Kinetic mechanism of 1-N6-etheno-2-aza-ATP hydrolysis by bovine ventricular myosin subfragment 1 and actomyosin subfragment 1. The nucleotide binding steps

The large change in fluorescence emission of 1-N6-etheno-2-aza-ATP (epsilon-aza-ATP) has been used to investigate the kinetic mechanism of etheno-aza nucleotide binding to bovine cardiac myosin subfragment 1 (myosin-S1) and actomyosin subfragment 1 (actomyosin-S1). The time course of nucleotide fluorescence enhancement observed during epsilon-aza-ATP hydrolysis is qualitatively similar to the time course of tryptophan fluorescence enhancement observed during ATP hydrolysis. In single turnover experiments, the nucleotide fluorescence rapidly increases to a maximum level, then decreases with a rate constant of 0.045 s-1 to a final level, which is about 30% of the maximal enhancement; a similar fluorescence enhancement is obtained by adding epsilon-aza-ADP to cardiac myosin-S1 or actomyosin-S1 under the same conditions (100 mM KCl, 10 mM 4-morpholinepropanesulfonic acid, 5 mM MgCl2, 0.1 mM dithiothreitol, pH 7.0, 15 degrees C). The kinetic data are consistent with a mechanism in which there are two sequential (acto)myosin-S1 nucleotide complexes with enhanced nucleotide fluorescence following epsilon-aza-ATP binding. The apparent second order rate constants of epsilon-aza-ATP binding to cardiac myosin subfragment 1 and actomyosin subfragment 1 are 2-12 times slower than those for ATP. Actin increases the rate of epsilon-aza-ADP dissociation from bovine cardiac myosin-S1 from 1.9 to 110 s-1 at 15 degrees C which can be compared to 0.3 and 65 s-1 for ADP dissociation under similar conditions. Although there are quantitative differences between the rate and equilibrium constants of epsilon-aza- and adenosine nucleotides to cardiac actomyosin-S1 and myosin-S1, the basic features of the nucleotide binding steps of the mechanism are unchanged.     

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.     

Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism.

The kinetics of the increase in protein fluorescence following the addition of ATP to subfragment-1 (SF-1) and acto-SF-1 have been reinvestigated. The concentration dependence of the rate obtained with SF-1 did not fit a hyperbola and at high ATP concentration, approximately 40% of the signal amplitude was lost due to a fast phase at the beginning of the transient (20 degrees C). At lower temperature (less than or equal to 10 degrees C) the fluorescence transient was biphasic, with a fast phase observed at high ATP concentration. These results indicate that there are two steps in the SF-1 pathway in which there is a change in protein fluorescence. Measurements of ATP binding and hydrolysis by chemical quench-flow methods indicate that the rate of ATP binding is correlated with the fast fluorescence step and hydrolysis is correlated with the slow fluorescence change. The SF-1 mechanism can thus be described as: (formula: see text) where M represents SF-1 and states of enhanced fluorescence are given by M (16%) and M (36% enhancement, relative to SF-1). Step 1 is a rapid equilibrium with K1 approximately 10(3) M-1. Tight binding of ATP occurs in step 2 and the loss of signal amplitude requires k2 greater than or approximately 1500--2000 s-1. The maximum observed fluorescence rate defines the rate of hydrolysis, k3 + k-3 = 125 s-1 (20 degrees C, 0.1 M KCl, pH 7.0). The steps in the mechanism correspond to the Bagshaw--Trentham scheme, with the important difference that the assignment of rate constant is altered. Formation of the acto-SF-1 complex gave a fluorescence enhancement of approximately 14% relative to SF-1. Dissociation of acto-SF-1 by ATP produced a 20--22% enhancement in fluorescence. There was no detectable fluorescence change during dissociation as evidenced by a lag in the fluorescence transient which corresponded to the kinetics of dissociation. The fluorescence change occurred at the same maximum rate as for SF-1 but there was no loss in signal amplitude at high ATP concentration. The kinetics of the fluorescence change corresponded to the rate of ATP hydrolysis, whereas tight ATP binding occurred at a much faster rate in approximate agreement with the rate of dissociation. Thus the fluorescence change in the acto-SF-1 pathway corresponds to step 3 in the SF-1 mechanism. The complete scheme can be described as follows: (formula: see text) where AM represents acto-SF-1. The tight binding step in the SF-1 pathway (k2) is sufficiently fast so that a similar step (k2') in the acto-SF-1 pathway could precede dissociation but the AM-ATP intermediate has not been detected. Following hydrolysis on the free SF-1, actin recombines with M.ADP.Pi or possibly with a second SF-1 product intermediate as proposed by Chock et al. (1976) and the fluorescence returns to the original AM level with product release.     

