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)

Purified troponin (Tn), the complex of the Ca-2+ binding subunit (TnC), the inhibitory subunit (TnI), and the tropomyosin binding subunit (TnT) binds 4 mol of Ca-2+ per mol. Two sites bind Ca-2+ with a binding constant of 5 times 10-8 M- minus 1, and two with a binding constant of 5 times 10-6 M- minus 1. In the presence of 2 mM MgCl2 the binding to four sites can be characterized with a single affinity constant of 5 times 10-6 M- minus 1. Purified TnC also binds 4 mol of Ca-2+ per mol; two sites have a binding constant of 2 times 10-7 M- minus 1 and two have one of 2 times 10-5 M- minus 1. In the presence of 2 mM MgCl2 the binding constant of the sites of higher affinity is reduced to 2 times 10-6 M- minus 1, while Ca-2+ binding to the sites of lower affinity is unaffected. Assuming competition between Mg-2+ and Ca-2+ for the high affinity sites on TnC and Tn, the changes in Ca-2+ binding can be accounted for with KMg values of 5 times 10-3 M- minus 1 and 5 times 10-4 M- minus 1, respectively. Tn and TnC bind 4 mol of Mg-2+ per mol in the absence of Cs-2+. The fact that at [Ca-2+] similar to 10- minus 5 M four Ca-2+ and only two Mg-2+ are bound per mol of TnC in the presence of 2 mM Mg-2+ further supports the view that there is direct competition between Mg-2+ and Ca-2+ for the high affinity Ca-2+ binding sites on TnC and Tn. These results then suggest that Tn and TnC contain six divalent cation binding sites: two high affinity Ca-2+ binding sites that also bind Mg-2+ competitively (Ca-2+-Mg-2+ sites); two sites with lower affinity for Ca-2+ that do not bind Mg-2+ (Ca-2+-specific sites); and two sites that bind Mg-2+ but not Ca-2+ (Mg-2+-specific sites). The complex of TnC and TnI (1:1 molar ratio) has the same binding properties as Tn, suggesting a conformational change in TnC upon interaction with TnI. Studies on myofibrillar ATPase activity as a function of free Ca-2+ concentration at two different free Mg-2+ concentrations suggest that full activation by Ca-2+ occurs only upon binding of Ca-2+ to the two Ca-2+-specific binding sites in Tn but does not require binding of Ca-2+ to the Ca-2+-Mg-2+ sites.
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PMID:The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. 12 31

In a search for an invertebrate muscle from which the muscle regulatory proteins could be obtained in a great quantity and at high homogeneity, the regulatory proteins, tropomyosin (Tm) and three subunits of troponin (Tn), have been isolated from the lobster tail muscle, purified and partially characterized. The calcium-sensitive ATPase of lobster myofibril was restored when purified lobster Tm and lobster Tn were added to actin. Quantitative SDS-polyacrylamide gel electrophoresis showed that the lobster muscle contains actin, Tm, Tn with a molar ratio 7:1:1 and that lobster Tn consists of three subunits, one of each I, C and T. Each subunit was identified according to its effect on the acto-S1 ATPase rate. The isomer composition in each fraction of purified Tn subunit and in Tm are different from the rabbit skeletal muscle proteins; Tm consists of a single species of polypeptide of M(r) 38,000; the TnT fraction appears to be homogeneous with M(r) 43,000; the TnI fraction contains five isomers, all showing similar isoelectric pH, differing in M(r) in the range from 28,000 to 31,000; two TnC fractions contain three isomers in total with a range of M(r) from 18,500 to 19,000. Further study of the lobster Tm elucidated that digestion by carboxypeptidase A gave rise to a homogeneous preparation of truncated and non-polymerizable Tm which is devoid of 11 residues at the C-terminus of the molecule. The C-terminal amino acid sequence of 11 residues is homologous to the thoracic isomer generated from Drosophila melanogaster Tm-I gene. The present study indicated that, despite heterogeneities owing to the occurrence of isomers, the lobster regulatory proteins serve as an invertebrate source of the proteins for structural and biophysical studies, alternative to vertebrate counterparts.
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PMID:Isolation, purification and partial characterization of tropomyosin and troponin subunits from the lobster tail muscle. 149 Oct 69

