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)

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.
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PMID:Kinetic characterization of a monomeric unconventional myosin V construct. 1048 77

Monocytes, separated from human peripheral blood, were preincubated with different polycyclic aromatic hydrocarbons (PAHs) for 24 h and the production of superoxide ions (O*2-) was then measured using as a stimulating agent phorbol 12-myristate 13-acetate. A significantly enhanced O*2- production is only observed when the cells are treated with benzo[a]pyrene (B[a]P); benzo[e]pyrene, benzo[a]anthracene and 3-methylcholanthrene induce a small but not significant increase of O*2-. Anthracene has no effect, while phenanthrene slightly inhibits. The priming activity of B[a]P is unrelated to variations in intracellular Ca2+ ([Ca2+]i), as demonstrated by the inability of B[a]P to increase [Ca2+]i concentration in both monocytes and the promonocytic cell line U937. Furthermore, in monocytes the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase inhibitor, thapsigargin, which can increase [Ca2+]i evokes a differentiation-like event associated with a decrease in the production of superoxide ions. These results further support that the enhancing activity of B[a]P on monocytes superoxide production is not mediated by an increase of [Ca2+]i. In contrast, the role of the aryl hydrocarbon receptor (AhR) in B[a]P-induced superoxide ion enhancement is suggested by the inhibitory effect of the specific antagonist alpha-naphthoflavone (alphaNF), while the tumor necrosis factor (TNF-alpha) is not involved in the phenomenon. Thus, the interaction of B[a]P with its cytosolic receptor and either the metabolism of the compound into reactive intermediates or the over-expression of some unknown genes seem to be involved in an essential step in this process.
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PMID:Priming effect of benzo[a]pyrene on monocyte oxidative metabolism: possible mechanisms. 1059 90

A comparison of sarcoplasmic reticulum (SR) preparations from skeletal muscles of ground squirrels Spermophilus undulatus, rats, and rabbits established that on the basis of protein yield and phospholipid/protein ratio these preparations are practically the same. Nevertheless, the specific activity of Ca-ATPase, the main protein component of SR membranes, in SR preparations of the ground squirrel skeletal muscles is only about half of the activity in SR preparations of rats and rabbits. Significant differences in protein composition of the preparations were detected: ground squirrel SR differed by an unusually high content of a 205 kD protein (probably myosin) and a number of low-molecular-weight SR protein components, and the SR preparations of rabbits are characterized by a high content of the Ca-binding proteins calsequestrin and sarcalumenin. Use of the anionic carbocyanine dye Stains-All established that all preparations contained only three proteins which are stained dark blue by this dye: calsequestrin, sarcalumenin, and a histidine-rich Ca-binding protein. The electrophoretic mobility of calsequestrin was identical in all preparations (molecular mass 63 kD), whereas sarcalumenin and histidine-rich Ca-binding protein are probably present in different isoforms with molecular masses of 130, 145, and 160 and 165, 155, and 170 kD, respectively, in SR preparations of ground squirrels, rats, and rabbits. Analysis of the fluorescence parameters of the fluorescent probes 8-anilino-1-naphthalene sulfonic acid and pyrene bound to SR membranes showed that the properties of the lipid bilayer in the SR membranes of the preparations differed considerably. It is suggested that the differences in protein composition and/or structural state of the ground squirrel SR membrane lipid bilayer could be the reason for the low Ca-ATPase activity in these preparations.
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PMID:Comparative characteristics of sarcoplasmic reticulum preparations from skeletal muscles of the ground squirrel Spermophilus undulatus, rats, and rabbits. 1061 29

Troponin I (TnI) is the component of the troponin complex, TnI, TnC, TnT, that is responsible for inhibition of actomyosin ATPase activity. Using the fluorescence of pyrene-labeled tropomyosin (Tm), we probed the interaction of TnI and TnIC with Tm on the reconstituted muscle thin filament. The results indicate that TnI and TnIC(-Ca(2+)) bind specifically and strongly to actin-Tm with a stoichiometry of 1 TnI or 1 TnIC/1 Tm/7 actin, in agreement with previous results. The binding of myosin heads (S1) to actin-Tm at low levels of saturation caused TnI and TnIC to dissociate from actin-Tm. These results are interpreted in terms of the S1-binding state allosteric-cooperative model of the actin-Tm thin filament, closed/open. Thus, TnI and TnIC(-Ca(2+)) bind to the closed state of actin-Tm and their binding is greatly weakened in the S1-induced open state, indicating that they act as allosteric inhibitors. The fluorescence change and the stoichiometry indicate that the TnI-binding site is composed of regions from both actin and Tm probably in the vicinity of Cys 190.
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PMID:Binding of troponin I and the troponin I-troponin C complex to actin-tropomyosin. Dissociation by myosin subfragment 1. 1065 59