Effects of tryptic digestion on myosin subfragment 1 and its actin-activated adenosinetriphosphatase

Myosin subfragment 1 (S-1) was fluorescently labeled at its rapidly reacting thiol ("SH1"). Short exposure to trypsin cuts the S-1 heavy chain into three still-associated fragments (20K, 50K, and 27K) [Balint, M., Wolf, L., Tarcsafalvi, A., Gergely, J., & Sreter, F.A. (1978) Arch. Biochem. Biophys. 190, 793-799] which bind F-actin to the same extent as does the uncut labeled S-1, as indicated by time-resolved fluorescence anisotropy decay (at 4 degrees C, pH 7, in 0.15 M KC1 and 5 mM MgC12, +/- 1 mM ADP). These results are thus in agreement with turbidity measurements on similar systems as reported by Mornet et al. [Mornet, D., Pantel, P., Audemard, E., & Kassab, R. (1979) Biochem. Biophys. Res. Commun. 89, 925-932]. The excited-state lifetime of the fluorescent label on cut S-1 is indistinguishable from that on normal S-1 (+/- ADP, +/- F-actin). F-Actin activation of MgATPase of cut S-1 is lower than that for normal S-1 at moderate concentrations of F-actin, as reported by Mornet et al. (1979). But as the F-actin concentration is increased, the MgATPase activities for cut S-1 approach those for uncut S-1. In terms of an eight-species steady-state kinetics scheme involving actin binding to free S-1, S-1 . ATP, S-1. ADP X P, and S-1 . ADP, actin affinity for the species S-1 . ADP X P was found to be 13.4 times greater for uncut S-1 than for cut S-1 [at 24 degrees C, pH 7.0, in 3 mM KC1, 1 mM ATP, 1 mM MgCl2, and 20 mM N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid].     

The effect of lithium and potassium on the transient state kinetics of the (Ca + Mg)-ATPase of cardiac sarcoplasmic reticulum.

KCl or LiCl, when added in 100 mM concentrations to cardiac sarcoplasmic reticulum incubated at 17 degrees C with 5 micron [gamma-32P]ATP, 1 mM MgCl2, and 9.1 micron M Ca2+, increased the apparent phosphorylation rate constant from 14.5 s-1 to 23.8 s-1 (100 mM LiCl) or to 44.1 s-1 (100 mM KCl). These same monovalent cations also increased the apparent rate constant for the hydrolysis of the phosphorylated sarcoplasmic reticulum from 0.51 s-1 to 1.12 s-1 (100 mM LiCl) or to 1.71 s-1 (100 mM KCl). Although there was a small burst in Pi production, rate constant of 0.97 s-1, when 100 mM KCl was added, the burst when LiCl or no monovalent cation was added was either nonexistent or so small as to make its detection unreliable. KCl thus appears to induce an intermediate which is either nonexistent when omitted or in such low concentration as not to be readily detected.     

The kinetics for the phosphoryl transfer steps of the sarcoplasmic reticulum calcium ATPase are the same with strontium and with calcium bound to the transport sites.

