Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

The effect of Mg2+ on the Ca2+ binding to troponin C in rabbit fast skeletal myofibrils.

The effect of Mg2+ on the Ca2+ binding to rabbit fast skeletal troponin C and the CA2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435-439). It was found that the Ca2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg2+ competitively and the Ca2(+)- and Mg2(+)-binding constants were 6.20 x 10(6) and 1.94 x 10(2) M-1, respectively, for the low affinity sites, and 1.58 x 10(8) and 1.33 x 10(3) M-1, respectively, for the high affinity sites. The Ca2+ dependence of myofibrillar ATPase was also affected by Mg2+, with the apparent Ca2(+)- and Mg2(+)-binding constants of 1.46 x 10(6) and 276 x 10(2) M-1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg2+ on Ca2+ binding to the low affinity sites, though the Ca2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.     

Effect of ethylene glycol and Ca2+ on the binding of Mg2+ x adenyl-5'-yl imidodiphosphate to rabbit skeletal myofibrils

The binding of Mg2+ X adenyl-5'-yl imidodiphosphate (Mg2+ X AMP-PNP) to rabbit skeletal myofibrils has been measured in aqueous solution and in 50% ethylene glycol in the presence and absence of Ca2+. In water, the observed binding was weak with less than half the calculated myosin active sites filled even at 1 mM Mg2+ X AMP-PNP. In 50% ethylene glycol, the binding is at least 100-fold tighter and extrapolates to the expected number of binding sites. This is contrasted to the small change seen for Mg2+ X ADP binding between the same sets of conditions. This difference between Mg2+ X AMP-PNP and Mg2+ X ADP is attributed to the strong coupling of Mg2+ X AMP-PNP binding to dissociation of myosin cross-bridges. The Ca2+ sensitivity of Mg2+ X AMP-PNP binding in 50% ethylene glycol is taken as further evidence of the thermodynamic coupling of Mg2+ X AMP-PNP binding to cross-bridge dissociation. In addition, the binding of Mg2+ X AMP-PNP in 50% ethylene glycol is biphasic while Mg2+ X ADP binding under the same conditions is not. The biphasic Mg2+ X AMP-PNP binding could be caused by either the presence of two or more classes of cross-bridges or by negative cooperativity, but the presence of only a single class of Mg2+ X ADP-binding sites implies that if multiple classes of sites are involved, they do not simply differ in steric hindrance or accessibility of the binding site as a whole. The importance of using purified AMP-PNP in the study of actomyosin X AMP-PNP complexes is discussed.     

Removal of troponin C and desensitization of myosin B from ascidian smooth muscle by treatment with ethylene diamine tetraacetate

On treatment with 10 mM EDTA at 30 degrees C, protein of 18,000 daltons was released from myofibrils, thin filaments and myosin B prepared from the smooth muscle of an ascidian, Halocynthia roretzi. This protein was purified from the EDTA extract of myofibrils by differential centrifugation, freeze-drying and gel-filtration. Based on its molecular weight, electrophoretic mobilities in the presence and absence of Ca2+ and other properties, it was identified as troponin C. By EDTA treatment, ascidian myosin B lost the Ca2+-sensitivity of Mg2+-ATPase, and EDTA-treated myosin B recovered the sensitivity by mixing with the EDTA extract of myosin B in the presence of Mg2+. Gel-electrophoretic patterns indicated that desensitization and resensitization of ascidian myosin B were accompanied by the removal and binding of troponin C. These results indicate that ascidian smooth muscle is regulated by a troponin-tropomyosin system, and desensitization induced by EDTA treatment is due to the removal of troponin C but not the release of the light chains of the myosin molecule. Based on these findings, we have established a simple method for the purification of troponin C from ascidian smooth muscle.     

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.     

