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)

Intracellular passive Ca2+, buffering was measured in voltage-clamped rat ventricular myocytes. Cells were loaded with indo-1 (K+ salt) to an estimated cytosolic concentration of 44 +/- 5 microM (Mean +/- SEM, n = 5), and accessible cell volume was estimated to be 24.5 +/- 3.6 pl. Ca2+ transport by the sarcoplasmic reticulum (SR) Ca-ATPase and sarcolemmal Na-Ca exchange was inhibited by treatment with thapsigargin and Na-free solutions, respectively. Extracellular [Ca2+] was maintained at 10 mM and, in some experiments, the mitochondrial uncoupler "1799" was used to assess the degree of mitochondrial Ca2+ uptake. To perform single cell titrations, intracellular Ca2+ ([Ca2+]i) was increased progressively by a train of depolarizing voltage clamp pulses from -40 to +10 mV. The total Ca2+ gain with each pulse was calculated by integration of the Ca current and then analyzed as a function of the rapid change in [Ca2+]i during the pulse. In the range of [Ca2+]i from 0.1 to 2 microM, overall cell buffering was well described as a single lumped Michaelis-Menten type species with an apparent dissociation constant, KD, of of 0.63 +/- 0.07 microM (n = 5) and a binding capacity, Bmax, of 162 +/- 15 mumol/l cell H2O. Correction for buffering attributable to cytosolic indo-1 gives intrinsic cytosolic Ca2+ buffering parameters of KD = 0.96 +/- 0.18 microM and Bmax = 123 +/- 18 mumol/l cell H2O. The fast Ca2+ buffering measured in this manner agrees reasonably with the characteristics of known rapid Ca buffers (e.g., troponin C, calmodulin, and SR Ca-ATPase), but is only about half of the total Ca2+ buffering measured at equilibrium. Inclusion of slow Ca buffers such as the Ca/Mg sites on troponin C and myosin can account for the differences between fast Ca2+ buffering in phase with the Ca current measured in the present experiments and equilibrium Ca2+ buffering. The present data indicate that a rapid rise of [Ca2+]i from 0.1 to 1 microM during a contraction requires approximately 50 microM Ca2+ to be added to the cytosol.
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PMID:Intrinsic cytosolic calcium buffering properties of single rat cardiac myocytes. 781 10

The Ca(2+)-binding properties of troponin C in the intact myofilament lattice and their relation to the activation of ATPase were investigated with isolated porcine cardiac myofibrils. Ca2+ binding, which is composed of two classes of binding sites with different affinities (classes 1 and 2), was clearly detected by a novel method for subtracting the large background activity of myofibrillar Ca2+ binding. The classes 1 and 2 were equivalent stoichiometrically to the two high-affinity sites (sites III and IV) and a single low-affinity site (site II) of troponin C. In the presence of ATP, positive cooperativity was observed in the Ca2+ binding of class-2 sites and the Hill equation parameters were in excellent agreement with those for the Ca(2+)-activated myofibrillar ATPase activity, which indicated that the activation of ATPase is a linear function of the Ca2+ occupancy of site II. In the absence of ATP, a marked increase in the affinity of only class-2 sites was observed while the cooperativity was lost. These results provide direct evidence that some feedback mechanism exists between myosin crossbridge attachment and the Ca2+ binding to site II of troponin C, which may thus confer positive cooperativity on the Ca2+ activation of myofibrillar ATPase activity.
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PMID:Ca2+ binding to cardiac troponin C in the myofilament lattice and its relation to the myofibrillar ATPase activity. 800 74

The most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N-terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC-extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N-terminal nine amino acids resulted in a 60% decrease in maximal Ca(2+)-dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N-terminal peptide did not appear to appreciably affect the Ca(2+)-binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N-terminal extension is important for the maximal activity of cTnC. The N-terminal helix may function in a relatively non-specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.
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PMID:The presence but not the sequence of the N-terminal peptide in cardiac TnC is important for function. 803 94

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

Increased ionic strength decreases maximal calcium-activated force (Fmax) of skinned muscle fibers via mechanisms that are incompletely understood. In detergent-skinned fibers from either rabbit (psoas) or lobster (leg or abdomen), Fmax in KCl-containing solutions was less than in potassium methanesulfonate (KMeSO3), which we showed previously was the least deleterious salt for adjusting ionic strength. In either salt, lobster fibers were considerably less sensitive to elevated ionic strength than rabbit fibers. Trimethylamine N-oxide (TMAO, a zwitterionic osmolyte found in high concentration in cells of salt-tolerant animals) increased Fmax, especially in high KCl solutions. In this regard, TMAO was more effective than a variety of other natural or synthetic zwitterions. In rabbit fibers, increasing ionic strength decreases Fmax but has little effect on contractile ATPase rate measured simultaneously using a linked-enzyme assay. Thus high salt increases the tension-cost of contraction (i.e. ratio ATPase/Fmax). At both high and low salt, TMAO decreases tension-cost. Given a simple two-state model of the cross-bridge cycle, these data indicate that ionic strength and TMAO affect the apparent detachment rate constant. High ionic strength KCl solutions extract myosin heavy- and light-chains, and troponin C from rabbit fibers. This extraction is virtually abolished by TMAO. Natural zwitterions, such as TMAO, have been shown to protect proteins against destabilization by high salt or other denaturatants. Our data indicate that, even in the best of salts, destabilization of the actomyosin complex may play a role in the effect of high ionic strength on the contractile process.
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PMID:Influence of ionic strength on contractile force and energy consumption of skinned fibers from mammalian and crustacean striated muscle. 810 86

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

Ca(2+)- and Sr(2+)-sensitive ATPase of myofibrillar preparations from slow skeletal (chicken anterior latissimus dorsi), fast skeletal (chicken breast) and porcine cardiac ventricular muscles was investigated. Sr2+ at concentrations about 6 times higher than Ca2+ was required for activating the ATPase of slow skeletal or cardiac myofibrils, while Sr2+ about 30 times higher than Ca2+ was required for activating fast skeletal myofibrils. The cooperativity of activation of slow skeletal myofibrils was almost the same as that of cardiac myofibrils, but lower than that of fast skeletal myofibrils. Sr2+/Ca2+ sensitivity ratio of the desensitized (troponin-tropomyosin-depleted) fast skeletal myofibrils reconstituted with troponin-tropomyosin from slow skeletal, fast skeletal, and cardiac muscles were 13, 34, and 5, respectively. The troponin C-depleted fast skeletal myofibrils showed the same Sr2+/Ca2+ sensitivity ratio and the same cooperativity of activation as those of the intact fast skeletal myofibrils when reconstituted with fast skeletal troponin C, and as those of the intact slow skeletal myofibrils when reconstituted with slow skeletal troponin C.
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PMID:Ca(2+)- and Sr(2+)-sensitive ATPase activity of slow skeletal myofibrils in comparison with fast skeletal and cardiac myofibrils. 816 63

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


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