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)

Inhibitory action of vanadate (orthovanadate and metavanadate) on ciliary dynein adenosinetriphosphatase (ATPase) from Tetrahymena was investigated. The apparent concentrations of vanadate giving half-maximal inhibition of Mg-ATPase activity of various dynein fractions were as follows: the axoneme-bound form of dynein at 100 nM, solubilized crude dynein at 50 nM, 14S dynein at 5 microM, and 30S dynein at 20 nM. The Ca-ATPase of 30S dynein was more than 30-fold less sensitive than its Mg-ATPase, and still less sensitive was the Ca-ATPase of 14S dynein. The Mg-ATPase of 30S dynein was most sensitive to vanadate at neutral pH, and the addition of KCl or NaCl into the assay mixture reduced its sensitivity. Varying the assay temperature between 0 and 37 degrees C affected the sensitivity to a slight extent. Metavanadate was as much a potent inhibitor of dynein ATPase as orthovanadate, but vanadium pentoxide was less potent. When the dynein ATPase activity was reciprocally plotted against the concentration of vanadate (the Dixon plot), the inhibition was proved to be biphasic. At lower concentrations of vanadate, the inhibition was more significant. Therefore the Dixon plot had a downward bent. Reexamination of the Lineweaver-Burk plot of 30S dynein Mg-ATP showed a downward bent, which indicates that 30S dynein may have at least two Km values, ca. 1 microM and 3 microM; or otherwise, 30S dynein might possibly have a negatively cooperative nature (Hill coefficient 0.67). The vanadate-induced inhibition of 30S dynein Mg-ATPase was noncompetitive in the entire range of ATP concentration examined. Since the vanadate-induced inhibition of 30S dynein Mg-ATPase could be classified into "tight-binding inhibition", we could estimate the dissociation constant of vanadate and the molecular weight per enzymatic active site according to the kinetics of tight-binding inhibition with several assumptions. Thus, the dissociation constant was 10-15 nM, depending on the ATPase assay condition, while the molecular weight per enzymatic active site was 420 000-480 000, independent of the assay condition with the assumption that the present 30S dynein preparation is totally pure. This value would be reduced about 20% when the purity was taken into consideration.
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PMID:Steady-state kinetic study of vanadate-induced inhibition of ciliary dynein adenosinetriphosphatase activity from Tetrahymena. 611 1

Hisanaga and Sakai [Hisanaga, S., & Sakai, H. (1983) J. Biochem. (Tokyo) 93, 87-98] demonstrated that cytoplasmic dynein could be purified, in part, by chromatography on a calmodulin-Sepharose 4B affinity column and that the adenosinetriphosphatase (ATPase) activity of the enzyme was stimulated by Ca2+-calmodulin. In the present study, we have investigated, in detail, the interaction of cytoplasmic and flagellar dynein from the sea urchin Hemicentrotus pulcherrimus with calmodulin (CaM) isolated from porcine brain or sea urchin egg. The dynein Mg2+-ATPase activity is stimulated 3-8-fold by calmodulin from either source. The stimulation is dependent on calcium ions and is inhibited by trifluoroperazine. CaM stimulation is sensitive to physiologically regulatory calcium ion concentrations around 1 microM. Activation is also sensitive to pH and occurs maximally at physiological pH near 7.0. Calmodulin binds directly to cytoplasmic dynein as judged by cosedimentation in a sucrose density gradient. The binding and enzymatic stimulation occur at calmodulin:dynein ratios of 150:1 to 300:1, which are consistent with estimates of in vivo ratios. Cytoplasmic and flagellar dynein ATPase activities are also stimulated by Triton X-100, a nonionic detergent, and by limited protolysis with trypsin. Both of these treatments abolish further activation by calmodulin. The possibility of a trypsin-labile, CaM binding subunit of the enzyme is discussed. In addition, since both CaM and dynein are localized in the mitotic apparatus, we suggest that CaM may regulate possible mitotic dynein activity.
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PMID:Calmodulin interaction with cytoplasmic and flagellar dynein: calcium-dependent binding and stimulation of adenosinetriphosphatase activity. 614 57

Demembranated cilia of Tetrahymena pyriformis were extracted with KCl or Tris-EDTA and the crude dyneins from each resolved by sucrose density gradient sedimentation into 14S-K, 30S-K, 14S-E and 30S-E dyneins, respectively. The calmodulin activation ratio (ATPase activity in presence of added calmodulin/ATPase activity in absence of added calmodulin) did not vary across the 30S dynein fractions regardless of the method of extraction nor did it vary across the 14S-E region. However, in going from the "heavy" fractions to the "light" fractions of the 14S-K region, it increased markedly. The concentration of calmodulin required for half-maximal activation did not differ appreciably in the "light" versus the "heavy" fractions of the 14S-K region, suggesting that the affinity for calmodulin does not vary in these fractions. SDS-polyacrylamide gel electrophoresis studies showed the presence of several polypeptides that varied in a systematic fashion across the 14S-K region and hence may be involved in regulating the sensitivity of 14S-K dynein ATPase activity to added calmodulin.
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PMID:Heterogeneity of 14S and 30S dynein ATPase activities with respect to activation by calmodulin. 621 63