The binding of the NH2-terminal region of troponin T (TnT) to the COOH-terminal region of tropomyosin (Tm) and the head-to-tail overlap between Tm molecules is thought to provide a pivotal link between troponin (Tn) and Tm (White, S.P., Cohen, C., and Phillips, G.N., Jr. (1987) Nature 325, 826-828). To further explore the structure-function relationship of the NH2-terminal region of TnT, we studied the binding of a 26,000-dalton TnT fragment (26K-TnT, Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M.J. (1984) J. Biochem. (Tokyo) 95, 1337-1342) which corresponds to residues 46-259 of TnT2f, the major isoform of TnT in rabbit fast twitch muscle, to immobilized alpha-Tm. Both 26K-TnT and TnT2f were retained by the alpha-Tm affinity column in the presence of 150 mM NaCl. However, upon increasing the NaCl concentration 26K-TnT was eluted from the column at a higher ionic strength than was TnT. When applied alone, the binary complex of TnI and TnC (TnC.TnI) was not retained by the alpha-Tm affinity column. When applied subsequently to prebound TnT2f or 26K-TnT, TnI.TnC was retained by the alpha-Tm affinity column and eluted together with TnT2f or 26K-TnT as ternary troponin complexes. Whether Ca2+ was present or not, Tn containing 26K-TnT was eluted at a higher ionic strength than was Tn containing TnT2f, indicating that removal of the first 45 residues of TnT2f strengthens the binding of Tn to Tm. In the presence of Tm, reconstituted Tn containing 26K-TnT conferred Ca2+ sensitivity on actomyosin-S1 MgATPase, and the steepness of the pCa-ATPase relation was unchanged with respect to the actoS1 ATPase regulated by TnT2f. It is concluded that the first 45 residues of TnT2f are not essential for anchoring the troponin complex to the thin filament and do not play a crucial role in the cooperative response of regulated actoS1 ATPase to Ca2+.
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PMID:Deletion of the first 45 NH2-terminal residues of rabbit skeletal troponin T strengthens binding of troponin to immobilized tropomyosin. 182 57

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.
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PMID:Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers. 186 27

We investigated the mechanism(s) responsible for differences in the effects of acidic pH on Ca2+ activation of the activity of adult and neonatal rat heart myofilaments. Studies on preparations of myofilaments reconstituted with adult troponin-tropomyosin (Tn-Tm) and either adult or neonatal thick filaments indicated that the difference in effect of acidic pH is related to differences in Tn-Tm and not other myofilament proteins. Immunoblotting analysis showed that development of the rat heart myofibrils is associated with isoform switching from slow skeletal TnI to cardiac TnI and from a slow mobility isoform of TnT (TnT1) to a faster Mr isoform (TnT2. Expression of slow skeletal TnI was associated with a relative insensitivity of myofilament Ca2+ activation to deactivation by acidic pH. Moreover, the effect of acidic pH on Ca2+ activation of ATPase activity of soleus myofibrils, which contain cardiac TnC and slow skeletal TnI, was essentially the same as the effect of acidic pH on rat cardiac myofibrils in the early neonatal period. Neonatal myofilaments also contained a relative abundance of a set of polypeptides copurifying with the thin filaments. We have identified these proteins as histones. The relative amount of histones among a variety of preparations from different species was not correlated with the pH sensitivity of myofibrillar Ca2+ activation. Shifts in TnT isoforms among these species were also not correlated with an altered response to acidic pH. Our data provide evidence in support of the hypothesis that the relative insensitivity of neonatal myofilament activity to acidic pH is due to the presence of slow skeletal TnI in the thin-filament regulatory complex.
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PMID:Identification and functional significance of troponin I isoforms in neonatal rat heart myofibrils. 193 54

Troponin was isolated from the abdominal muscle of the American lobster (Homarus americanus) by essentially the same method as used for akazara scallop troponin [J. Biol. Chem. 261, 16749-16754 (1986)]. The thus isolated troponin together with lobster tropomyosin confers high Ca2(+)-sensitivity to rabbit reconstituted actomyosin. The troponin consists of components having Mr of about 42,000, 32,000, 30,000, and 17,000, but not the Mr 52,000-59,000 component previously reported to be present in several crustacean troponins. These troponin components were separated from each other by DEAE-Toyopearl column chromatography in the presence of 6 M urea. The Mr 17,000 component was further separated into one major and two minor components by the same chromatography, but each of them was confirmed to be a Ca2+ binding component, TnC. The Mr 32,000 and 30,000 components were both regarded as inhibitory subunits, TnIs, since the Mg-ATPase activity of actomyosin in the presence of tropomyosin was strongly inhibited by the addition of the components, and the inhibition was reversed by the further addition of TnC. Finally, the Mr 42,000 component was regarded as TnT, since this component formed stoichiometic complex with TnC and TnI, and was indispensable for Ca2+ regulation of the actomyosin-tropomyosin system.
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PMID:American lobster troponin. 214 56

