Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of 2,3-butanedione monoxime on myosin and myofibrillar ATPases. An example of an uncompetitive inhibitor.

2,3-Butanedione monoxime (BDM) reversibly inhibits force production in muscle. At least part of its action appears to be directly on the contractile apparatus. To understand better its mechanism of action, we studied the effect of BDM on the steps of myosin subfragment 1 Mg(2+)-ATPase in 0.1 M potassium acetate, pH 7.4. Because of the rapidity of certain processes, we experimented at 4 degrees C and our main technique was the rapid flow quench method. By varying the experimental conditions (relative concentrations of reagents, time scale, quenching agent), it was possible to study selectively the different steps of the S1 Mg(2+)-ATPase: [formula: see text] At saturation (20 mM), BDM had two major effects on the ATPase. First, it increased the equilibrium constant of the cleavage step (K3) from 2 to > 10. Second, it slowed the kinetics of the release of Pi by an order of magnitude (k4; from 0.054 to 0.004 s-1). By contrast, the kinetics of the binding of ATP (k) and the release of ADP (k6) were little affected by BDM. Thus, the oxime appears to interact specifically with M**.ADP.Pi, and it is a rare example of an uncompetitive inhibitor. Its effect is to reduce the steady-state concentration of the "strong" actin binding state M*.ADP and to increase that of the "weak" binding state, M**.ADP.Pi. The effect of BDM on the initial ATPase of Ca2+ activated myofibrils was very similar to that on S1 ATPase. Thus, with myofibrils too BDM seems to exert its main effect subsequent to the initial binding and cleavage steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide turnover rate measured in fully relaxed rabbit skeletal muscle myofibrils

Steady state measurements of the ATP turnover rate of myosin crossbridges in relaxed living mammalian muscle or in in vitro systems are complicated by other more rapid ATPase activities. To surmount these problems we have developed a technique to measure the nucleotide turnover rate of fully relaxed myosin heads in myofibrils using a fluorescent analogue of ATP (mant-ATP). Rabbit myofibrils, relaxed in 1.6 mM ATP, were rapidly mixed with an equal volume of solution containing 80 microM mant-ATP and injected into a fluorimeter. As bound ADP is released, a fraction of the myosin active sites bind mant-ATP and fluorescence emission rises exponentially, defining a rate of nucleotide turnover of 0.03 +/- 0.001 s-1 at 25 degrees C (n = 17). This rate was approximately equal to one half that of purified myosin. The turnover rates for myosin and myofibrils increased between 5 degrees and 42 degrees C, reaching 0.16 +/- 0.04 s-1 and 0.06 +/- 0.005 s-1, respectively, at 39 degrees C, the body temperature of the rabbit. If the rate observed for purified myosin occurred in vivo, it would generate more heat than is observed for resting living muscle. When myosin is incorporated into the myofilament lattice, its ATPase activity is inhibited, providing at least a partial explanation for the low rate of heat production by living resting muscle.     

Kinetic mechanism of 1-N6-etheno-2-aza-ATP and 1-N6-etheno-2-aza-ADP binding to bovine ventricular actomyosin-S1 and myofibrils

The fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) at 410-460 nm is enhanced approximately 8-fold upon mixing substoichiometric concentrations of epsilon-aza-ATP with bovine cardiac actomyosin-S1 or myofibrils. The time course of nucleotide fluorescence measured in a front face stopped flow cell upon mixing epsilon-aza-ATP with bovine cardiac myofibrils ([Ca2+] less than 10(-7) M) is essentially the same as that with bovine cardiac actomyosin subfragment-1. In single turnover experiments, the fluorescence rapidly rises to a maximum value, then decreases with a rate constant of 0.04 s-1 at 0 degree C to a final value that is approximately twice the level of the unbound nucleotide. At concentrations of epsilon-aza-ATP greater than 40 microM the kinetics of epsilon-aza-ATP binding is clearly biphasic for both actomyosin-S1 and myofibrils. At 0 degree C, the rate of the more rapid phase is proportional to nucleotide concentration and has a second order rate constant of 1.7 X 10(5) M-1 s-1; the rate of the slower phase extrapolates to a maximum of 4-5 s-1 at high nucleotide concentration. The rate constants for dissociation of epsilon-aza-ADP from bovine cardiac actomyosin-S1 and myofibrils were measured from the decrease in epsilon-aza-ADP fluorescence enhancement observed upon displacement by ATP to be 20 and 18 s-1, respectively, at 0 degree C. These results indicate that most of the cross-bridges in cardiac myofibrils are bound to actin and that the geometric constraints imposed upon the interaction of actin and myosin by the three-dimensional structure of the myofibril do not modify the kinetics of epsilon-aza-ATP binding or epsilon-aza-ADP dissociation.     

