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)

To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Stimulation of human platelet Ca(2+)-ATPase and Ca2+ restoration by calpain. 839 39

Fura-2-loaded platelets were immobilized on fibrinogen, and cytosolic free calcium concentration ([Ca2+]i) was measured by video imaging of the fluorescence signal. In the immobilized, single platelets, ADP and low doses of thrombin evoked repetitive spikes of [Ca2+]i, whereas higher thrombin concentrations gave elevated plateaus in [Ca2+]i. Stimulation of the cells with thrombin after the addition of ADP changed the frequency of spiking, but not the maximal levels of [Ca2+]i reached. In suspensions of platelets, ADP and low doses of thrombin evoked transient elevation of myo-inositol phosphates, suggesting that the initiation of spiking was due to stimulation of phospholipase C. In platelet suspensions, the Ca(2+)-ATPase inhibitor thapsigargin (TG) evoked a gradual rise in [Ca2+]i, which was potentiated by preactivation of the platelets and inhibited by prostaglandin E1 and nitroprusside. In single platelets, TG induced a sudden increase in [Ca2+]i after a long but variable delay, followed by a phase of slow oscillations. The effects of preactivation with ADP were 2-fold: the delay time before the response to TG was shortened and the maximal level of [Ca2+]i reached with TG was higher than the level of the preceding Ca2+ spikes. Apparently, Ca2+ responses induced by the inhibition of Ca(2+)-ATPases are potentiated by prior elevation of [Ca2+]i and reduced by substances that inhibit agonist-evoked increases in [Ca2+]i. The data point to a mechanism of Ca(2+)-induced Ca2+ release and the presence of TG-sensitive and TG-insensitive Ca2+ stores in platelets. Rapid spiking may involve both pools, whereas the latter alone may account for the slow TG-evoked oscillations.
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PMID:Roles of phospholipase C and Ca(2+)-ATPase in calcium responses of single, fibrinogen-bound platelets. 841 43

The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors tert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmidazolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.
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PMID:Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells. 858 30

The N-terminal 28-residue peptide of actin whose Cys10 was labeled with 5-iodoacetamido-fluorescein (F3K peptide) was isolated from the fluorescently labeled thrombin digest of actin. The effect of myosin subfragment 1 (S1) on the fluorescence of F3K peptide was examined in the absence of ATP. With increasing concentration of S1 added, the fluorescence intensity of F3K peptide increased by maximally 7.3% with an apparent dissociation constant of 5.7 microM, suggesting a role of this peptide region of actin in acto-S1 binding in rigor. F3K peptide was crosslinked with S1 at 10 mM NaCl using a zero-length crosslinker by the method of Grabarek and Gergely [Anal. Biochem. 185, 131-135 (1990)]. The crosslinking was greatly inhibited by the presence of either 0.2 M NaCl or 5 mM MgATP. The analyses of amino acid compositions and sequences of the fluorescent peptides isolated from a lysylendopeptidase digest of the crosslinked S1 indicated that F3K peptide was mainly crosslinked to residues 637-642 of the S1 heavy chain. The crosslinked S1 was isolated by selectively pelleting the uncrosslinked S1 with F-actin. ATPase activity of the isolated crosslinked S1 alone was twice as high as that of control S1. The actin-activated ATPase activity of the crosslinked S1 was much lower than that of uncrosslinked S1. The estimated Vm and Km values were 1.72 s-1 and 125 microM, respectively. The Vm decreased to less than 1/8, while Km increased only twofold. The results suggest that the N-terminal 28-residue segment of actin may be implicated in the rigor binding of actomyosin and in the actin-activation of myosin ATPase, but may not be the main determinant of actomyosin binding in the presence of ATP.
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PMID:Crosslinking of a 28-residue N-terminal peptide of actin to myosin subfragment 1. 872 Jan 41

We have related the release of procoagulant microparticles from platelets to calcium movement and the activation of the Ca(2+)-dependent protease calpain. The effects of the Ca(2+)-ATPase inhibitors thapsigargin, cyclopiazonic acid and 2.5-di-(t-butyl)-1,4-benzohydroquinone were compared with those of the Ca2+ ionophore A23187. Whereas all three Ca(2+)-ATPase inhibitors induced aminophospholipid exposure on platelets, only thapsigargin and cyclopiazonic acid promoted microparticle formation and only when strong Ca2+ influx, calpain activation and proteolysis of cytoskeletal proteins occurred concomitantly. Preincubation with dibutylbenzohydroquinone inhibited the responses to thapsigargin and cyclopiazonic acid but not to A23187. When platelets were suspended in a Ca(2+)-free medium, calpain activation and microparticle formation were not observed, even with maximum mobilisation of internal Ca2+ stores by A23187. Incubation of fluo-3-loaded plateters with A23187 in 0.1 mM EGTA followed by the sequential addition of 25 microM Ca2+ increments to the medium showed that calpain activation occurred when the intraplatelet [Ca2+] reached 3-8 microM. To assess the physiologic significance of these results, the subpopulation of platelets that expressed procoagulant activity after stimulation by a thrombin/collagen mixture was isolated by means of annexin-V-coupled magnetic beads. Subsequent western blotting experiments confirmed that this subpopulation contained activated calpain. Overall, our results provide evidence that microparticle formation and calpain activation require an elevated intraplatelet [Ca2+] that is brought about by influx across the plasma membrane.
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PMID:Calcium influx is a determining factor of calpain activation and microparticle formation in platelets. 877 8

