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)

alpha 1-Adrenergic agonists have negative inotropic effects on mammalian myocardium under some conditions, and biochemical experiments measuring the Ca(2+)-activated actomyosin ATPase activity of myofibrillar preparations suggest that this may result from a decrease in cross-bridge cycling rate caused by phosphorylation of myofilament proteins. Experiments with intact ventricular preparations, however, have failed to demonstrate a mechanical manifestation of a decrease in cycling rate. The present study examined the effect of alpha 1-adrenergic receptor stimulation on maximum shortening velocity in skinned single ventricular myocytes from rats. Enzymatically isolated myocytes were incubated with the beta-receptor antagonist propranolol in the presence or absence of the alpha 1-adrenergic receptor agonist phenylephrine and were then rapidly skinned to preserve the phosphorylation state of myofilament proteins. The velocity of unloaded shortening (Vo) was determined by use of the slack-test method and compared between skinned control and phenylephrine-treated cells. The relationship between isometric tension and [Ca2+] was also assessed for each myocyte. Vo was significantly lower in the alpha 1-adrenergic receptor agonist-treated cells than in the control cells, but there was no effect on Ca2+ sensitivity of isometric tension. In addition, the myosin heavy chain isoform composition accounted for a significant amount of the variation in Vo within the treatment groups. On the basis of these and previous results we propose that alpha 1-adrenergic receptor stimulation inhibits cross-bridge cycling rate at the level of myofilament proteins by a mechanism that may involve phosphorylation of troponin I by protein kinase C.
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PMID:Alpha 1-adrenergic receptor stimulation decreases maximum shortening velocity of skinned single ventricular myocytes from rats. 778 69

Troponin I is a myofibrillar protein involved in the Ca(2+)-mediated regulation of actomyosin ATPase activity. We report here the isolation and characterization of the gene coding for the slow-muscle-specific isoform of the rat troponin I polypeptide (TpnI). Using restriction mapping, PCR mapping and partial DNA sequencing, we have determined the exon/intron arrangement of this gene. The transcription unit is 10.5-kb long and contains nine exons ranging in size from 4 bp to 330 bp. The rat TpnI(slow) gene is interrupted by large intervening sequences; a 3.3-kb intron separates the 5' untranslated exons from the protein-coding exons. Comparison of the structure of rat TpnI(slow) with that of quail TpnI(fast) reveals that they have a similar intron/exon organization. The 5' untranslated region of the rat gene contains an additional exon, otherwise, the positions of introns and coding exons map to essentially identical regions in both genes.
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PMID:Isolation and structure of the rat gene encoding troponin I(slow). 805 38

The plant phenol tannin stimulated severalfold the Ca(2+)-dependent ATPase and Ca(2+)-uptake activities of dog cardiac sarcoplasmic reticulum (SR) with an EC50 value of 0.6 microM. The stimulation was due to a marked increase in the apparent affinity of the cardiac SR ATPase for Ca2+ ions while the Vmax was not affected. No stimulation of skeletal muscle SR preparations could be observed. The characteristics of stimulation were similar to those observed after phosphorylation of the regulatory protein phospholamban (PLN) by protein kinase A. The ability of protein kinase A to phosphorylate PLN was prevented by tannin with an IC50 of 3 microM. Phosphorylation of troponin I, another physiological substrate of protein kinase A, was resistant to tannin inhibition. The data show that submicromolar concentrations of tannin prevent PLN phosphorylation by interacting with the cytosolic portion of PLN. The specific binding of tannin reverses the inhibition that PLN exerts on cardiac SR ATPase.
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PMID:Reversal of phospholamban-induced inhibition of cardiac sarcoplasmic reticulum Ca(2+)-ATPase by tannin. 806 Mar 55

The myocardium is a highly adaptive tissue, as evidenced by phenotypic alterations throughout development and under conditions of altered hemodynamic load. With pressure overload, the myocardium displays adult-to-fetal transitions in expression of contractile and non-contractile proteins. Most intriguing is the fact that many of these transitions are also observed in the senescent heart. The purpose of this work was to establish if the thin filament regulatory proteins, troponin I and troponin T, exhibit reexpression of early developmental isoforms, suggestive of coordinate reprogramming of contractile protein isoform expression. As a functional index of reexpression of the early isoform of troponin I, slow skeletal troponin I, myofibrils were isolated from 12 and 24-month-old Fischer 344 rat ventricles and assayed for myofibrillar ATPase activity at pH 7.0 and 6.5. Both preparations displayed rightward shifts in Ca-ATPase relationships with no differences between groups. SDS-PAGE and Western blot analysis showed that whereas myosin heavy chain expression underwent a transition to predominance of the early development isoform, beta-myosin heavy chain, there was no reexpression of the fetal isoforms of either troponin I or troponin T in the rat heart at 24 months of age. Northern blot analysis using cDNA probes specific for cardiac or slow skeletal troponin I also confirmed the lack of slow skeletal reexpression in the 24-month ventricle. These results are significant in that they demonstrate a lack of coordinate expression of contractile protein isoforms under myocardial adaptation to the aging process.
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PMID:Discoordinate regulation of contractile protein gene expression in the senescent rat myocardium. 807 7