1. The calcium sensitivity of force production of cardiac muscle fibres is altered by certain drugs. The sites of action of three such compounds (pimobendan, sulmazole, isomazole) within the myofibril have been investigated. Calmodulin antagonists, perhexilene and bepridil, which have been shown to alter the calcium dependence of myofibrillar ATPase activity and oxmetidine, an H2-receptor antagonist which binds to calmodulin, were also studied. 2. The rates of dissociation of calcium from both the regulatory and high affinity sites on bovine isolated cardiac troponin C (cTnC) were measured in a stopped-flow fluorimeter. The rates of dissociation were found to be 136.5 +/- 16 s-1 and 1.3 +/- 0.20 s-1 (mean +/- s.e.mean, n = 11 determinations; conditions: 100 mM KCl, 10 mM MOPS, 3 mM MgCl2, 0.1 mM dithriothreitol, pH 7.0, 15 degrees C). Sulmazole, isomazole and perhexiline (final concentration of 50 microM) had no effect on the rate of Ca2+ dissociation from the regulatory Ca2+ site, indicating that these compounds do not act on cTnC directly. 3. The rate of dissociation of Ca2+ from the regulatory site was slightly reduced (approximately 20%) by pimobendan (50 and 100 microM) and was somewhat increased by oxmetidine (28% at 100 microM). 4. Bepridil (25 microM) reduced the rate of dissociation by 50%, indicating a direct effect of bepridil on TnC. 5. Sulmazole, isomazole, perhexiline, pimobendan (50 microM) and bepridil (25 microM) were without effect on the rate of dissociation of Ca2+ from the high affinity Ca2+/Mg2+ sites. Oxmetidine caused 24% decrease in the rate of Ca2+ dissociation from these sites. 6. The rate of dissociation of Ca2+ from the regulatory site on the complex of troponin-tropomyosin (TnTm) was measured. Sulmazole and pimobendan (50 microM) were without effect on the rate of dissociation of Ca2+ from the regulatory site in the protein complex, and isomazole (50 microM) caused only a slight reduction (23%). Perhexiline (50 microM) or bepridil (10 microM) reduced the rate of Ca2 dissociation by about 50%. The rate of dissociation of Ca2+ from the high affinity Ca2 +/Mg2 + sites was not altered by sulmazole, isomazole, or pimobendan (50 microM), but was decreased - 35% by perhexiline (50 microM) or bepridil (10 microM).
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PMID:The effects of reported Ca2+ sensitisers on the rates of Ca2+ release from cardiac troponin C and the troponin-tropomyosin complex. 220

A micro cuvette is described, which allows to measure mechanical, optical and energetic parameters of single skinned muscle fibres or small fibre bundles. Two examples for possible applications are given: The measurement of ATPase activity of single muscle fibres. The detection of fluorescence changes due to Ca binding to fluorescence labeled TnC, which was incorporated into small bundles of rabbit psoas fibres before the experiment.
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PMID:Perfusion cuvette for the simultaneous measurement of mechanical, optical and energetic parameters of skinned muscle fibres. 294 40

The cardiac troponin (Tn) complex, consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT), has been reconstituted from purified troponin subunits isolated from bovine heart muscle. The Ca2+-binding properties of cardiac Tn were determined by equilibrium dialysis using either EGTA or EDTA to regulate the free Ca2+ concentration. Cardiac Tn binds 3 mol Ca2+/mol and contains two Ca2+-binding sites with a binding constant of 3 X 10(8) M-1 and one binding site with a binding constant of 2 X 10(6) M-1. In the presence of 4 mM MgC12, the binding constant of the sites of higher affinity is reduced to 3 X 10(7) M-1, while Ca2+ binding to the site at the lower affinity is unaffected. The two high affinity Ca2+-binding sites of cardiac Tn are analogous to the two Ca2+-Mg2+ sites of skeletal Tn, while the single low affinity site is similar to the two Ca2+-specific sites of skeletal Tn (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4625-5633). The Ca2+-binding properties of the complex of TnC and TnI (1:1 molar ratio) were similar to those of Tn. Cardiac TnC also binds 3 mol of Ca2+/mol and contains two sites with a binding constant of 1 X 10(7) M-1 and a single site with a binding constant of 2 X 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the high affinity sites of TnC and Tn, the binding constants for Mg2+ were 0.7 and 3.0 X 10(3) M-1, respectively. The Ca2+ dependence of cardiac myofibrillar ATPase activity was similar to that of an actomyosin preparation regulated by the reconstituted troponin complex. Comparison by the Ca2+-binding properties of cardiac Tn and the cardiac myofibrillar ATPase activity as a function of [Ca2+] and at millimolar [Mg2+] suggests that activation of the ATPase occurs over the same range of [Ca2+] where the Ca2+-specific site of cardiac Tn binds Ca2+.
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PMID:The calcium and magnesium binding sites on cardiac troponin and their role in the regulation of myofibrillar adenosine triphosphatase. 644 12

Fragments of rabbit skeletal muscle Ca2+-binding subunit of troponin (TnC), obtained by cleavage with trypsin, thrombin, and CNBr, were tested for their ability to form binary and ternary complexes with ATPase inhibitory subunit (TnI) and tropomyosin-binding subunit (TnT) and their ability to replace TnC in reversing TnI inhibition of actomyosin ATPase activity. Three regions of TnC were found to be involved in interaction with TnI. Regions near Ca2+-binding sites II and III require Ca2+ for the interaction, while a third region near Ca2+-binding site IV binds TnI whether or not Ca2+ is present. The TnT binding site has been localized in the NH2-terminal half of TnC. Several of the TnC fragments form soluble ternary complexes with TnI and TnT. Fragments that contain amino acid residues 89-100 and at least one pair of Ca2+-binding sites are able to reverse the TnI inhibition of actomyosin ATPase activity, which exhibits the same [Ca2+]1/2 regardless of which of the Ca2+-binding sites are present in the fragment.
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PMID:Proteolytic fragments of troponin C. Interactions with the other troponin subunits and biological activity. 645 9


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