A cosegregation analysis of sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase (SERCA) II genotype, systolic blood pressure and platelet intracellular Ca2+ concentration was performed to dissect polygenic hypertensive traits in spontaneously hypertensive rats. Backcross analysis between spontaneously hypertensive rats and normotensive. Donryu rats demonstrated the existence of an inferred single major gene locus (ht). Thrombin-stimulated intraplatelet Ca2+ concentration was significantly higher in the SERCA II homozygotes than in the heterozygotes. The SERCA II genotype did not cosegregate with the blood pressure level. The SERCA II gene was assigned to rat chromosome 12. These results suggest that the SERCA II gene on rat chromosome 12 contributes to increased thrombin-stimulated intraplatelet Ca2+ concentration and that the SERCA II gene is not identical to ht.
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PMID:Genetic linkage of the sarco(endo)plasmic reticulum Ca(2+)-dependent ATPase II gene to intracellular Ca2+ concentration in the spontaneously hypertensive rat. 888 11

The release of the vasoactive peptide endothelin-1 (ET-1) is Ca2+ dependent after thrombin stimulation; however, little is known about the pathways involved. We studied the importance of Ca(2+)-dependent signal transduction pathways on preproET-1 mRNA induction in human endothelial cells. Thrombin-mediated preproET-1 mRNA induction was inhibited after clamping of cytosolic free CA2+ concentration ([Ca2+]i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Chelation of extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid also had a significant inhibitory effect on the induction of preproET-1 mRNA. The Ca2+ ionophore A23187 induced constitutive as well as thrombin-stimulated preproET-1 mRNA expression. Mobilization of Ca2+ stores into the cytosol by inhibition of endoplasmic reticulum Ca(2+)-adenosinetriphosphatase with thapsigargin was effective also in inducing preproET-1 mRNA. Calmodulin antagonists W-7 and calmidazolium, as well as Ca2+/calmodulin-dependent kinase II inhibitor KN-62, significantly reduced thrombin-induced preproET-1 mRNA. Inhibition by cyclosporin A of the Ca(2+)-calmodulin-dependent phosphatase calcineurin potentiated constitutive preproET-1 mRNA. These data suggest that, in human endothelial cells, thrombin-mediated preproET-1 gene induction is regulated by a stimulatory Ca2+/calmodulin kinase II-dependent pathway.
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PMID:Roles of calcium and kinases in regulation of thrombin-stimulated preproendothelin-1 transcription. 894 10

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.
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PMID:Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins. 894 71

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.
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PMID:Purification of porcine phospholamban expressed in Escherichia coli. 895 94

1. Nitric oxide (NO) donors inhibit platelet function and Ca2+ mobilization evoked by different agonists. This led us to investigate the direct effects of authentic NO on basal cytosolic Ca2+ concentration ([Ca2+]i) and on Ca2+ mobilization induced by thrombin or by two inhibitors of intracellular Ca(2+)-ATPases, thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone (t-BuBHQ). 2. Cytosolic Ca2+ concentration was evaluated with Fura-2, in the absence of Ca2+ influx. Addition of 5 microM NO increased by 48% the basal cytosolic [Ca2+] of resting human platelets whereas a lower concentration (0.1 microM) did not induce significant modifications. This NO-induced Ca2+ increase was inversely correlated with the basal level of cytosolic [Ca2+]. 3. NO pretreatment for 30 to 120 s decreased by 42 to 57% the transient [Ca2+]i peak evoked by 0.10 u ml-1 thrombin and strongly attenuated the initial rate of [Ca2+]i rise induced by 1 microM thapsigargin or by 20 microM t-BuBHQ. The two components of the thapsigargin response, the Ca2+ release due to inhibition of Ca2+ pumps and the thromboxane A2-dependent self-amplification mechanism, were inhibited by NO. The observation that a subsequent stimulation was still capable of eliciting Ca2+ release suggests the presence of NO-insensitive Ca2+ stores. 4. These findings indicate that nitric oxide can modulate basal cytosolic [Ca2+] in unstimulated human platelets and decrease the Ca2+ mobilization from NO-sensitive internal stores evoked by stimulation of receptors or by Ca(2+)-ATPase inhibitors. This underlines the important role of nitric oxide in the modulation of platelet Ca2+ handling.
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PMID:Influence of authentic nitric oxide on basal cytosolic [Ca2+] and Ca2+ release from internal stores in human platelets. 896 44


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