Calmodulin and troponin C structures studied by Fourier transform infrared spectroscopy: effects of Ca2+ and Mg2+ binding

Fourier transform infrared (FTIR) spectroscopy has been used to examine the conformationally sensitive amide I' bands of calmodulin and troponin C. These are observed to undergo a sequence of spectroscopic changes which reflect conformational rearrangements that take place when Ca2+ is bound. Calmodulin and troponin C show similar though not identical changes on Ca2+ binding, and the effect of Mg2+ on troponin C is quite different from that of Ca2+. Both proteins show absorption maxima in the amide I' region at 1644 cm-1 which is significantly lower in frequency than has been generally observed for proteins that contain a high percentage of alpha-helix. It is proposed that an unusually high proportion of the helices in the structures of these proteins are distorted from the normal alpha-helical configuration such that the carbonyl stretching frequencies are lowered. It is further proposed that the shift to lower frequency is due to backbone carbonyl groups in the distorted helices that form strong hydrogen bonds with solvent molecules. A decrease in intensity at 1654 cm-1, the normal frequency assignment for alpha-helical structure, is observed as Ca2+ binds to calmodulin and troponin C. This suggests that Ca2+ binding results in a net decrease in "normal" alpha-helix conformation. There is a corresponding increase in intensity of the band at 1644 cm-1, possibly due to an increase in distorted helix content, allowing for a net increase in helix consistent with circular dichroism estimates of the Ca2+-dependent changes in helix content in calmodulin.     

Effect of substitution of troponin C in cardiac myofibrils with skeletal troponin C or calmodulin on the Ca2+- and Sr2+-sensitive ATPase activity

Troponin C was removed almost completely from the porcine cardiac myofibrils by the same extraction procedure using CDTA as that previously reported for the rabbit skeletal myofibrils (Morimoto, S. & Ohtsuki, I. (1987) J. Biochem. 101, 291-301), and the effects of substitution of troponin C in cardiac myofibrils with rabbit skeletal troponin C or bovine brain calmodulin were examined. While the ATPase activity of intact cardiac myofibrils or cardiac troponin C-reconstituted cardiac myofibrils was activated at only a little higher concentration of Sr2+ than Ca2+, the skeletal troponin C-substituted cardiac myofibrils, as well as intact rabbit skeletal myofibrils, required more than 10 times higher concentration of Sr2+ than Ca2+ for activation of the myofibrillar ATPase activity. However, the concentrations of Ca2+ and Sr2+ required for the activation of the ATPase activity of the skeletal troponin C-substituted cardiac myofibrils were both about 5 times higher than those of intact skeletal myofibrils. The skeletal troponin C-substituted cardiac myofibrils, as well as intact skeletal myofibrils, also showed higher cooperativity in the Ca2+-activation of the ATPase activity than intact or cardiac troponin C-reconstituted cardiac myofibrils. The ATPase activity of calmodulin-substituted cardiac myofibrils was activated at a several times lower concentration of Ca2+ or Sr2+ than that of calmodulin-substituted skeletal myofibrils, while the ratios of the concentration of Sr2+ to Ca2+ required for activation were almost the same in both cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.     

Effect of myosin cross-bridge interaction with actin on the Ca2(+)-binding properties of troponin C in fast skeletal myofibrils

Ca2+ binding to fast skeletal muscle troponin C reincorporated into troponin C-depleted (CDTA-treated) myofibrils has been measured directly by using 45Ca and indirectly by using a fluorescent probe. Direct Ca2(+)-binding measurements have shown that the Ca2+ affinity of the low-affinity sites is enhanced in the absence of ATP and conversely reduced when myosin is selectively extracted from myofibrils, compared to the Ca2+ affinity in the presence of ATP. Fluorescence intensity changes of a dansylaziridine label at the Met-25 residue of troponin C have shown the same Ca2(+)-sensitivity whether or not ATP is present, while much lower Ca2(+)-sensitivity is seen in the myosin-extracted myofibrils. Since the Met-25 residue is in the amino terminal side alpha-helix of Ca2(+)-binding site I and far from Ca2(+)-binding site II in the primary structure, Ca2+ binding to site II has been evaluated by assuming that the fluorescence change monitors Ca2+ binding to site I alone. Ca2+ binding to site II thus estimated has shown high positive cooperativity only in the presence of ATP and has been found to be nearly proportional to the activation of myofibrillar ATPase, suggesting that Ca2(+)-binding site II is directly involved in the activation of myofibrillar ATPase activity. On the other hand, Ca2(+)-binding site I has been suggested to regulate the interaction of weakly binding cross-bridges with the thin filament, since the fluorescence change in the presence of ATP is saturated at the free Ca2+ concentration required for the activation of myofibrillar ATPase.     

