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PubMed2000

Measurement of nucleotide exchange rate constants in single rabbit soleus myofibrils during shortening and lengthening using a fluorescent ATP analog.

The kinetics of displacement of a fluorescent nucleotide, 2'(3')-O-[N[2-[[Cy3]amido]ethyl]carbamoyl]-adenosine 5'-triphosphate (Cy3-EDA-ATP), bound to rabbit soleus muscle myofibrils were studied using flash photolysis of caged ATP. Use of myofibrils from this slow twitch muscle allowed better resolution of the kinetics of nucleotide exchange than previous studies with psoas muscle myofibrils (, Biophys. J. 73:2033-2042). Soleus myofibrils in the presence of Cy3-EDA-nucleotides (Cy3-EDA-ATP or Cy3-EDA-ADP) showed selective fluorescence staining of the A-band. The K(m) for Cy3-EDA-ATP and the K(d) for Cy3-EDA-ADP binding to the myofibril A-band were 1.9 microM and 3.8 microM, respectively, indicating stronger binding of nucleotide to soleus cross-bridges compared to psoas cross-bridges (2.6 microM and 50 microM, respectively). After flash photolysis of caged ATP, the A-band fluorescence of the myofibril in the Cy3-EDA-ATP solution under isometric conditions decayed exponentially with a rate constant of 0.045 +/- 0.007 s(-1) (n = 32) at 10 degrees C, which was about seven times slower than that for psoas myofibrils. When a myofibril was allowed to shorten with a constant velocity, the nucleotide displacement rate constant increased from 0.066 s(-1) (isometric) to 0.14 s(-1) at 20 degrees C with increasing shortening velocity up to 0.1 myofibril length/s (V(max), the shortening velocity under no load was approximately 0. 2 myofibril lengths/s). The rate constant was not significantly affected by an isovelocity stretch of up to 0.1 myofibril lengths/s. These results suggest that the cross-bridge kinetics are not significantly affected at higher strain during lengthening but depend on the lower strain during shortening. These data also indicate that the interaction distance between a cross-bridge and the actin filament is at least 16 nm for a single cycle of the ATPase.

Adenosine Triphosphate↗

PubMed1986

Alteration of actin-tropomyosin interaction in 2,4-pentanedione-treated rabbit skeletal myofibrils.

In previous work, we (El-Saleh, S., Theiret, R., Johnson, P., and Potter, J. D. (1984) J. Biol. Chem. 259, 11014-11021) presented evidence that Ca2+ activation of skeletal myofilaments depends on a specific actin domain. We showed that rabbit skeletal thin filaments reconstituted with actin modified at Lys-237 activate heavy meromyosin X Mg2+-ATPase activity independently of the Ca2+ ion concentration. The modification, which apparently blocks the inhibitory effects of troponin-tropomyosin (Tn X Tm), on acto-heavy meromyosin X Mg2+-ATPase activity, consisted of conversion of Lys-237 to an enamine by reaction of purified actin with 2,4-pentanedione (PD). In experiments reported here, we have treated myofibrils with PD with the idea of altering actin in its native state within the myofilament lattice. Preparations of native and Tn X Tm free ("desensitized") myofibrils were incubated with PD (100 mol/mol of actin lysine) under rigorous conditions (10 mM 4-morpholinepropanesulfonic acid, pH 7.0, 2.0 nM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 0.4 mM dithiothreitol, and 0.15 mM NaN3). Actin isolated from PD X myofibrils contained 0.5 mol of enamine/mol. In the presence of Ca2+, the Mg2+-ATPase activity of PD-treated myofibrils was 110-120% of the maximum Ca2+-stimulated Mg2+-ATPase activity of untreated control myofibrils. In low free Ca2+ (pCa greater than 8), the Mg2+-ATPase activity of the PD-treated myofibrils was not suppressed and remained at 100-106% of the maximum activity of the control myofibrils. Ca2+ sensitivity of the PD-treated myofibrils was restored following treatment with hydroxylamine, which hydrolyzes enamine's products. Preparations of desensitized myofibrils reconstituted with PD-modified or unmodified Tn X Tm demonstrated the same Ca2+-sensitive ATPase activities. On the other hand, preparations reconstituted with unmodified or PD-modified Tn X Tm and PD-modified desensitized myofibrils were insensitive to Ca2+ ion concentration. The Mg2+-ATPase activity of preparations of myosin treated with PD was not activated by modified or unmodified actin. Our results indicate that is is possible to produce an active state(s) of the myofibrils in the absence and presence of Ca2+ by specific alteration of the actin X Tm interaction following modification of myofibrillar actin most likely at Lys-237.

