PMID:Inhibition of flagellar motility of fowl spermatozoa by L-carnitine: its relationship with respiration and phosphorylation of axonemal proteins. 791 83

Fowl sperm motility was measured by altering the extracellular pH (pHe) at 30 degrees C and 40 degrees C. At 30 degrees C, the motility of intact spermatozoa was vigorous in a medium in the wide pHe range of 7.3-10.1. In contrast, intact spermatozoa were almost immotile at 40 degrees C in medium below a pHe of 8.1. However, the motility could be restored by increasing the pHe; maximum motility was obtained in medium at pHe 9.4. Stimulation of the motility of demembranated spermatozoa at 40 degrees C was also observed with an increased pHe. However, demembranated spermatozoa at 40 degrees C that had been stimulated by increasing the pHe lost their motility when 1 mmol CaCl2 l-1 was added. Motility was restored by the subsequent addition of 2 mmol EGTA l-1. At a high pHe at 40 degrees C, the flagellar ATPase activity of crude dynein extract was not affected, regardless of the addition of CaCl2 or EGTA. The intracellular pH (pHi) of intact spermatozoa, estimated by measuring the accumulation of 9-aminoacridine fluorescence, increased with increasing pHe at both 30 degrees C and 40 degrees C. These results demonstrate that the reversible temperature-dependent immobilization of fowl spermatozoa at 40 degrees C is inhibited by an increased pHi. Furthermore, it is possible that the effects of the increased pHi may not act directly on dynein ATPase activity, but are mediated by a Ca(2+)-related substance(s) on the axoneme.
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PMID:Inhibition of temperature-dependent immobilization of fowl spermatozoa at body temperature by an increased intracellular pH. 796 13

The treatment of bull spermatozoa from epididymal cauda with 5 micrograms digitonin per microliters cells led to the destruction of the plasma membrane which was shown by electron microscopy. Structural changes of mitochondria and of the axoneme were not found. The functional intactness was maintained. The plasma membranes of hypotonically treated spermatozoa were permeable for succinate and the mitochondria showed a diminished functional intactness. For the total ATPase activity of digitonin-treated cells, mainly representing the dynein ATPase, a maximal activity of 20.3 nmol ATP x min-1 x microliters cells-1 (mean +/- S.D.; n = 8) and an apparent half saturation constant of 0.29 mM ATP were determined. The portions of Na(+)-K(+)-ATPase and mitochondrial ATPase in the total ATP-splitting activity were found to be less than 15%. In comparison to the capacity of mitochondrial ATP production, the maximal activity of ATP consuming reactions amounted to about 70%. These results support findings that the ATP turnover of spermatozoa is more limited by the ATP consumption than by the ATP production.
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PMID:[Morphologic characteristics and ATPase activity in permeabilized epididymal bull sperm]. 833 8

The binding of nucleoside triphosphates and related ligands to dynein ATPase from sea urchin sperm flagella has been studied by equilibrium partition analysis in an aqueous biphasic system containing dextran and poly(ethylene glycol). The stoichiometry of binding and the corresponding stepwise binding constants are obtained from direct binding isotherms fitted to the primary data. The results suggest that dynein possesses four different binding sites for nucleoside triphosphates per mole of heavy chain. The stepwise binding constants for MgATP range from approximately 10(4) M-1 to approximately 10(5) M-1. The isolated alpha and beta heavy chains have binding parameters similar to intact dynein. The amount of ADP bound normally is approximately 75% that of ATP, both for the intact dynein and for the separated heavy chains, although full saturation is achieved at high nucleotide concentrations. In the presence of the ATPase inhibitor vanadate, ADP binds with affinities similar to those of ATP, with binding constants close to those of ATP in the absence of vanadate. No appreciable binding of AMP or EDTA/ATP is observed. The substitution of Ca2+ or Fe3+ for Mg2+ does not significantly alter the amount of ATP bound; however, CaATP is bound with a somewhat lower affinity. Scatchard and Hill plots of the binding data and the calculated site-binding constants suggest that ATP and ADP bind in a weakly cooperative manner. These results suggest that the multiple binding of nucleotide to dynein heavy chains occurs at physiological concentrations, putatively at the four binding sites predicted earlier on the basis of their amino acid sequences. The data are consistent with a model in which, in addition to a single catalytic site, nucleotide binding occurs at additional noncatalytic sites that represent an as yet unknown functional aspect of dynein.
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PMID:Phase partition analysis of nucleotide binding to axonemal dynein. 870 26