Replacement of troponin components in myofibrils.

The Ca(2+)-sensitive ATPase activity of rabbit skeletal myofibrils was desensitized by treatment with excess troponin T and was found to be activated irrespective of the Ca2+ concentrations. A SDS-gel electrophoretic study showed that both troponin C and troponin I were removed from the myofibrils on treatment with troponin T. The Ca(2+)- and Sr(2+)- sensitivities of the ATPase of troponin T-treated myofibrils reconstituted with troponin C. I were the same as in the intact myofibrils. The Ca(2+)-activated ATPase of rabbit skeletal myofibrils was also desensitized on treatment with chicken breast troponin T or its 26K fragment. The SDS-gel electrophoretic study revealed that troponin T, in addition to troponin C and troponin I, was also removed from the myofibrils and, instead, chicken breast troponin T or its 26K fragment was incorporated into the myofibrils. The Ca(2+)- sensitivity of myofibrils treated with chicken breast troponin T or its 26K fragment was then regained on reconstitution with troponin C.I. These findings indicate that the change in composition of myofibrils on treatment with troponin T or its 26K fragment is due to the selective replacement of the troponin C.I.T complex in the myofibrils as a whole with troponin T or its 26K fragment.     

Coordination structures of Ca2+ and Mg2+ in Akazara scallop troponin C in solution. FTIR spectroscopy of side-chain COO- groups.

FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO- antisymmetric stretch provides information about the coordination modes of COO- groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 in the Mg2+-bound state was identical to that in the apo state. Therefore, the side-chain COO- group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO- antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO- groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I' region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO- groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Studies on (K+ + H+)-ATPase III. Binding of adenylyl imidodiphosphate

1. Adenylyl imidodiphosphate (AMPPNP) binds to (K+ + H+)-ATPase from pig gastric mucosa with a dissociation constant (Kd) of 50 microM for the AMPPNP-enzyme complex. 2. Monovalent cations reduce the amount of AMPPNP bound in the following order of effectiveness Tl+ greater than K+ greater than Rb+ greater than Cs+ greater than Na+, Li+, choline+. 3. AMPPNP binding to the enzyme has a pH optimum at pH 7.0--7.5 in the absence of added ions, which is shifted to pH 8 upon addition of MgCl2. 4. Cyclodiaminotetraacetic acid (CDTA, Tris salt) inhibits binding of AMPPNP. This inhibition is not due to chelation of Mg2+. It may be due to direct binding of CDTA to the enzyme or to removal of stabilizing cations other than Mg2+. 5. Binding curves determined in the presence of various concentrations of Mg2+ show that at low Mg2+ concentrations (less than 0.5 mM), the apparent number of binding sites is reduced, while at higher Mg2+ concentrations (greater than or equal to 0.5 mM), the binding of AMPPNP is inhibited in a competitive way. 6. From these observations it is concluded that the enzyme has two binding sites for AMPPNP and only one for Mg-AMPPNP (or two with strong anti-cooperativity), and that Mg2+ inhibits binding of Mg-AMPPNP. This finding is interpreted in terms of a model involving a dimeric form of the enzyme.     

The effects of Mg2+ on the Ca2+-binding properties and Ca2+-induced tyrosine-fluorescence changes of calmodulin isolated from rabbit skeletal muscle