Actins↗

PubMed1980

Properties of phosphorylated myofibrils from gizzard smooth muscle.

Phosphorylation of chicken gizzard myosin light chain in myofibril and its effect on myofibrillar ATPase activity were investigated in the contracted state of myofibrils. When myofibrils were incubated for two hours at 30 degreeds C with ATP, magnesium and calcium, the myosin light chain was phosphorylated by endogenous light-chain kinase. Standing overnight, the phosphorylated light chain was dephosphorylated by endogenous light-chain phosphatase. Control myofibril had much higher ATPase activity than phosphorylated and phosphorylated-dephosphorylated myofibrils. It was very interesting that the phosphorylated and phosphorylated-dephosphorylated myofibrils were quite similar in ATPase activity. However, phosphorylated myofibril differed from phosphorylated-dephosphorylated myofibril in Ca2+ dependency of Mg2+-ATPase activity. The phosphorylated-dephosphorylated myofibril was not affected by the presence or absence of Ca2+. In contrast, phosphorylated myofibril apparently showed a negative Ca2+-sensitivity. On the other hand, the results indicating that the superprecipitation gel formed by phosphorylated-dephosphorylated myosin could not be dissolved in 0.6 M NaCl, suggest that the phosphorylation-dephosphorylation process of the actomyosin system in gizzard myofibril results in stronger actin-myosin interaction.

Actins↗

PubMed1982

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells.

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.

Animals↗

PubMed1987

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.

Adenosine Triphosphatases↗

PubMed1989

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.

Adenosine Triphosphatases↗

PubMed1992

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.

Animals↗

PubMed2005

Differential effects of Latrunculin-A on myofibrils in cultures of skeletal muscle cells: insights into mechanisms of myofibrillogenesis.

To test different models of myofibrillogenesis, we followed live cells expressing Green Fluorescent Proteins ligated to either actin or alpha-actinin and analyzed stress fibers, premyofibrils, and myofibrils in quail myotube cultures. Actin filaments in the three types of fibers were compared by analyzing the effects of Latrunculin-A (Lat-A), a monomeric actin binding macrolide drug (M.W. = 422 Daltons), on stress fibers in fibroblasts and on myofibrils in skeletal myotubes in the same culture. Lat-A, at low concentrations (0.2 microM), induced the loss of stress fibers in fibroblasts within a few hours and within 10 min when Lat-A was increased to 1.0 microM. The effect was reversible with reformation of the stress fibers when the drug was removed. In contrast to the Lat-A induced disassembly of stress fibers in fibroblasts, assembling myofibrils in the skeletal muscle cells were not affected by 1.0-microM concentrations of Lat-A. With increasing concentrations of Lat-A (up to 5 microM), and increasing incubation times, however, the drug induced premyofibrils, the precursors of mature myofibrils, to disassemble and the accumulation of mature myofibrils to be halted. Removal of the drug led to the reformation of premyofibrils and the resumption of myofibrillogenesis in the spreading edges of the myotubes. In contrast, the mature myofibrils in the central shaft of the myotubes were stable in doses of Lat-A as high as 50 microM. The newly assembled mature myofibrils located adjacent to the premyofibrils at the ends and sides of the myotube were intermediate in sensitivity to Lat-A, disassembling when exposed to 10 microM Lat-A for one hour. To determine how a change in the actin filaments during myofibrillogenesis might confer greater resistance to depolymerization by Lat-A, we stained the myotubes with an antibody directed against CapZ, a protein that blocks the release of monomer actin from the barbed ends of actin filaments. CapZ was absent from premyofibrils. It was distributed uniformly along nascent myofibrils where F-actin was unstriated, and was localized in a clearly striated Z-band pattern in the mature myofibrils where F-actin patterns were fully striated. These Lat-A and CapZ results are discussed in the context of various models of myofibrillogenesis.

Actinin↗

PubMed1994

The effect of troponin C substitution on the Ca(2+)-sensitive ATPase activity of vertebrate and invertebrate myofibrils by troponin Cs with various numbers of Ca(2+)-binding sites.

