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)

Monoclonal antibodies that react with defined regions of the heavy and light chains of chicken skeletal muscle myosin have been used to provide a correlation between the primary and the tertiary structures of the head. Electron microscopy of rotary shadowed antibody-myosin complexes shows that the sites for three epitopes in the 25,000 Mr tryptic fragment (25k) of subfragment-1, including one within 4000 Mr of the amino terminus of the myosin heavy chain, are clustered 145(+/- 20) A from the head-rod junction. An epitope in the 50,000 Mr fragment maps even further out on the head. These antibodies bind to the head in several orientations, suggesting that each of the heads can rotate can rotate 180 degrees about the head-rod junction. The epitopes are accessible on subfragment-1 bound to actin when they were probed with Fab fragments; therefore, none of these heavy chain sites is is on the contact surface between the head and actin. Two of the anti-25k antibodies affect the K+-EDTA-and Ca2+-ATPase activities of myosin in a manner that mimics the effect on activity of the modification of the reactive thiol, SH-1. These two antibodies also inhibit the actin-activated ATPase non-competitively with respect to actin. None of the other eight antibodies tested had any marked effect on activity. A monoclonal antibody that reacts with an epitope in the amino-terminal third of myosin light chain 2 maps close to the head-rod junction. A polyclonal antibody specific for the amino terminus of light chain 3 binds further up in the "neck region" of the head, indicating that these portions of the two classes of light chains are located at different sites.
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PMID:Probing myosin head structure with monoclonal antibodies. 294

The Ca2+-sensitive ATPase activity of rabbit skeletal myofibrils disappeared completely after treatment with a solution containing CDTA, a strong divalent cation chelator, at a low ionic strength. A gel electrophoretic study revealed that all troponin C and about half of myosin light chain 2 were removed from the myofibrils by the CDTA treatment. The CDTA-treated myofibrils, when reconstituted with skeletal troponin C, showed almost exactly the same Ca2+- or Sr2+-sensitive ATPase activity as that of intact myofibrils. The CDTA-treated myofibrils reconstituted with porcine cardiac troponin C showed the same Ca2+- or Sr2+-sensitivity of the ATPase as that of porcine cardiac myofibrils; Sr2+-sensitivity relative to Ca2+-sensitivity was about ten times higher than, and the maximal slope of the activation curve was about half that of skeletal myofibrils. These findings indicate that these characteristic features of divalent cation regulation in the contraction of skeletal and cardiac muscles are determined solely by the species of troponin C. Bovine brain calmodulin hardly activated the ATPase activity of the CDTA-treated myofibrils even in the presence of Ca2+. Excess calmodulin, however, was found to give Ca2+- or Sr2+-sensitivity to the ATPase activity of the CDTA-treated myofibrils. Frog skeletal parvalbumins 1 and 2, even in excess, did not affect the ATPase activity of the CDTA-treated myofibrils.
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PMID:Ca2+- and Sr2+-sensitivity of the ATPase activity of rabbit skeletal myofibrils: effect of the complete substitution of troponin C with cardiac troponin C, calmodulin, and parvalbumins. 295 10

Interchain disulfide crosslinks between the heavy-chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5'-dithiobis(2-nitrobenzoic acid (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2-nitro-5-thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2-modified light chain 2 by 32 - 39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2-modified light chain 2 attaches to the heavy chain in the region of the 21-kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/subfragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793-799]. Modification of thiol-1 groups with iodoacetamide as well as crosslinking the thiol-1 and thiol-2 groups by the bifunctional reagent p-N,N'-phenylenedimaleimide prior to incubation with Nbs2-modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2-modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin-activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2-modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2-modified light chain 2 to light-chain-2-deficient heavy meromyosin restored the original light-chain-2-dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light 2.
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PMID:Crosslinking by thiol disulfide interchange of 5,5'-dithiobis(2-nitrobenzoic acid)-treated light chain and heavy chain of rabbit skeletal myosin. 675 73