Rate constants for most of the steps of the reaction cycle of the sarcoplasmic reticulum calcium-ATPase are similar or identical with Ca2+ or Sr2+ as the transported ions in spite of the large differences in the size and affinity of Ca2+ and Sr2+ (5 mM MgCl2, 100 mM KCl, pH 7.0, 25 degrees C). Phosphorylation of cE.Sr2 and cE.Ca2 by ATP occurs with kp = 220-235 s-1, whereas phosphorylation of E.ATP+Ca2+ or Sr2+ is consistent with kb = 50-70 s-1. Hydrolysis of E approximately P.Sr2 and E approximately P.Ca2 occurs with kt = 20 s-1, and the addition of 7 mM ADP to E approximately P.Sr2 or to E approximately P.Ca2 gives a burst of approximately 43% dephosphorylation, followed by dephosphorylation with k = 46 s-1. However, one Sr2+ ion dissociates from cE.Sr2 and from cE.ATP.Sr2 with k congruent to 120 s-1, whereas one Ca2+ ion dissociates from cE.Ca2 with k = 38 s-1 and from cE.ATP.Ca2 with k = 80 s-1.     

Transient phase of adenosine triphosphate hydrolysis by myosin, heavy meromyosin, and subfragment 1.

The transient phase of adenosine triphosphate (ATP) hydrolysis (early burst) was investigated for myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) over a range of temperatures and pH's. The burst size at pH 8,20 degrees C, is 0.8-0.85, based on steady-state and transient measurements. The equilibrium constant for the enzyme-substrate to enzyme-product transition is 0.85 +/- 0.05. It is concluded that both myosin heads undergo the rapid hydrolysis step and that there are no significant differences for S-1 vs. HMM or myosin. The transient data are fitted reasonably well by a single rate process, but available evidence is consistent with some heterogeneity and a range of rate constants differing by a factor of two. At pH 6.9 and 3 degrees C, the burst size is 0.5 and the hydrolysis is slower than the configuration change measured by fluorescence. The results are consistent with the kinetic scheme (see article). The lower burst at low temperature and pH can be partly explained by a reduction in the equilibrium constant, K3, and ATP can be synthesized on the enzyme by a pH-temperature jump.     

Slow dissociation of ATP from the calcium ATPase

The acyl-phosphate intermediate of the sarcoplasmic reticulum calcium ATPase reaction, formed in a brief incubation of vesicular enzyme with 5 microM [gamma-32P]ATP and calcium, reacts biphasically with added ADP (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4). Both the burst size and the rate constant for the slow phase increase with increasing ADP concentration in the way that is expected if the burst represents very rapid formation of an equilibrium amount of enzyme-bound ATP and the slow phase represents rate-limiting dissociation of ATP. Also consistent with this interpretation are the slow labeling of phosphoenzyme under conditions in which unlabeled ATP must dissociate first and the observation of a burst of ATP formation on ADP addition to phosphoenzyme. Values of the equilibrium constants for ADP dissociation from phosphoenzyme (0.75 mM), for ATP formation on the enzyme (2.3), and for the ATP dissociation rate constant (37 s-1) were obtained from a quantitative analysis of the data.     

Demembranated muscle fibers catalyze a more rapid exchange between phosphate and adenosine triphosphate than actomyosin subfragment 1

The rate of ATP in equilibrium with Pi exchange, that is, the incorporation of medium Pi into ATP during the net hydrolysis of ATP, has been measured for rabbit psoas muscle fibers, myofibrils, and actomyosin subfragment 1 (acto-S1). The maximum exchange rate in fibers at saturating [Pi] is 0.04 s-1 per myosin head at 8 degrees C, pH 7, and an ionic strength of 0.2 M. The dependence of the rate on Pi concentration can be approximated by a hyperbola with an apparent dissociation constant (Km) of 3 mM. Myofibrils catalyze ATP in equilibrium with Pi exchange with a similar Km but at a slightly lower rate. In contrast, the soluble acto-S1 system, in which ATP hydrolysis is not coupled to tension generation, catalyzes exchange at a rate 500 times lower than that of fibers at low Pi concentration, and the Km for Pi is greater than 50 mM. The difference between the ATP in equilibrium with Pi exchange of fibers and of acto-S1 is discussed in terms of a model in which Pi binds to a force-generating state AM'-ADP and, due to mechanical constraint, the average free energy of this state is higher in the fiber than in acto-S1.     