The effect of four different classes of troponin C with different numbers of Ca(2+)-binding sites was investigated on the Ca(2+)-activation profiles of the ATPase of troponin C-depleted myofibrils prepared from vertebrate fast skeletal (rabbit), vertebrate cardiac (bovine) and invertebrate crustacean tail striated (crayfish, lobster) muscles. Troponin C from vertebrate sources [fast skeletal (rabbit, chicken) with four Ca(2+)-binding sites, and cardiac (bovine, chicken) with three Ca(2+)-binding sites] activated the Ca(2+)-ATPase of troponin C-depleted myofibrils from the vertebrate skeletal or cardiac muscles, but did not activate the invertebrate troponin C-depleted crustacean myofibrils. On the other hand, two kinds of crustacean (crayfish, lobster) troponin C with two Ca(2+)-binding sites activated only crustacean troponin C-depleted myofibrils. One invertebrate molluscan (Akazara scallop) troponin C with one Ca(2+)-binding site did not activate the Ca(2+)-ATPase of the troponin C-depleted myofibrils from either vertebrate or crustacean striated muscles. The results obtained from the four kinds of combinations of troponin C and troponin C-depleted myofibrils from vertebrate skeletal and cardiac muscles demonstrated that the characteristic cooperativity of the Ca(2+)-activation profiles of both vertebrate skeletal and cardiac myofibrils was determined by the skeletal or cardiac origin of troponin C molecules, irrespective of the animal species, and the Ca(2+)-affinity of the myofibrillar ATPase was related to the skeletal or cardiac origin of both the troponin C and myofibrils. These findings indicated that each of the four classes of troponin C has its own characteristic Ca(2+)-activation profile for each kind of myofibril examined in the present study.

Animals↗

PubMed2000

Binding of filamin isoforms to myofibrils.

Two filamin isoforms were purified from bovine tissues and characterized. Muscle filamin and nonmuscle filamin had different SDS gel mobilities, proteolytic digestion patterns, myofibrillar binding distributions and myofibril binding affinities. The muscle specific filamin had an apparent molecular weight of 265 kDa and bound primarily to the Z-lines of myofibrils but also to the I-bands near the Z-lines. The nonmuscle specific filamin had an apparent molecular weight of 275 kDa and bound exclusively to the Z-lines of myofibrils. The filamin myofibril binding was studied quantitatively. Plotting bound fraction (mg filamin/mg myofibril) vs. equilibrium concentration of free filamin yielded a biphasic binding curve. The first hyperbolic binding phase described the binding of filamin to myofibrils but the second phase appeared to be nonspecific due to filamin aggregation. The muscle filamin had a significantly lower (P < 0.05) apparent binding affinity to myofibrils than nonmuscle filamin. However, the muscle filamin showed a significantly higher (P < 0.05) saturation value for myofibrils than nonmuscle filamin. The binding of phosphorylated filamin to myofibrils was significantly lower (P < 0.05) than the corresponding native proteins for both filamin isoforms.

Animals↗

PubMed1996

Myofibril-bound muscle phosphofructokinase is less sensitive to inhibition by ATP than the free enzyme, but retains its sensitivity to stimulation by bisphosphorylated hexoses.

Phosphofructokinase activity is modulated by allosteric effectors and macromolecular interactions (e.g. binding to myofibrillar components). The aim of this study was to determine the effects of ATP and bisphosphorylated sugars upon phosphofructokinase in the presence of myofibrils. Myofibrils were prepared from resting and electrically stimulated rat muscle. Dephosphorylation of myofibrils was performed with alkaline phosphatase acid. Purified rabbit skeletal muscle phosphofructokinase was used for all experiments. Myofibrils from resting muscle showed a higher capacity to bind phosphofructokinase and a lower phosphate content than myofibrils from stimulated muscle. Dephosphorylation of myofibrils did not increase their binding capacity. Myofibrils greatly counteracted the inhibition of phosphofructokinase by high concentrations of ATP, without affecting maximum activity. In the presence of myofibrils, both glucose 1,6-bisphosphate and fructose 2,6-bisphosphate additionally activated muscle phosphofructokinase. We suggest that the binding of phosphofructokinase to myofibrils in combination with increasing glucose 1,6-bisphosphate concentration could be important in the enhancement of the glycolytic flux that takes place during muscle contraction.