We investigated the rate of tension development (kappa td) after photolytical release of ATP from P3-1-(2-nitrophenyl)-ethyladenosine-5'-triphosphate ('caged ATP') of atrial and ventricular fibre bundles from pig. Contraction was initiated from high-tension (HT) and low-tension (LT) rigor at maximal Ca2+ activation (pCa 4.5). The kappa td of atrial fibre bundles was 6.8 s-1 from LT and 6.9 s-1 from HT rigor. Rate of tension development of ventricular fibre bundles was significantly lower (P < 0.001) being 1.06 s-1 and 0.94 s-1 from LT and HT rigor, respectively. The kappa td of skinned ventricular fibre bundles incubated in a high [K+], low [Ca2+] (cardioplegic) solution prior to the skinning procedure decreased significantly (P < 0.05) to 0.73 s-1 and 0.63 s-1 from LT and HT rigor, respectively, whereas that of skinned atrial fibre bundles remained at 7.1 s-1 and 6.9 s-1 from LT and HT rigor, respectively. Phosphorylation levels of the myosin light chain 2 isoform in the atrial fibre bundles (ALC-2) was 15.6 +/- 2.7%. The corresponding values for the two ventricular isoforms, VLC-2 and VLC-2*, were 31.2 +/- 0.4% and 25.1 +/- 2.1%, respectively. Phosphorylation levels of fibre bundles incubated in cardioplegic solution prior to skinning were 11.6%, 18.9%, and 15.4% of the ALC-2, VLC-2 and VLC-2*, respectively. The results show that the rate of tension development is more than seven-fold higher in the atrial compared with ventricular fibre bundles. These results correlate with the differences in ATPase activity of the contractile proteins in solution and, most likely, reflect differences in the myosin isoform composition. In ventricular fibre bundles the increased levels of light chain phosphorylation were associated with increased rate of contraction.
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PMID:Rate of active tension development from rigor in skinned atrial and ventricular cardiac fibres from swine following photolytic release of ATP from caged ATP. 757 32

Partial inhibition of cardiac Na/K-ATPase by digitalis drugs such as ouabain is the initial event leading to positive inotropy in the heart. We showed recently that exposure of rat cardiac myocytes to ouabain concentrations that produce positive inotropy, but no overt toxicity, caused inductions of some early response genes and hypertrophy of these myocytes. The aim of this work was to determine if ouabain also affects the expressions of certain late response genes that are regulated by other hypertrophic stimuli. Non-toxic concentrations of ouabain (5-100 microM) increased mRNAs of skeletal alpha-actin, atrial natriuretic factor, myosin light chain 2, and transforming growth factor beta: indicating that ouabain's effects on these marker genes are similar to those of hypertrophic stimuli that mimic the effects of pressure overload. Expression of skeletal alpha-actin was more sensitive to ouabain than that of atrial natriuretic factor, suggesting significant differences in the ouabain-specific pathways of the induction of these fetal genes. The effects of ouabain on skeletal alpha-actin gene were transcriptional, and required an increase in net influx of extracellular Ca2+. Protein kinase C and Ca(2+)-calmodulin kinases, but not protein kinase A, were involved in the signal pathways leading to the induction of skeletal alpha-actin gene. These data and our prior findings indicate that an increase in net influx of Ca2+ through partial inhibition of Na/K-ATPase initiates protein kinase-dependent pathways resulting in alterations in cardiac growth and expressions of both early and late response genes.
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PMID:Ouabain-induced hypertrophy in cultured cardiac myocytes is accompanied by changes in expression of several late response genes. 914 Aug 3

We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.
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PMID:Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation. 932 36