The interdependence between motility, respiration, ATP production, and utilization was investigated in intact spermatozoa of turbot (Psetta maxima), a marine teleost. When spermatozoa were diluted in a hyperosmotic medium (>300 mOsmol/kg), they immediately became motile, and the intracellular concentration of ATP as well as the adenylate energy charge ratio dropped concomitant with the straight-line velocity. The ADP and AMP levels increased from 1.4 to 8.0 nmole/10(8) cells and from 0.6 to 6.0 nmole/10(8) cells, respectively. Moreover, 31P-NMR spectra recorded prior to the swimming phase revealed the presence of phosphomonoesters (PMEs) and phosphodiesters (PDEs), intracellular inorganic phosphate (Pi), and phosphocreatine (PCr). At the end of the motility period, PCr, PDE, and PME decreased, while the Pi level increased markedly. Following initiation of motility, O2 consumption of spermatozoa increased from 34.9 to 124.8 O2 nmole/10(9) spermatozoa/min. FCCP, an uncoupler of oxidative phosphorylation, did not significantly affect the respiratory rate of motile spermatozoa. Ouabain, a specific inhibitor of (Na+/K+)/ATPase, slightly decreased the respiration rate of motile spermatozoa, indicating that the major part of ATP catabolism was linked to dynein ATPase. Inhibitors of the respiratory chain (KCN, NaN3, NaHCO3-, oligomycin) reduced sperm respiration, percentage of motile cells, velocity, and adenylate contents. Following the reactivation of motility of demembranated spermatozoa, KCN, NaN3, NaHCO3- altered the flagellar beat frequency, demonstrating that these respiratory inhibitors possess action sites other than mitochondria. Mitochondrial oxidative phosphorylation is highly requested to produce energy required during motion. Nevertheless it is insufficient to maintain endogenous ATP stores. A second phase of motility was induced by a transfer of exhausted spermatozoa into an ionic medium of low osmolality (200 mOsmol/kg) for 30 min. Spermatozoa, once reactivated in AM, recovered 55% of initial motility and 31% of initial fertilization rate. In hypo-osmotic medium, mitochondrial oxidative phosphorylation also induced ATP regeneration. Following activation of movement, several morphological changes were observed in the mitochondria and the midpiece.
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PMID:Nucleotide content, oxidative phosphorylation, morphology, and fertilizing capacity of turbot (Psetta maxima) spermatozoa during the motility period. 1033 61

bis-cyclopentadienyl [Cp] complexes of vanadium(IV) or vanadocenes are rapid and potent inhibitors of human sperm motility with potential as a new class of contraceptive agents. We investigated the utility of boar sperm as a model system to study the mechanisms of drug action because boar sperm lacks phosphocreatine and creatine kinase activity, the essential components of the "phosphagen shuttle" system for human sperm motility. Two representative vanadocenes, vanadocene dichloride [VDC] and bis[pentamethylcyclopentadienyl] vanadium dichloride [VPMDC], in which the bis-Cp rings were substituted with five electron-donating methyl groups were evaluated. The concentration-dependent effects of VDC and VPMDC on spermicidal activity, axonemal dynein adenosine triphosphatase (ATPase) activity, and tyrosine phosphorylation of global sperm proteins were assessed by computer-assisted sperm analysis, spectrophotometry, and immunoblotting, respectively. Both the unsubstituted and the pentamethyl-substituted vanadocene induced rapid sperm immobilization (T(1/2) < 15 s). Substitution of the bis-Cp rings by five methyl groups augmented the SIA of VDC threefold. The EC(50) values for VDC and VPMDC were 2.1 and 0.76 microM, respectively. Spermicidal activity of vanadocenes was not associated with the inhibition of dynein ATPase(s) or increase in tyrosine phosphorylation of sperm proteins. These results suggest that the potent spermicidal activity of vanadocenes against boar sperm is mediated by a unique mechanism that is independent of dynein ATPase activity, phosphatase activity, and phosphocreatine/creatine kinase system. Therefore, boar sperm is a suitable model for further investigating the molecular mechanism of spermicidal action of vanadocenes.
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PMID:Evaluation of boar sperm as a model system to study the mechanism of spermicidal activity of vanadocenes. 1077 10

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.
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PMID:Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa. 1086 6


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