Adenosine Triphosphate↗

PubMed1993

Probing actin incorporation into myofibrils using Asp11 and His73 actin mutants.

We used a cell free system Bouché et al.: J. Cell Biol. 107:587-596, 1988] to study the incorporation of actin into myofibrils. We used alpha-skeletal muscle actin and actins with substitutions of either His73 [Solomon and Rubenstein: J. Biol.Chem. 262:11382, 1987], or Asp11 [Solomon et al.: J. Biol. Chem. 263:19662, 1988]. Actins were translated in reticulocyte lysate and incubated with myofibrils. The incorporated wild type actin could be cross-linked into dimers using N,N'-1,4-phenylenebismaleimide (PBM), indicating that the incorporated actin is actually inserted into the thin filaments of the myofibril. The His73 mutants incorporated to the same extent as wild type actin and was also cross-linked with PBM. Although some of the Asp11 mutants co-assembled with carrier actin, only 1-3% of the Asp11 mutant actins incorporated after 2 min and did not increase after 2 hr. Roughly 17% of wild type actin incorporated after 2 min and 31% after 2 hr. ATP increased the release of wild type actin from myofibrils, but did not increase the release of Asp11 mutants. We suggest that (1) the incorporation of wild type and His73 mutant actins was due to a physiological process whereas association of Asp11 mutants with myofibrils was non-specific, (2) the incorporation of wild type actin involved a rapid initial phase, followed by a slower phase, and (3) since some of the Asp11 mutants can co-assemble with wild type actin, the ability to self-assemble was not sufficient for incorporation into myofibrils. Thus, incorporation probably includes interaction between actin and a thin filament associated protein. We also showed that incorporation occurred at actin concentrations which would cause disassembly of F-actin. Since the myofibrils did not show large scale disassembly but incorporated actin, filament stability and monomer incorporation are likely to be mediated by actin associated proteins of the myofibril.

Actins↗

PubMed1993

Infection with the avian polyomavirus, BFDV, selectively affects myofibril structure in embryonic chick ventricle cardiomyocytes.

Embryonic cardiomyocytes can both beat and divide. They assemble cardiac muscle-specific proteins into sarcomeric myofibrils and contract. In addition, they periodically synthesize DNA, complete mitosis, disassemble sarcomeric myofibrils in the area of the mitotic spindle, assemble cytoplasmic isoform-specific proteins into a cleavage furrow contractile ring, undergo cytokinesis, and then reform sarcomeric myofibrils in daughter cells. Little is known about how embryonic cardiomyocytes disassemble their myofibrils as they traverse the cell cycle and divide. In the present study, beating embryonic avian ventricular cardiomyocytes in primary culture were stimulated to initiate DNA synthesis without subsequent mitosis or cytokinesis by infection with the lytic avian polyomavirus, Budgerigar Fledgling Disease Virus (BFDV). Within 48 hours, infected, adherent cardiomyocytes disassemble most of their sarcomeric myofibrils, retaining cardiac myosin only in thin myofibrils with disrupted sarcomeric periodicity and in amorphous nonfibrillar pools. By 72 hours, infected cardiomyocytes contain no myofibrils and no longer react with antibodies to cardiac myosin. In contrast, infected cardiomyocytes continue to display cytoplasmic myosin localized in stress-fiber-like-structures in adherent cells, or in disrupted fibers and dispersed pools in detaching cells. Infected cardiomyocytes also continue to display interphase-like arrays of polymerized microtubules, even when rounded-up just prior to lysis. These results suggest that polyomavirus infection may provide a useful model system for further study of the regulation of myofibrils disassembly in embryonic cardiomyocytes.

Animals↗

PubMed1984

Structural change of myofibrils during mitosis of newt embryonic myocardial cells in culture.