Calcium binding to regulatory sites located in the NH2-terminal domain of troponin C (TnC) induces a conformational change that blocks the inhibitory action of troponin I (TnI) and triggers muscle contraction. We used deletion mutants of TnI in conjunction with a series of TnC mutants to understand the structural and functional relationship between different TnI regions and TnC domains. Our results indicate that TnI is organized into structural and regulatory regions which interact in an antiparallel fashion with the corresponding structural and regulatory regions of TnC. Functional studies show that the COOH-terminal region of TnI, when linked to the inhibitory region (TnI103-182) can regulate actomyosin ATPase. A TnI lacking the first 57 amino acids (TnId57) has been shown to have similar properties (Sheng, Z., Pan, B.-S., Miller, T. E., and Potter, J. D. (1992) J. Biol. Chem. 267, 25407-25413). Regulation was not observed with the COOH-terminal region alone (TnI120-182), with the NH2-terminal region alone (TnI1-98), or with the NH2-terminal linked to the inhibitory region (TnI1-116). Binding studies show that the NH2-terminal region of TnI interacts with the COOH-terminal domain of TnC in the presence of Ca2+ or Mg2+ and that the inhibitory plus COOH-terminal region of TnI (TnI103-182) interacts with the NH2-terminal domain of TnC in a Ca(2+)-dependent manner. Based on these results we propose a model for the Ca(2+)-induced conformational change. In our model the NH2-terminal domain of TnI is anchored strongly to the COOH-terminal domain of TnC in the absence and presence of Ca2+ while the inhibitory and COOH-terminal regions of TnI switch between actin-tropomyosin in the absence of Ca2+ to binding sites in both NH2- and COOH-terminal domains of TnC in the presence of Ca2+.
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PMID:Structural and regulatory functions of the NH2- and COOH-terminal regions of skeletal muscle troponin I. 810 6

The effects of Ca2+, Mg2+, and troponin I (TnI) inhibitory peptide (Ip) binding on the spectral properties of a Phe-154 to Trp mutant (F154W) of chicken recombinant troponin C (rTnC) have been examined. Residue 154 is positioned in the final flanking helix H of metal binding site IV. Since there are no other Tyr or Trp residues in the protein, spectral properties can be unambiguously assigned. No significant differences in the far UV CD spectra of rTnC and F154W were observed in either the absence or presence of Ca2+. When reconstituted into whole Tn the ATPase specific activities (+/- Ca2+) of the troponin-tropomyosin-actomyosin subfragment 1 system were the same for both proteins. A 2-fold reduction in Ca2+ affinity of C domain sites III/IV but not of N domain sites I/II in isolated F154W is explicable in terms of the environment of residue 154 in the relatively disordered apo-C domain and its buried position in the known ordered 2Ca2+ crystal structure. Filling of sites III/IV by divalent cations was accompanied by a number of spectral changes which were different for Ca2+ and Mg2+. Binding of Ip peptides (residues 96-116 and 104-115(116)) elicited fluorescence emission spectral alterations in the presence of Ca2+. These were not observed in its absence nor in the presence of Mg2+ even though binding occurs under these conditions. Since Ca2+ affinity to C domain but not to N domain sites was increased by Ip at the low concentrations of protein and Ip tested, Ip binding appears to be stronger with C domain.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ca2+, Mg2+, and troponin I inhibitory peptide binding to a Phe-154 to Trp mutant of chicken skeletal muscle troponin C. 813 Feb 10