Force and motion generation by actomyosin involves the cyclic formation and transition between weakly and strongly bound complexes of these proteins. Actin's N-terminus is believed to play a greater role in the formation of the weakly bound actomyosin states than in the formation of the strongly bound actomyosin states. It has been the goal of this project to determine whether the interaction of actin's N-terminus with myosin changes upon transition between these two states. To this end, a yeast actin mutant, Cys-1, was constructed by the insertion of a cysteine residue at actin's N-terminus and replacement of the C-terminal cysteine with alanine. The N-terminal cysteine was labeled stoichiometrically with pyrene maleimide, and the properties of the modified mutant actin were examined prior to spectroscopic measurements. Among these properties, actin polymerization, strong S1 binding, and the activation of S1 ATPase by pyrenyl-Cys-1 actin were not significantly different from those of wild-type yeast actin, while small changes were observed in the weak S1 binding and the in vitro motility of actin filaments. Fluorescence changes upon binding of S1 to pyrenyl-Cys-1 actin were measured for the strongly (with or without ADP) and weakly (with ATP and ATPgammaS) bound acto-S1 states. The fluorescence increased in each case, but the increase was greater (by about 75%) in the presence of MgATP and MgATPgammaS than in the rigor state. This demonstrates a transition at the S1 contact with actin's N-terminus between the weakly and strongly bound states, and implies either a closer proximity of the pyrene probe on Cys-1 to structural elements on S1 (most likely the loop of residues 626-647) or greater S1-induced changes at the N-terminus of actin in the weakly bound acto-S1 states.
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PMID:Structural transition at actin's N-terminus in the actomyosin cross-bridge cycle. 1067 29

Polycyclic aromatic hydrocarbons are environmental pollutants known to be carcinogenic and immunotoxic. In intact cell assays, benzo[a]pyrene (B[a]P) disrupts Ca(2+) homeostasis in both immune and nonimmune cells, but the molecular mechanism is undefined. In this study, B[a]P and five metabolites are examined for their ability to alter Ca(2+) transport across microsomal membranes. Using a well-defined model system, junctional SR vesicles from skeletal muscle, we show that a single o-quinone metabolite of B[a]P, B[a]P-7,8-dione, can account for altered Ca(2+) transport across microsomal membranes. B[a]P-7,8-dione induces net Ca(2+) release from actively loaded vesicles in a dose-, time-, and Ca(2+)-dependent manner. In the presence of 5 microM extravesicular Ca(2+), B[a]P-7,8-dione exhibited threshold and EC(50) values of 0.4 and 2 microM, respectively, and a maximal release rate of 2 micromol of Ca(2+) min(-1) mg(-1). The mechanism by which B[a]P-7,8-dione enhanced Ca(2+) efflux was further investigated by measuring macroscopic fluxes and single RyR1 channels reconstituted in bilayer lipid membranes and direct measurements of SERCA catalytic activity. B[a]P-7,8-dione (< or = 20 microM) had no measurable effect on initial rates of Ca(2+) accumulation in the presence of ruthenium red to block ryanodine receptor (RyR1), nor did it alter Ca(2+)-dependent (thapsigargin-sensitive) ATPase activity. B[a]P-7,8-dione selectively altered the function of RyR1 in a time-dependent diphasic manner, first activating then inhibiting channel activity. Considering that RyR1 and its two alternate isoforms are broadly expressed in mammalian cells and their important role in Ca(2+)-signaling, the present results reveal a mechanism by which metabolic bioactivation of B[a]P may mediate RyR dysfunction of pathophysiological significance.
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PMID:A bioactive metabolite of benzo[a]pyrene, benzo[a]pyrene-7,8-dione, selectively alters microsomal Ca2+ transport and ryanodine receptor function. 1117 46

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.
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PMID:Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions. 1127 30

Gold-lined sea bream (Rhabdosargus sarba) were intraperitoneally injected with 35 mg/kg benzo[a]pyrene (in DMSO), with fish administered DMSO as the solvent control. Fish were sacrificed 3 days post injection and their livers dissected for the analysis of adenosine triphosphates (ATPase), glutathione-S-transferase (GST), DNA adducts and ethyoxyresorufin-O-deethylase (EROD). Exposure to B[a]P resulted in reduced ATPase activity, elevated GST activity and DNA adduct level, but no apparent change in EROD activity.
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PMID:Biomarker studies on gold-lined sea bream (Rhabdosargus sarba) exposed to benzo[a]pyrene. 1138 Jan 74

Brain myosin-Va consists of two heavy chains, each containing a neck domain with six tandem IQ motifs that bind four to five calmodulins and one to two essential light chains. Previous studies demonstrated that myosin-Va exhibits an unusually high affinity for F-actin in the presence of ATP and that its MgATPase activity is stimulated by micromolar Ca(2+) in a highly cooperative manner. We demonstrate here that Ca(2+) also induces myosin-Va binding to and cosedimentation with F-actin in the presence of ATP in a similar cooperative manner and calcium concentration range as that observed for the ATPase activity. Neither hydrolysis of ATP nor buildup of ADP was required for Ca(2+)-induced cosedimentation. The Ca(2+)-induced binding was inhibited by low temperature or by 0.6 m NaCl, but not by 1% Triton X-100. Tight binding between myosin-Va and pyrene-labeled F-actin in the presence of ATP and Ca(2+) was also detected by quenching of the pyrene fluorescence. Negatively stained preparations of actomyosin-Va under Ca(2+)-induced binding conditions showed tightly packed F-actin bundles cross-linked by myosin-Va. Our data demonstrate that high affinity binding of myosin-Va and F-actin in the presence of ATP or 5'-O-(thiotriphosphate) is induced by micromolar concentrations of Ca(2+). Since Ca(2+) regulates both the actin binding properties and actin-activated ATPase of myosin-Va over the same concentration range, we suggest that the calcium signal may regulate the mechanism of processivity of myosin Va.
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PMID:High affinity binding of brain myosin-Va to F-actin induced by calcium in the presence of ATP. 1151 16

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is approximately 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with approximately 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still approximately 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased approximately 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased approximately 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric-phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.
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PMID:Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus. 1156 64


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