Calcium regulation in the human heart is impaired during idiopathic dilated cardiomyopathy (IDC). Here, we analyze the structural basis for impairment in the regulatory mechanism. Regulation of contractility was monitored by MgATPase and Ca2+-binding assays as a function of calcium. Myofibrillar proteolysis and expression of troponin T isoforms were established by gel electrophoresis and by Western blots. Myofibrillar ATPase assays in low salt however, revealed a drastic lowering of calcium sensitivity in IDC myofibrils as indicated by reductions in both activation by high calcium and in EGTA-mediated inhibition of MgATPase. Structural changes in myofilament proteins were found in most IDC hearts, specifically proteolysis of myosin light chain 2 (LC2), troponin T and I (TnT and TnI), and sometimes a large isoform shift in TnT. IDC did not induce mutations in LC2 and troponin C (TnC), as established by cDNA sequence data from IDC cases, thus, calcium binding to IDC myofibrils was unaffected. Reassociation of IDC myofibrils with native LC2 raised MgATPase activation at high Ca2+ to control levels, while repletion with intact, canine TnI/TnT restored inhibition at low Ca2+. A model, identifying possible steps in the steric blocking mechanism of regulation, is proposed to explain IDC-induced changes in Ca2+-regulation. Moreover, shifts in TnT isoforms may imply either a genetic or a compensatory factor in the development and pathogenesis of some forms of IDC.
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PMID:Calcium regulation in the human myocardium affected by dilated cardiomyopathy: a structural basis for impaired Ca2+-sensitivity. 1039 Nov 53

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.
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PMID:The calcineurin-NFAT pathway and muscle fiber-type gene expression. 1100 71

Evidence suggests that p38 mitogen-activated protein kinase (MAPK) activation influences cardiac function on an acute basis. The characterization and mechanisms by which this occurs were investigated in the present study. Adult rat ventricular myocytes treated with 1 mM arsenite for 30 min had a 16-fold increase in p38 MAPK phosphorylation that was attenuated by SB-203580 (a p38 MAPK inhibitor). Extracellular signal-regulated protein kinase (ERK) and c-Jun NH2-terminal kinase (JNK) were also minimally activated, but this activation was not sensitive to SB-203580. In addition, arsenite caused a p38 MAPK-independent translocation/activation of protein phosphatase 2a (PP2a) and decrease in phosphorylation of myosin light chain 2 (LC2). Arsenite-p38 MAPK activation led to translocation of heat shock protein 27 but not alpha B-crystallin to the myofilaments. Using isolated cardiomyocytes, we determined that arsenite reduces isometric tension without a change in Ca2+ sensitivity of tension via p38 MAPK and lowers myofibrillar actomyosin Mg2+-ATPase activity in a p38 MAPK-independent manner. Thus arsenite induces a p38 MAPK-independent change in PP2a and LC2 that may account for the arsenite-dependent decrease in ATPase and a p38 MAPK-dependent modification of the myofilaments that decreases myocardial force development.
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PMID:Acute p38 MAPK activation decreases force development in ventricular myocytes. 1288 Dec 12

We tested the hypothesis that activation of Rho-A-dependent kinase (ROCK-II) alters cardiac myofilament response to Ca2+ by mechanisms involving phosphorylation of thin filament proteins. We determined effects of a constitutively active form of ROCK-II on ATPase activity and tension development in detergent-extracted (skinned) fiber bundles isolated from mouse left ventricular papillary muscles. ROCK-II induced a depression in maximum ATPase rate and tension, which was associated with phosphorylation of troponin T (TnT), troponin I (TnI), and myosin-binding protein C (C-protein). This effect of ROCK-II was retained in fiber bundles isolated from transgenic (TG) mice in which phosphorylation sites (S14, S15, and S19) of myosin light chain 2 were mutated to alanine. Moreover, exchange of ROCK-II-phosphorylated Tn complex with the native Tn complex in the fiber bundles resulted in inhibition of maximal Ca2+ activation of tension and ATPase activity. Mass spectrometric analysis demonstrated that ROCK-II phosphorylated cardiac TnI (cTnI) at S23, S24, and T144 and cardiac TnT (cTnT) at S278 and T287. An important role for these cTnT sites is indicated by results demonstrating that ROCK-II induced a depression in tension and ATPase activity in skinned fiber bundles from a TG model in which cTnI is replaced by slow skeletal TnI, which lacks S23 and S24 and in which T144 is replaced by proline. Our data provide the first evidence that ROCK-II phosphorylation of the Tn complex, most likely at cTnT, has an important role in functional effects of signaling through the Rho-A pathway.
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PMID:Functional effects of rho-kinase-dependent phosphorylation of specific sites on cardiac troponin. 1577 59


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