ATPase of 14S dynein, extracted from spermatozoa of the sea urchin, Hemicentrotus pulcherrimus, and partially purified by sucrose density gradient centrifugation, was inhibited non-competitively by palmitoyl CoA at concentrations higher than 20 microns, and was stimulated at concentrations between 2 microns and 10 microns. The effects of palmitoyl CoA on dynein ATPase were reversed by bovine serum albumin (1 mg/ml) and spermine (0.1 and 1 mM). Myristoyl CoA exerted effects similar to those of palmitoyl CoA. Short chain fatty acyl CoAs, such as butyryl CoA, propionyl CoA and acetyl CoA, CoA, Na-palmitate, Na-myristate, and palmitoyl carnitine had hardly any effect on dynein ATPase. Palmitoyl CoA failed to inhibit purified CF1 ATPase from chloroplasts of spinach, ATPase of rat liver mitochondria and alkaline phosphatase from calf intestine.
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PMID:Inhibition by palmitoyl CoA of dynein ATPase from sea urchin spermatozoa. 621

We recently demonstrated that elevated concentrations (greater than 20 microM) of the dynein substrate MgATP2- inhibit the spontaneous ATP-induced sliding disintegration of isolated, Triton-demembranated Tetrahymena cilia. We have used a turbidimetric assay (delta A350 nm) and electron microscopy to examine the effect of ATP on sliding disintegration when activated by other divalent cations. Mg2+, Ca2+, and Mn2+ are each capable of activating sliding, but only with Mg2+ and Mn2+ is disintegration inhibited by elevated ATP concentrations (greater than or equal to 1 mM). The two major ATPase activities obtained by KCl extraction of Tetrahymena axonemes differ in their cation specificities such that Mg2+ and Ca2+ activate the 21S dynein ATPase with equal efficiency, whereas the 13S axonemal ATPase activity is reduced by approximately 50% when CaATP2- replaces MgATP2- as substrate. With 1 mM MgATP2- as substrate, 10(-7) to 10(-2) M added CaCl2 alleviates the ATP-dependent inhibition of disintegration and likewise represses 13S MgATPase activity. In contrast, free Ca2+ has no effect on either the disintegration response or Mg-ATPase activity. In contrast to Triton-treated cilia, glycerinated cilia, which beat in 1 mM MgATP2-, are inhibited from beating by high CaATP2- concentrations. These substrate specificities suggest that concentration-dependent, substrate inhibition of sliding disintegration may be a manifestation of a physiological mechanism that is mediated by the 13S axonemal ATPase and that may function to modulate sliding during bend formation. However, the effects of added CaCl2 probably do not reflect a physiological mechanism for regulating beat parameters, but rather may result from CaATP2- competing for MgATP2- binding sites on the 13S ATPase, thereby blocking expression of the 13S ATPase.
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PMID:Evidence for a role of 13S axonemal ATPase in modulation of ciliary microtubule sliding. 622 Aug 5

Extraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77-92% of the total axonemal Mg++ or Ca++-ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of approximately 2.0 +/- 0.5 mumoles Pi hydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of approximately 310,000-340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800-19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of approximately 0.6 +/- 0.3 mumoles Pi hydrolyzed min/mg and associated with approximately 330,000-, 21,700-, and 18,100-dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, one HMW polypeptide and one 28,700-dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents approximately 9.2% of the total axonemal protein; 12S dynein represents approximately 4.7% of the axonemal protein.
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PMID:Purification and polypeptide composition of dynein ATPases from Chlamydomonas flagella. 622 Aug 6

Mitochondria of sperm of the sea urchin Strongylo-centrotus purpuratus are tightly coupled before induction of the acrosomal reaction. When the sperm are diluted at low external pH (pH 5.5) or in high potassium (200 mM) or in the absence of sodium, their internal pH is acidic (6.2-7.0) as measured by amine accumulation. Under these conditions the internal ATPase activity (primarily the dynein ATPase) is inhibited, sperm are immotile, and mitochondria are in respiratory state 4 (ATP concentration is maximal). When the internal pH is alkalinized, the internal ATPase activity is increased, as estimated either in vivo by measuring the decrease in ATP concentration after addition of oligomycin to prevent ATP synthesis, or in vitro using Triton X-100 permeabilized cells. This increase in ATPase activity correlates with an increase of up to 50-fold in respiratory rates and a mitochondrial transition to state 3. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone-uncoupled respiration is also sensitive to the internal pH for it is inhibited at acidic internal pH. However, since nonmotile, nonrespiring sperm that are obtained when the internal pH is acidic have high concentrations of ATP, we conclude that in vivo the internal pH controls the rate of dynein ATPase and that this ATPase activity is limiting for the respiration of tightly coupled mitochondria. The redox state of the respiratory chain may also be under the direct influence of the internal pH.
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PMID:Metabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration. 622 53