Binding and hydrolysis of ATP by cardiac myosin subfragment 1: effect of solution parameters on transient kinetics

Transient kinetic data of ATP binding and cleavage by cardiac myosin subfragment 1 (S1) were obtained by fluorescence stopped flow and analyzed by using computer modeling based on a consecutive, reversible two-step mechanism: (formula: see text) where M1 and M12 denote myosin species with enhanced fluorescence and K'O = K0/(K0[ATP] + 1). The kinetic constants K0, k12, k23, and k32 and the fractional contributions of M1 and M12 to the total fluorescence are analyzed over a range of systematically varied solution parameters. The initial ATP binding equilibrium (K0), which decreases with increasing pH, is facilitated by a positively charged protein residue with a pK of 7.1. An active-site charge of +1.5 is determined from the ionic strength dependence. The rate constants k12, k23, and k32 also exhibit pK's near neutrality but increase with increasing pH. The majority of the large (-54 kJ/mol) negative free energy of ATP binding occurs upon S1 isomerization, k12, and a large increase in entropy (183 J/kmol at 15 degrees C) is associated with the cleavage step. The equilibrium constant for the cleavage step, K2, is determined as 3.5 at pH 7.0, 15 degrees C, and 200 mM ionic strength. There are no significant changes in fractional contributions to total fluorescence enhancement due to solvent-dependent conformational changes of S1 in these data. When values for the combined rate constants are calculated and compared with those determined by graphical analysis, it is observed that graphical analysis overestimates the binding rate constant (K0k12) by 25% and the hydrolysis rate constant (k23 + k32) by as much as 30%.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. Occurrence of two types of phosphoenzyme intermediates in the presence of KCl.

The steady state kinetics of ATP hydrolysis by partially purified adenosine triphosphatase preparations of sarcoplasmic reticulum was investigated at 0 degrees C and pH 7.0 in 2.0 mM MgCl2, 20 microM [gamma-32P]ATP, 20 microM CaCl2, and various concentrations of KCl in the presence and absence of 12% dimethyl sulfoxide. The steady state phosphoenzyme formed under these conditions could be resolved kinetically into ADP-sensitive and ADP-insensitive forms. These steady state kinetic data were analyzed according to a scheme in which the ADP-sensitive and ADP-insensitive phosphoenzymes occur sequentially, and Pi is derived from the latter. The KCl-dependent turnover rate of the ADP-insensitive phosphoenzyme that was estimated according to this scheme was in good agreement with the directly measured hydrolysis rate constant of the ADP-insensitive phosphoenzyme. In addition, the time course of the decomposition of the total amount of phosphoenzyme, measured after a steady state level was reached in 20 mM KCl and further phosphorylation was prevented by addition of excess ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid, was also in agreement with that calculated according to this scheme using values of the rate constants estimated from the amounts of the ADP-sensitive and ADP-insensitive phosphoenzymes and the rate of ATP hydrolysis. These results, together with our previous findings, support the view that this scheme describes the mechanism of ATP hydrolysis in the presence of KCl.     

Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kb = 70 +/- 7 s-1 after free enzyme was mixed with saturating ATP and 50 microM Ca2+; this is one-third the rate constant of 220 s-1 for phosphorylation of enzyme preincubated with calcium, cE.Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KCl, 5 mM Mg2+, 25 degrees C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives approximately 50% E approximately P.Ca2 with kobsd = 77 s-1, not the sum of the forward and reverse rate constants, kobsd = kf + kr = 140 s-1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kb represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of cE.Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions and phosphoryl transfer, have rate constants of greater than 500 s-1. Chase experiments with unlabeled ATP or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of [gamma-32P]ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s-1 from E.ATP.Ca2+ and at 24 s-1 from E.ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 microM ATP and 50 microM Ca2+ to enzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (kb). The fraction of Ca2+-free E.[gamma-32P]ATP that is trapped to give labeled phosphoenzyme after the addition of Ca2+ and a chase of unlabeled ATP is half-maximal at 6.8 microM Ca2+, with a Hill slope of n = 1.8. The calculated dissociation constant for Ca2+ from E.ATP.Ca2 is approximately 2.2 X 10(-10) M2 (K0.5 = 15 microM). The rate constant for the slow phase of the biphasic reaction of E approximately P.Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 microM to 10 nM, with half-maximal increase at 1.7 microM Ca2+. This shows that Ca2+ is dissociating from a different species, aE.ATP.Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k less than or equal to 45 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reactions of 1-N6-ethenoadenosine nucleotides with myosin subfragment 1 and acto-subfragment 1 of skeletal and smooth muscle