Structure and function of the two heads of the myosin molecule. I. Binding of adenosine diphosphate to myofibrils during the adenosinetriphosphatase reaction.

1. The myosin content of myofibrils was found to be 51% by SDS-gel electrophoresis. 2. The initial burst of Pi liberation of the ATPase [EC 3.6.1.3] of a solution of myofibrils in 1 M KCl was measured in 0.5 M KCl, and found to be 0.93 mole/mole of myosin. 3. The amount of ADP bound to myofibrils during the ATPase reaction and the ATPase activity were measured by coupling the myofibrillar ATPase reaction with sufficient amounts of pyruvate kinase [EC 2.7.1.40] and PEP to regenerate ATP. The maximum amount of ADP bound to myofibrils in 0.05M KCl and in the relaxed state was about 1.5 mole/mole of myosin. On the other hand, the ATPase activity exhibited substrate inhibition, and the amount of ATP required for a constant level of ATPase activity was smaller than that required for the maximum binding of ADP to myofibrils. 4. The maximum amount of ADP bound to myofibrils in 0.5 M KCl was about 1.9 mole/mole of myosin. When about one mole of ADP was found to 1 mole of myosin in myofibrils, the myofibrillar ATPase activity reached the saturated level, and with further increase in the concentration of ATP one more mole of ADP was found per mole of myosin.     

Reaction intermediates formed by myofibrils during the ATPase reaction under relaxed conditions

The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, M*ATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or M*ATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.     

Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

Submillisecond rotational dynamics of spin-labeled myosin heads in myofibrils.

The rotational motion of crossbridges, formed when myosin heads bind to actin, is an essential element of most molecular models of muscle contraction. To obtain direct information about this molecular motion, we have performed saturation transfer EPR experiments in which spin labels were selectively and rigidly attached to myosin heads in purified myosin and in glycerinated myofibrils. In synthetic myosin filaments, in the absence of actin, the spectra indicated rapid rotational motion of heads characterized by an effective correlation time of 10 microseconds. By contrast, little or no submillisecond rotational motion was observed when isolated myosin heads (subfragment-1) were attached to glass beads or to F-actin, indicating that the bond between the myosin head and actin is quite rigid on this time scale. A similar immobilization of heads was observed in spin-labeled myofibrils in rigor. Therefore, we conclude that virtually all of the myosin heads in a rigor myofibril are immobilized, apparently owing to attachment of heads to actin. Addition of ATP to myofibrils, either in the presence or absence of 0.1 mM Ca2+, produced spectra similar to those observed for myosin filaments in the absence of actin, indicating rapid submillisecond rotational motion. These results indicate that either (a) most of the myosin heads are detached at any instant in relaxed or activated myofibrils or (b) attached heads bearing the products of ATP hydrolysis rotate as rapidly as detached heads.     

Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.

Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.     

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.     

Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.

The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

Characterization of the myosin adenosine triphosphate (M.ATP) crossbridge in rabbit and frog skeletal muscle fibers.

In the presence of ATP and absence of Ca2+, muscle crossbridges have either MgATP or MgADP.Pi bound at the active site (S. B. Marston and R. T. Tregear, Nature [Lond.], 235:22:1972). The behavior of these myosin adenosine triphosphate (M.ATP) crossbridges, both in relaxed skinned rabbit psoas and frog semitendinosus fibers, was analyzed. At very low ionic strength, T = 5 degrees C, mu = 20 mM, these crossbridges spend a large fraction of the time attached to actin. In rabbit, the attachment rate constants at low salt are 10(4) - 10(5) s-1, and the detachment rate constants are approximately 10(4) s-1. When ionic strength is increased up to physiological values by addition of 140 mM potassium propionate, the major effect is a weakening of the crossbridge binding constant approximately 30-40-fold. This effect occurs because of a large decrease, approximately 100-fold, in the crossbridge attachment rate constants. The detachment rate constants decrease only 2-3-fold. The effect of ionic strength on crossbridge binding in the fiber is very similar to the effect of ionic strength on the binding of myosin subfragment-1 to unregulated actin in solution. Thus, the effect of increasing ionic strength in fibers appears to be a direct effect on crossbridge binding rather than an effect on troponin-tropomyosin. The finding that crossbridges with ATP bound at the active site can and do attach to actin over a wide range of ionic strengths strongly suggests that troponin-tropomyosin keeps a muscle relaxed by blocking a step subsequent to crossbridge attachment. Thus, rather than troponin-tropomyosin serving to keep a muscle relaxed by inhibiting attachment, it seems quite possible that the main way in which troponin-tropomyosin regulates muscle activity is by preventing the weakly-binding relaxed crossbridges from going on through the crossbridge cycle into more strongly-binding states.     

Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils.

We have used saturation transfer electron paramagnetic resonance (ST-EPR) to measure the microsecond rotational motion of actin-bound myosin heads in spin-labeled myofibrils in the presence of the ATP analogs AMPPNP (5'-adenylylimido-diphosphate) and ATP gamma S (adenosine-5'-O-(3-thiotriphosphate)). AMPPNP and ATP gamma S are believed to trap myosin in two major conformational intermediates of the actomyosin ATPase cycle, respectively known as the weakly bound and strongly bound states. Previous ST-EPR experiments with solutions of acto-S1 have demonstrated that actin-bound myosin heads are rotationally mobile on the microsecond time scale in the presence of ATP gamma S, but not in the presence of AMPPNP. However, it is not clear that results obtained with acto-S1 in solution can be extended to actomyosin constrained within the myofibrillar lattice. Therefore, ST-EPR spectra of spin-labeled myofibrils were analyzed explicitly in terms of the actin-bound component of myosin heads in the presence of AMPPNP and ATP gamma S. The fraction of actin-attached myosin heads was determined biochemically in the spin-labeled myofibrils, using the proteolytic rates actomyosin binding assay. At physiological ionic strength (mu = 165 mM), actin-bound myosin heads were found to be rotationally mobile on the microsecond time scale (tau r = 24 +/- 8 microseconds) in the presence of ATP gamma S, but not AMPPNP. Similar results were obtained at low ionic strength, confirming the acto-S1 solution studies. The microsecond rotational motions of actin-attached myosin heads in the presence of ATP gamma S are similar to those observed for spin-labeled myosin heads during the steady-state cycling of the actomyosin ATPase, both in solution and in an active isometric muscle fiber. These results indicate that weakly bound myosin heads, in the pre-force phase of the ATPase cycle, are rotationally mobile, while strongly bound heads, in the force-generating phase, are rotationally immobile. We propose that force generation involves a transition from a dynamically disordered crossbridge to a rigid and stereospecific one.     

Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate

The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent Kd of 3.8 X 10(-5) M. The binding of ANS- had no effect on Ca2+ transport or Ca2+-dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS-with half-maximal effect at 4 X 10(-5) M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+, Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).     

Effects of removal and reconstitution of myosin regulatory light chain and troponin C on the Ca(2+)-sensitive ATPase activity of myofibrils from scallop striated muscle

In order to examine the involvement of troponin-linked Ca(2+)-regulation, in addition to well-known myosin-linked Ca(2+)-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca(2+) by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca(2+)-concentration at 25 and 15 degrees C. In the absence of Ca(2+), the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca(2+)-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25 degrees C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15 degrees C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca(2+)-regulation, and that the troponin-linked Ca(2+)-regulation is more significant at lower temperature.     

Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.

The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.     

Rate of ATP synthesis by dynein

The rates of ATP synthesis and release by the dynein ATPase were determined in order to estimate thermodynamic parameters according to the pathway: (Formula: see text). Dynein was incubated with high concentrations of ADP and Pi to drive the net synthesis of ATP, and the rate of ATP production was monitored fluorometrically by production of NADPH through a coupled assay using hexokinase and glucose-6-phosphate dehydrogenase. The turnover number for the rate of release of ATP from 22S dynein was 0.01 s-1 per site at pH 7.0, 28 degrees C, assuming a molecular weight of 750 000 per site. The same method gave a rate of ATP synthesis by myosin subfragment 1 of 3.4 X 10(-4) s-1 at pH 7.0, 28 degrees C. The rate of ATP synthesis at the active site was estimated from the time dependence of medium phosphate-water oxygen exchange. Dynein was incubated with ADP and [18O] Pi, and the rate of loss of the labeled oxygen to water was monitored by 31P NMR. A partition coefficient of 0.31 was determined, which is equal to k-2/(k-2 + k3). Assuming k3 = 8 s-1 [Johnson, K.A. (1983) J. Biol. Chem. 258, 13825-13832], k-2 = 3.5 s-1. From the rates of ATP binding and hydrolysis measured previously (Johnson, 1983), the equilibrium constants for ATP binding and hydrolysis could be calculated: K1 = 5 X 10(7) M-1 and K2 = 14.(ABSTRACT TRUNCATED AT 250 WORDS)