Troponin C has a 14-residue alpha-helix at the extreme amino terminus (the N-helix) which is absent in calmodulin. To learn the significance of this region in troponin C, residues 1-14 were deleted using site-directed mutagenesis. Analysis of the mutant troponin C (delta 14-TnC) showed that deletion of the N-helix did not alter the secondary structure of troponin C. Like wild type troponin C, it exhibited Ca(2+)-dependent conformational changes based on electrophoretic mobility and increases in alpha-helix content. The thermal stability of delta 14-TnC, however, was 20 degrees C lower than wild type troponin C in the presence or absence of divalent cations because of destabilization of the amino-terminal domain. To determine the functional consequences of the deletion, its ability to relieve troponin I and IT inhibition of the actomyosin ATPase was assayed. The results show that the mutant could relieve troponin I inhibition in the presence and absence of Ca2+ but could relieve troponin IT inhibition only to 45-50% of the wild type level, even at high concentrations. Also, the calcium affinity of the low affinity sites is reduced as evidence by the 2.4-2.8-fold increase in Ca2+ concentration required to achieve half-maximal activation of the MgATPase and calcium titration of the metal-induced conformation monitored by far UV circular dichroism measurements. In addition, the N-helix is required for the full conformational change to take place upon the binding of Ca2+, but not Mg2+, to the high affinity sites. The results indicate that the N-helix of troponin C is important for the stability of troponin C and may play a vital role in the Ca(2+)-switching mechanism.
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PMID:The effects of deletion of the amino-terminal helix on troponin C function and stability. 814 78

The effect of partial removal of troponin I and C on the profiles of Ca(2+)-sensitive ATPase activity in rabbit skeletal myofibrils was investigated by replacing the troponin C.I.T-complex in the myofibrils with exogenously added troponin T under the same conditions as those reported previously [Shiraishi et al. (1992) J. Biochem. 111, 61-65]. During the course of the troponin T treatment, the level of the ATP hydrolysis at low Ca2+ concentrations was elevated and the pCa for half maximum activation increased, while the cooperativity decreased. These changes in the parameters of the ATPase were correlated with the extent of the troponin I removal from myofibrils.
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PMID:The effect of partial removal of troponin I and C on the Ca(2+)-sensitive ATPase activity of rabbit skeletal myofibrils. 818 28

To assess the structural and functional significance of the N helix (residues 3-13) of avian recombinant troponin C (rTnC), we have constructed NHdel, in which residues 1-11 have been deleted, both in rTnC and in the spectral probe mutant F29W (Pearlstone, J. R., Borgford, T., Chandra, M., Oikawa, K., Kay, C. M., Herzberg, O., Moult, J., Herklotz, A., Reinach, F. C., and Smillie, L. B. (1992) Biochemistry 31, 6545-6553). Comparison of the far- and near-UV CD spectra (+/- Ca2+) of F29W and F29W/NHdel and titration of the Ca(2+)-induced ellipticity and fluorescence changes indicates that the deletion has little effect on the global fold of the molecule but reduces the Ca2+ affinity of the N domain, but not the C domain, by 1.6-1.8-fold. Comparisons of the mutants NHdel, F29W, and F29W/NHdel with rTnC have been made using several functional assays. In reconstituted troponin-tropomyosin actomyosin subfragment 1 and myofibrillar ATPase systems, both F29W and NHdel have significantly reduced Ca(2+)-activated enzymatic activities. These effects are cumulative in the double mutant F29W/NHdel. On the other hand, maximal isometric tension development in Ca(2+)-activated reconstituted skinned fibers is not affected with F29W and NHdel, although the Ca2+ sensitivity of NHdel in this system is markedly reduced. We conclude that both mutations, NHdel and F29W, are functionally deleterious, possibly affecting interactions of the N domain with troponin I and/or T.
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PMID:The effects of N helix deletion and mutant F29W on the Ca2+ binding and functional properties of chicken skeletal muscle troponin. 819 34

Three subunits of rabbit skeletal muscle troponin were expressed in and purified from Escherichia coli. The procedures were optimized, and the reconstituted troponin complex is highly homogeneous, stable, and obtainable in large quantities, allowing us to conduct crystallization studies of the troponin complex. The three subunits expressed and purified are beta-TnT(N'-208), TnI(C64A, C133S), and the wild type TnC. beta-TnT(N'-208) is a 25 kDa fragment of beta-troponin T, which consists of 208 amino acids and lacks 58 residues in the N-terminal variable region. TnI(C64A, C133S) is a mutant troponin I, in which Cys-64 and Cys-133 are replaced by Ala and Ser, respectively. Each subunit was separately expressed in E. coli, purified by column chromatography including HPLC, and reassembled to form troponin complex. The reconstituted troponin complex was not distinguishable from authentic troponin prepared from rabbit skeletal muscle; the acto-S1 ATPase rate, as well as the superprecipitation, was calcium-sensitive. Small flat crystals up to 0.2 mm long have been reproducibly obtained in preliminary crystallization trials.
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PMID:Reconstitution of rabbit skeletal muscle troponin from the recombinant subunits all expressed in and purified from E. coli. 828 38


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