The fluorescent nucleotides epsilon ADP and epsilon ATP were used to study the binding and hydrolysis mechanisms of subfragment 1 (S-1) and acto-subfragment 1 from striated and smooth muscle. The quenching of the enhanced fluorescence emission of bound nucleotide by acrylamide analyzed either by the Stern-Volmer method or by fluorescence lifetime measurements showed the presence of two bound nucleotide states for 1-N6-ethenoadenosine triphosphate (epsilon ATP), 1-N6-ethenoadenosine diphosphate (epsilon ADP), and epsilon ADP-vanadate complexes with S-1. The equilibrium constant relating the two bound nucleotide states was close to unity. Transient kinetic studies showed two first-order transitions with rate constants of approximately 500 and 100 s-1 for both epsilon ATP and epsilon ADP and striated muscle S-1 and 300 and 30 s-1, respectively, for smooth muscle S-1. The hydrolysis of [gamma-32P] epsilon ATP yielded a transient phase of small amplitude (less than 0.2 mol/site) with a rate constant of 5-10 s-1. Consequently, the hydrolysis of the substrate is a step in the mechanism which is distinct from the two conformational changes induced by the binding of epsilon ATP. An essentially symmetric reaction mechanism is proposed in which two structural changes accompany substrate binding and the reversal of these steps occurs in product release. epsilon ATP dissociates acto-S-1 as effectively as ATP. For smooth muscle acto-S-1, dissociation proceeds in two steps, each accompanied by enhancement of fluorescence emission. A symmetric reaction scheme is proposed for the acto-S-1 epsilon ATPase cycle. The very similar kinetic properties of the reactions of epsilon ATP and ATP with S-1 and acto-S-1 suggest that two ATP intermediate states also occur in the ATPase reaction mechanism.     

MgATPase activity of myosin subfragment 1. The dimer is more active than the monomer

The MgATPase activity of the rabbit skeletal myosin subfragment 1 (S1), in the steady state, was measured by means of the intrinsic fluorescence of tryptophan. This technique gave results similar to those obtained by other methods (linked or radioactive assays). The activity was measured under conditions that effect the monomer/dimer ratio. It is shown that there is a close correlation between MgATPase activity and the proportion of dimer. At 20 degrees C, for pH 6.9 to 8.1 and for [KCl] less than or equal to 1 M, the observed activity (kobs) can be linearly related to the proportion of dimer (Ed/Eo) by: kobs(s-1) = 0.016-7 X 10(-3)[KCl] + 0.031(Ed/Eo), where [KCl] is expressed in M. We deduce that, at 20 degrees C and for [KCl] = 0 M, the activity of the monomer is kmobs = 0.016 s-1 (Ed/Eo = 0) and that of the dimer kdobs = 0.047 s-1 (Ed/Eo = 1), i.e. a ratio kdobs/kmobs approximately equal to 3. Beyond pH approximately equal to 8.3, the activities of both the monomer and the dimer increased steeply with increasing pH value. In the standard conditions (pH 8.0, [KCl] = 0 to 100 mM), S1 is mainly in the form of a dimer, and such conditions are not appropriate for study of the S1 monomer. For studying the pure monomer, the conditions required at 20 degrees C and in bis-Tris-propane are: S1 concentration approximately equal to 0.2 mg/ml, pH 6.9 to 7.8, [KCl] approximately equal to 300 mM. For studying the pure dimer, the conditions required are: S1 concentration greater than or equal to 0.2 mg/ml, pH 7.8 to 8.1 and [KCl] approximately equal to 0. In both cases the MgATP concentration is about 50 microM. Finally, if great care is taken concerning the age of the S1 solutions and the evaluation of the proportion of dimer, the values of kobs are extremely precise: the uncertainty regarding the values of kobs, as determined by means of intrinsic fluorescence, does not exceed +/- 0.001 s-1. Beyond this error bar conditions are uncontrolled.     