Newt embryonic myocardial cells can undergo mitosis in culture. The successive changes in the striation pattern of sarcomeres of myofibrils during mitosis were studied by polarization microscopy without fixing or killing the cells. Birefringence of well-organized striation patterns, i.e., bright A-bands and dark I-bands, was clearly visible in interphase cells and did not show any detectable changes during incubation for 3 h or more. Electron microscopy showed the presence of well-organized myofibrils with Z-bands in these interphase cells. When myocardial cells entered the mitotic stage, the birefringence of striation pattern of their myofibrils gradually changed with the pattern in small parts of the myofibrils gradually becoming indistinct (called 'indistinct striation' in this paper). These indistinct regions increased in size during the mitotic stage. In addition, in some regions of the indistinct striation, the birefringence of sarcomeres gradually decreased and finally disappeared (called 'disappearance of sarcomeres' in this paper). No myocardial cells underwent mitosis without these disruptive changes of the myofibril striation patterns. In the post-mitotic stage, the well-organized striation of the myofibrils reappeared. Electron microscopy showed disorganized sarcomeres without Z-bands in the regions of indistinct striation, and no well-organized myofibrils in the regions where the sarcomeres had disappeared. Thus the well-organized myofibrils with Z-bands became transiently disorganized at least in some parts, during mitosis. They were then reorganized into daughter myocardial cells.

Animals↗

PubMed1993

Rotational dynamics of actin-bound myosin heads in active myofibrils.

We have used saturation-transfer electron paramagnetic resonance (ST-EPR) to measure the submillisecond rotational motions of actin-bound myosin heads in active myofibrils. The cross-bridges were spin-labeled with a maleimide nitroxide derivative (MSL) that has previously been shown to undergo microsecond rotational motions on actin-bound myosin heads in solution during steady-state ATPase activity at low ionic strength [Berger, C. L., Svensson, E. C., & Thomas, D. D. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 8573]. To determine whether this is also true for cross-bridges in the myofibrillar lattice under physiological buffer conditions, we have performed ST-EPR experiments during the brief steady state following photolysis of caged ATP in a suspension of spin-labeled myofibrils. The myofibrils were partially cross-linked with EDC [1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide] to prevent their shortening upon activation. The fraction of actin-attached myosin heads was determined biochemically at physiological ionic strength in the active myofibrils, using the proteolytic rates acto-myosin binding assay [Duong, A. M., & Reisler, E. (1989) Biochemistry 28, 3502]. These data were then used to correct the ST-EPR spectra of active myofibrils for the presence of unattached myosin heads, which were assumed to undergo the same motions as in relaxation. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to have considerable microsecond rotational motion (tau r = 3.5 +/- 1.1 microseconds) in the active myofibrils. Similar results (tau r = 3.2 +/- 0.8 microseconds) were obtained with active myofibrils at low ionic strength (mu = 45 mM), confirming the work done in solution. Thus, under physiological conditions and even within the constraints of the myofibrillar lattice, actively cycling actin-attached myosin heads are rotationally mobile on the microsecond time scale. Since partially EDC-fixed myofibrils are an excellent analog of isometrically contracting muscle fibers in solution, it is likely that these microsecond rotational motions are directly related to the molecular mechanism of muscle contraction in vivo.

Actins↗

PubMed1989

Binding of myosin to actin in myofibrils during ATP hydrolysis.

Measurements of cross-bridge attachment to actin in myofibrils during ATP hydrolysis require prior fixation of myofibrils to prevent their contraction. The optimal cross-linking of myofibrils was achieved by using 10 mM carbodiimide (EDC) under rigor conditions and at 4 degrees C. The fixed myofibrils had elevated MgATPase activity (150%) and could not contract. As judged by chymotryptic digestions and subsequent SDS gel electrophoresis analysis, less than 25% of myosin heads were cross-linked in these myofibrils. The isolated, un-cross-linked myosin heads showed pH-dependent Ca2+- and EDTA(K+)-ATPase activities similar to those of standard intact S-1. For measurements of myosin binding to actin, the modified myofibrils were digested with trypsin at a weight ratio of 1:50 under rigor, relaxed, and active-state conditions. Aliquots of tryptic digestion reactions were then cleaved with chymotrypsin to yield isolated myosin heads and their fragments. Analysis of the decay of myosin heavy-chain bands on SDS gels yielded the rates of myosin cleavage under all conditions and enabled the measurements of actomyosin binding in myofibrils in the presence of MgATP. Using this approach, we detected rigorlike binding of 25 +/- 6% of myosin heads to actin in myofibrils during ATP hydrolysis.

Actins↗

PubMed2003

Why choose myofibrils to study muscle myosin ATPase?

