Cross-bridge kinetics, cooperativity, and negatively strained cross- bridges in vertebrate smooth muscle. A laser-flash photolysis study

The effects of laser-flash photolytic release of ATP from caged ATP [P3-1(2-nitrophenyl)ethyladenosine-5'-triphosphate] on stiffness and tension transients were studied in permeabilized guinea pig protal vein smooth muscle. During rigor, induced by removing ATP from the relaxed or contracting muscles, stiffness was greater than in relaxed muscle, and electron microscopy showed cross-bridges attached to actin filaments at an approximately 45 degree angle. In the absence of Ca2+, liberation of ATP (0.1-1 mM) into muscles in rigor caused relaxation, with kinetics indicating cooperative reattachment of some cross-bridges. Inorganic phosphate (Pi; 20 mM) accelerated relaxation. A rapid phase of force development, accompanied by a decline in stiffness and unaffected by 20 mM Pi, was observed upon liberation of ATP in muscles that were released by 0.5-1.0% just before the laser pulse. This force increment observed upon detachment suggests that the cross-bridges can bear a negative tension. The second-order rate constant for detachment of rigor cross-bridges by ATP, in the absence of Ca2+, was estimated to be 0.1-2.5 X 10(5) M-1s-1, which indicates that this reaction is too fast to limit the rate of ATP hydrolysis during physiological contractions. In the presence of Ca2+, force development occurred at a rate (0.4 s-1) similar to that of intact, electrically stimulated tissue. The rate of force development was an order of magnitude faster in muscles that had been thiophosphorylated with ATP gamma S before the photochemical liberation of ATP, which indicates that under physiological conditions, in non-thiophosphorylated muscles, light-chain phosphorylation, rather than intrinsic properties of the actomyosin cross-bridges, limits the rate of force development. The release of micromolar ATP or CTP from caged ATP or caged CTP caused force development of up to 40% of maximal active tension in the absence of Ca2+, consistent with cooperative attachment of cross-bridges. Cooperative reattachment of dephosphorylated cross-bridges may contribute to force maintenance at low energy cost and low cross-bridge cycling rates in smooth muscle.     

Myokinase and contractile function of glycerinated muscle fibers

Glycerinated rabbit psoas muscle fibers containing native CPK, ATPase, and myokinase activities were used and isometric contraction and relaxation responses to either ADP or ATP + CP or to ATP alone in the presence and absence of P1, P5-di(adenosine-5'-pentaphosphate), a myokinase inhibitor, were compared. In previous (14) work it was shown that CP generated more efficient and faster contraction and relaxation of glycerinated muscle fibers than ATP. The present work deals with the role of myokinase in the differential response of fibers to CP and ATP. Inhibition of the myokinase activity of these fibers caused slight diminution of the rate of contraction at physiological concentrations of ATP. Uninhibited fibers were not able to reach maximum contraction, because the tension began to drop gradually even in the presence of Ca2+. Addition of Ap5A permitted maximum contraction and the ability to stay at the contracted state. In the case of CP + adenosine nucleotides (ATP or ADP), myokinase activity decreased the rate of tension development which was statistically significant after 5-7 sec of contraction. Thus, a higher tension was obtainable when myokinase was inhibited. At high concentration of adenine nucleotides (greater than 2 mM) and in the absence of Ap5A, not only the maximum tension never was reached, but a spontaneous drop in tension was observed before addition of EGTA, as was seen with ATP alone. Relaxation was faster and more complete in the presence of uninhibited myokinase activity except that the ADP was low (125 mM). These observations provide further evidence for a close functional interaction of these three enzymes in the mechanism of contraction and relaxation, giving further support to the notion of the creatine-phosphocreatine energy shuttle.     

Interaction of myosin with thin filaments during contraction and relaxation: effect of ionic strength

The influence of ionic strength on the isometric tension, stiffness, shortening velocity and ATPase activity of glycerol-treated rabbit psoas muscle fiber in the presence and the absence of Ca2+ has been studied. When the ionic strength of an activating solution (containing Mg2+-ATP and Ca2+) was decreased by varying the KCl concentration from 120 to 5 mM at 20 degrees C, the isometric tension and stiffness increased by 30% and 50%, respectively. The ATPase activity increased 3-fold, while the shortening velocity decreased to one-fourth. At 6 degrees C, similar results were obtained. These results suggest that at low ionic strengths ATP is hydrolyzed predominantly without dissociation of myosin cross-bridges from F-actin. In the absence of Ca2+, with decreasing KCl concentration the isometric tension and stiffness developed remarkably at 20 degrees C. However, the ATPase activity and shortening velocity were very low. At low ionic strength, even in the absence of Ca2+ myosin heads are bound to thin filaments. The development of the tension and stiffness were greatly reduced at 6 degrees C or at physiological ionic strength.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Butanedione monoxime suppresses contraction and ATPase activity of rabbit skeletal muscle