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.     

Heterogeneity in the actin activation of myosin

The soluble proteolytic fragments of myosin, heavy meromyosin and subfragment 1, were prepared with varying amounts of the proteases chymotrypsin and papain, respectively. The actin-activated ATP hydrolysis were examined with oxygen-18-labeled ATP. Each preparation of heavy meromyosin and subfragments 1 displayed two pathways of ATP hydrolysis, called respectively the high and low oxygen exchange mechanisms. The contributions of the two mechanisms were found to be sensitive to the potassium chloride concentration. With a fixed concentration of actin (300 microM), the contribution of the low-exchange mechanism decreased from a maximum of 90% of the ATP hydrolysis at 10 and 20 mM KCl to 12% at 180 mM KCl. The results suggested that the two mechanisms were competing reactions catalyzed by a single species of myosin.     

Mechanism of actomyosin adenosine triphosphatase. Evidence that adenosine 5'-triphosphate hydrolysis can occur without dissociation of the actomyosin complex.

We have investigated the steps in the actomyosin ATPase cycle that determine the maximum ATPase rate (Vmax) and the binding between myosin subfragment one (S-1) and actin which occurs when the ATPase activity is close to Vmax. We find that the forward rate constant of the initial ATP hydrolysis (initial Pi burst) is about 5 times faster than the maximum turnover rate of the actin S-1 ATPase. Thus, another step in the cycle must be considerably slower than the forward rate of the initial Pi burst. If this slower step occurs only when S-1 is complexed with actin, as originally predicted by the Lymn-Taylor model, the ATPase activity and the fraction of S-1 bound to actin in the steady state should increase almost in parallel as the actin concentration is increased. As measured by turbidity determined in the stopped-flow apparatus, the fraction of S-1 bound to actin, like the ATPase activity, shows a hyperbolic dependence on actin concentration, approaching 100% asymptotically. However, the actin concentration required so that 50% of the S-1 is bound to actin is about 4 times greater than the actin concentration required for half-maximal ATPase activity. Thus, as previously found at 0 degrees C, at 15 degrees C much of the S-1 is dissociated from actin when the ATPase is close to Vmax, showing that a slow first-order transition which follows the initial Pi burst (the transition from the refractory to the nonrefractory state) must be the slowest step in the ATPase cycle. Stopped-flow studies also reveal that the steady-state turbidity level is reached almost instantaneously after the S-1, actin, and ATP are mixed, regardless of the order of mixing. Thus, the binding between S-1 and actin which is observed in the steady state is due to a rapid equilibrium between S-1--ATP and acto--S-1--ATP which is shifted toward acto-S-1--ATP at high actin concentration. Furthermore, both S-1--ATP and S-1--ADP.Pi (the state occurring immediately after the initial Pi burst) appear to have the same binding constant to actin. Thus, at high actin concentration both S-1--ATP and S-1--ADP.Pi are in rapid equilibrium with their respective actin complexes. Although at very high actin concentration almost complete binding of S-1--ATP and S-1--ADP.Pi to actin occurs, there is no inhibition of the ATPase activity at high actin concentration. This strongly suggests that both the initial Pi burst and the slow rate-limiting transition which follows (the transition from the refractory to the nonrefractory state) occur at about the same rates whether the S-1 is bound to or dissociated from actin. We, therefore, conclude that S-1 does not have to dissociate from actin each time an ATP molecule is hydrolyzed.     

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.