Our objective is to propose an overview of the usefulness of skeletal myofibril as an experimental system for studying mechanochemical coupling of skeletal muscles and myosin ATPase activity. The myofibril is a true functional mini-muscle that is able to contract in the presence of ATP. It also contains the machinery necessary for the calcium sensitivity of the contraction. In the absence of calcium, myofibrillar ATPase activity is basal, no shortening occurs and no active force is developed. In the presence of calcium, myofibrillar ATPase is activated and myofibrils either shorten with no external load (native myofibrils) or contract isometrically (cross-linked myofibrils). With this organised system, both chemical and mechanical studies can be carried out. For a decade, our laboratory has been using rabbit psoas myofibrils for exploring myosin ATPase activity. The first challenge was to successfully apply rapid kinetic approaches, such as rapid-flow-quench, to this organised system. Another challenge was to work with myofibrils in cryoenzymic conditions, i.e. in the presence of organic solvents and at sub-zero temperatures. In this overview, we highlight differences between the myosin ATPase in organised systems (myofibrils or fibres) and that of contractile proteins in solution (S1 or actoS1) that we observed using these approaches. We discuss the importance of these differences in terms of mechanochemical coupling. It is concluded that great care should be taken when extrapolating mechanochemical properties of the contractile proteins in solution to the whole muscle.

Actins↗

PubMed2001

The effect of Mg2+ on cardiac muscle function: Is CaATP the substrate for priming myofibril cross-bridge formation and Ca2+ reuptake by the sarcoplasmic reticulum?

Kinetics are established for the activation of the myofibril from the relaxed state [Smith, Dixon, Kirschenlohr, Grace, Metcalfe and Vandenberg (2000) Biochem. J. 346, 393-402]. These require two troponin Ca2+-binding sites, one for each myosin head, to act as a single unit in initial cross-bridge formation. This defines the first, or activating, ATPase reaction, as distinct from the further activity of the enzyme that continues when a cross-bridge to actin is already established. The pairing of myosin heads to act as one unit suggests a possible alternating mechanism for muscle action. A large positive inotropic (contraction-intensifying) effect of loading the Mg2+ chelator citrate, via its acetoxymethyl ester, into the heart has confirmed the competitive inhibition of the Ca2+ activation by Mg2+, previously seen in vitro. In the absence of a recognized second Ca2+ binding site on the myofibril, with appropriate binding properties, the bound ATP is proposed as the second activating Ca2+-binding site. As ATP, free or bound to protein, can bind either Mg2+ or Ca2+, this leads to competitive inhibition by Mg2+. Published physico-chemical studies on skeletal muscle have shown that CaATP is potentially a more effective substrate than MgATP for cross-bridge formation. The above considerations allow calculation of the observed variation of fractional activation by Ca2+ as a function of [Mg2+] and in turn reveal simple Michaelis-Menten kinetics for the activation of the ATPase by sub-millimolar [Mg2+]. Furthermore the ability of bound ATP to bind either cation, and the much better promotion of cross-bridge formation by CaATP binding, give rise to the observed variation of the Hill coefficient for Ca2+ activation with altered [Mg2+]. The inclusion of CaADP within the initiating cross-bridge and replacement by MgADP during the second cycle is consistent with the observed fall in the rate of the myofibril ATPase that occurs after two phosphates are released. The similarity of the kinetics of the cardiac sarcoplasmic reticulum ATPase to those of the myofibril, in particular the positive co-operativity of both Mg2+ inhibition and Ca2+ activation, leads to the conclusion that this ATPase also has an initiation step that utilizes CaATP. The first-order activation by sub-millimolar [Mg2+], similar to that of the myofibril, may be explained by Mg2+ involvement in the phosphate-release step of the ATPase. The inhibition of both the myofibril and sarcoplasmic reticulum Ca2+ transporting ATPases by Mg2+ offers an explanation for the specific requirement for phosphocreatine (PCr) for full activity of both enzymes in situ and its effect on their apparent affinities for ATP. This explanation is based on the slow diffusion of Mg2+ within the myofibril and on the contrast of PCr with both ATP and phosphoenolpyruvate, in that PCr does not bind Mg2+ under physiological conditions, whereas both the other two bind it more tightly than the products of their hydrolysis do. The switch to supply of energy by diffusion of MgATP into the myofibril when depletion of PCr raises [ATP]/[PCr] greatly, e.g. during anoxia, results in a local [Mg2+] increase, which inhibits the ATPase. It is possible that mechanisms similar to those described above occur in skeletal muscle but the Ca2+ co-operativity involved would be masked by the presence of two Ca2+ binding sites on each troponin.

Adenosine Triphosphatases↗