Calmodulin from phosphorylase kinase (the delta subunit) was obtained as a homogeneous protein in a spectroscopically pure form, and its interaction with Ca2+ and Mg2+ was studied. 1. Determination of the binding of Ca2+ to calmodulin in a buffer of low ionic strength (0.001 M) show that it contained six binding sites for this divalent cation. 2. Employment of a buffer of high ionic strength (0.18 M) allowed two Ca2+/Mg2+-binding sites (KdCa2+ = 4.0 microM), which showed Ca2+ - Mg2+ competition (KdMg2+ = 0.75 mM), to be distinguished from two Ca2+-specific binding sites (KdCa2+ = 40 microM). The remaining two Ca2+-binding sites are not observed under these conditions and are probably Mg2+-specific binding sites. Thus, the binding sites on calmodulin are remarkably similar to those of the homologous Ca2+-binding protein, troponin C [Potter and Gergely (1975) J. Biol. Chem. 250, 4628, 4633]. 3. The conformational states of calmodulin are defined by Ca2+, Mg2+ and salt concentrations, which can be differentiated by their Ca2+ affinity and their relative tyrosine fluorescence intensity. In a buffer of high ionic strength, Mg2+ induces a conformation which enhances the apparent affinity for Ca2+. Addition of Ca2+ leads to an enhancement of the tyrosine fluorescence intensity, which remains enhanced even upon removal of Ca2+ by chelation with EGTA. Only additional chelation of Mg2+ with EDTA reduces the tyrosine fluorescence intensity. 4. Comparison of the Ca2+-binding parameters of phosphorylase kinase, which were previously determined under identical experimental conditions [Kilimann and Heilmeyer (1977) Eur. J. Biochem. 73, 191-197], with those reported here on calmodulin isolated from this enzyme, allows the conclusion that Ca2+ binding to the holoenzyme occurs by binding to the delta subunit exclusively. 5. Ca2+ binding and Ca2+ activation of phosphorylase kinase are compared and discussed in relation to the Ca2+ and Mg2+-induced conformation changes of calmodulin.     

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.     

Myofibrillar Ca2+-stimulated Mg2+-ATPase from chronically ischemic canine heart

Functional properties of myofibrils from chronically ischemic canine myocardium were evaluated. Ischemia was produced by tight stenosis of left anterior descending artery (LAD), followed by 40 min acute ischemia with prior preconditioning. Animals of the first group were sacrificed after 8 weeks. In the second group, angioplasty of LAD was performed after 8 weeks of ischemia and animals were kept alive for other 4 weeks. Control animals were sham operated. Activity and kinetic parameters of myofibrillar Ca2+-stimulated Mg2+-ATPase were measured in myofibrils isolated from anterior and posterior parts of all hearts. We did not find any differences in maximal velocity (Vmax), half-maximal activation constant for calcium (K(Ca2+)50) and cooperativity coefficient (n(hill)) of myofibrils from different experimental groups as compared to controls, either at pH 7, pH 6.5 (acidosis) or pH 7.5 (alkalosis). K(Ca2+)50 increased in medium simulated acidosis (12.6-33.5 times) and n(hill) decreased significantly in all groups as compared with values obtained at pH 7. These results indicate that activity and Ca2+-sensitivity of myofibrillar Mg2+-ATPase remain unchanged despite deteriorated heart function 8 weeks after LAD obstruction. Experiments have confirmed that Ca2+-stimulated-ATPase from canine heart myofibrils responded to pH decrease by a decreased sensitivity to Ca2+ and a decreased cooperativity. However, sensitivity of the enzyme to the pH changes is unaltered by 8 weeks of chronic ischemia.     

Calcium regulation in the human myocardium affected by dilated cardiomyopathy: A structural basis for impaired Ca2+-sensitivity

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 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.     

A laser Raman spectroscopic study of Ca2+ binding to troponin C.

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.     

Effects of ADP and AMPPNP on the hydrogen-deuterium exchange kinetics in Ca2+, Mg2+-ATpase of sarcoplasmic reticulum

The kinetics of the hydrogen-deuterium exchange reaction in sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle was followed by infrared absorption measurements. The exchange rate in SR was much lower than that in the soluble proteins reported so far. When adenylyl-imidophosphate (AMPPNP, an TP analog) was present, the exchange rate was lower than that in free SR and it was the lowest when ADP was present. The effect of the nucleotides on the exchange rate reflects the conformational change of the Ca2+, Mg2+-ATpase of SR membranes on binding the nucleotides. The structure of e Ca2+, Mg2+-ATPase is more restricted in the following order: SR + ADP greater than SR + AMPPNP greater than free SR.