21S Dynein ATPase [EC 3.6.1.3] from axonemes of a Japanese sea urchin, Pseudocentrotus depressus, and its subunit fractions were studied to determine their kinetic properties in the steady state, using [gamma-32P]ATP at various concentrations, 5 mM divalent cations, and 20 mM imidazole at pH 7.0 and 0 degrees C. The following results were obtained. 1. 21S Dynein had a latent ATPase activity of about 0.63 mumol Pi/(mg . min) in 1 mM ATP, 100 mM KCl, 4 mM MgSO4, 0.5 mM EDTA, and 30 mM Tris-HCl at pH 8.0 and 25 degrees C. Its exposure to 0.1% Triton X-100 for 5 min at 25 degrees C induced an increase in the ATPase activity to about 3.75 mumol Pi/(mg . min) and treatment at 40 degrees C for 5 min also induced a similar activation. 2. The double-reciprocal plot for the ATPase activity of dynein activated by the treatment at 40 degrees C consisted of two straight lines, while that of nonactivated 21S dynein fitted a single straight line. 3. In low ionic strength solution, the Mg- and Mn-ATPase of 21S dynein showed substrate inhibition at ATP concentrations above 0.1 mM; the inhibition decreased with increasing ionic strength. Ca- and Sr-ATPase showed no substrate inhibition. 4. Both the Vmax and Km values of dynein ATPase decreased reversibly upon addition of about 40% (v/v) glycerol. In the presence of glycerol, the dynein ATPase showed an initial burst of Pi liberation. The apparent Pi-burst size was 1.0 mol/(10(6) g protein) and the true size was calculated to be 1.6 mol/1,250 K after correcting for the effect of Pi liberation in the steady state and the purity of our preparation. 5. One of the subunit fractions of 21S dynein which was obtained by the method of Tang et al. showed substrate inhibition and an initial burst of Pi liberation of 1.4 mol/(10(6) g protein) in the presence of 54% (v/v) glycerol.
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PMID:Reaction mechanism of 21S dynein ATPase from sea urchin sperm. I. Kinetic properties in the steady state. 622 78

The amounts of ATP and ADP bound to 21S dynein during the ATPase reaction were measured in the presence of 2.83 mg/ml 21S dynein, 2 mM PEP, 4 mg/ml PK, 0.1 M KCl, 5 mM MgCl2, 1 mM DTT, 0.1 mM PMSF, 50% [2-3H]glycerol, and 20 mM imidazole at pH 7.0 and 0 degrees C. The maximum amounts of ATP and ADP bound to 21S dynein were 0.29 and 0.55 mol/(10(6) g protein), respectively. The dissociation constants of ATP for the ATP and ADP binding (4 microM) were almost equal to the Km value (3.7 microM) of dynein-ATPase in the steady state. The amount of bound ADP during the initial phase showed an overshoot, which reached 0.6-0.8 mol/10(6) g protein at 5 s, then decreased to the steady state level within 20 s. Furthermore, the rate of TCA-Pi liberation during the initial 5 s was 6 times the steady-state rate. The apparent Pi-burst size, estimated by extrapolating the steady-state Pi liberation to zero time, was 1.33 mol/(10(6) g protein). The true Pi-burst size was calculated to be 1.56 mol/(10(6) g protein) by correcting for the effect of Pi liberation at steady state. All these findings could be explained quantitatively by the following reaction scheme for 21S dynein ATPase in the presence of glycerol: (formula; see text) where K1 = 25.5 microM, and k2, k3, and k4 were 0.39, 0.21, and 0.11 s-1, respectively.
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PMID:Reaction mechanism of 21S dynein ATPase from sea urchin sperm. II. Formation of reaction intermediates. 622 79

Cilia from the protozoan Tetrahymena pyriformis were demembranated and then extracted for 5 min with a buffer containing 0.5 M NaCl. The briefly extracted axonemal pellet was then reextracted for about 20 hr. The soluble material obtained from each extraction was resolved into 14S and 30S dynein ATPases by sedimentation on sucrose density gradients and tested for sensitivity to added calmodulin. The 14S dynein obtained by a 5-min extraction was generally insensitive to added calmodulin, whereas that obtained by 20-hr extraction of the 5-min extracted axonemes was activated by calmodulin, the activation being much larger in the "light" 14S fractions than in the "heavy" fractions. The 30S dynein ATPase obtained by a 5-min extraction was generally activated over 1.6-fold by added calmodulin, whereas that obtained by the subsequent long extraction was usually activated only 1.3-fold. After further purification of the 5-min extracted 30S dynein and of the 5-min to 20-hr-extracted 14S dynein on DEAE-Sephacel, these dyneins retained much of their calmodulin activatability. The ATPase activity of both 14S and 30S dyneins was inhibited more strongly by erythro-9-[3-(2-hydroxynonyl)] adenine and by vanadate in the presence of added calmodulin than in its absence. These data suggest that the only ATPase activity present in the fractions studied is that of the dyneins and demonstrate that both the 14S and 30S dynein ATPases may be obtained in forms that are activated by added calmodulin as well as in forms that are insensitive to added calmodulin.
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PMID:Effect of extraction time on ability of calmodulin to activate 30S and 14S dynein ATPases. 623 29


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