The effects of 2,3-butanedione 2-monoxime (BDM) on mechanical responses of glycerinated fibers and the ATPase activity of heavy meromyosin (HMM) and myofibrils have been studied using rabbit skeletal muscle. The mechanical responses and the ATPase activity were measured in similar conditions (ionic strength 0.06-0.2 M, 0.4-4 mM MgATP, 0-20 mM BDM, 2-20 degrees C and pH 7.0). BDM reversibly reduced the isometric tension, shortening speed, and instantaneous stiffness of the fibers. BDM also inhibited myofibrillar and HMM ATPase activities. The inhibitory effect on the relative ATPase activity of HMM was not influenced by the addition of actin or troponin-tropomyosin-actin. High temperature and low ionic strength weakened BDM's suppression of contraction of the fibers and the ATPase activity of contracting myofibrils, but not of the HMM, acto-HMM and relaxed myofibrillar ATPase activity. The size of the initial phosphate burst at 20 degrees C was independent of the concentration of BDM. These results suggest that the suppression of contraction of muscle fibers is due mainly to direct action of BDM on the myosin molecules.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.     

Changes in the ATPase activity of insect fibrillar flight muscle during calcium and strain activation probed by phosphate-water oxygen exchange

During ATP hydrolysis by Ca2+-activated chemically skinned fibers from the flight muscle of the giant waterbug Lethocerus indicus, there is extensive phosphate-water oxygen exchange. For unstrained fibers the pattern of exchange shows that there is more than one pathway for hydrolysis, due to the ATPase activity of cross-bridges. Multiple pathways are an established property of both vertebrate actomyosin and fibers. The pattern of exchange can be fitted by two pathways: one with low exchange because the step(s) controlling Pi release are rapid, the other with high exchange and slow Pi release. The high-exchange pathway is responsible for most of the increase in ATPase activity on Ca2+ activation. On strain activation, only the high-exchange pathway is present, accounting for all the ATPase increase and responsible for force generation. In fully activated fibers, the cross-bridges which hydrolyze ATP and generate force behave uniformly with respect to oxygen exchange. The exchange pattern shows that the rate of Pi release changes dramatically over a very narrow strain increase. Step(s) controlling Pi release are at least partially rate-limiting for the overall ATPase reaction. The results are discussed in relation to models for strain activation and the identity of force-generating states.     

Activation of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP.

The kinetics of force production in chemically skinned trabeculae from the guinea pig were studied by laser photolysis of caged ATP in the presence of Ca2+. Preincubation of the tissue during rigor with the enzyme apyrase was used to reduce the population of MgADP-bound cross-bridges (Martin and Barsotti, 1994). In untreated tissue, tension remained constant or dipped slightly below the rigor level immediately after ATP release, before increasing to the maximum measured in pCa 4.5 and 5 mM MgATP. The in-phase component stiffness, which is a measure of cross-bridge attachment, exhibited a large decrease before increasing to 55% of that measured in rigor. Neither the rate of the decline nor of the rise in tension was sensitive to the concentration of photolytically released ATP. The rate of the decline in stiffness was found to be dependent on [ATP]: 1.8 x 10(4) M-1/s-1, a value more than four times higher than that previously measured in similar experiments in the absence of Ca2+. The rate of tension development averaged 14.9 +/- 2.5 s-1. Preincubation with apyrase altered the mechanical characteristics of the early phase of the contraction. The rate and amplitude of the initial drop in both tension and stiffness after caged ATP photolysis increased and became dependent on [ATP]. The second-order rate constants measured for the initial drop in tension and stiffness were 8.4 x 10(4) M-1 s-1 and 1.5 x 10(5) M-1 s-1. These rates are more than two times faster than those previously measured in the absence of Ca2+. The effects of apyrase incubation on the time course of tension and stiffness were consistent with the hypothesis that during rigor, skinned trabeculae retain a significant population of MgADP-bound cross-bridges. These in turn act to attenuate the initial drop in tension after caged ATP photolysis and slow the apparent rate of rigor cross-bridge detachment. The results also show that Ca2+ increases the rate of cross-bridge detachment in both untreated and apyrase-treated tissue, but the effect is larger in untreated tissue. This suggests that in cardiac muscle Ca2+ modulates the rate of cross-bridge detachment.     

Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.     

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.     

Ca2+ uptake by corpus-luteum plasma membranes. Evidence for the presence of both a Ca2+-pumping ATPase and a Ca2+-dependent nucleoside triphosphatase.

Plasma-membrane vesicles from rat corpus luteum showed an ATP-dependent uptake of Ca2+. Ca2+ was accumulated with a K1/2 (concn. giving half-maximal activity) of 0.2 microM and was released by the bivalent-cation ionophore A23187. A Ca2+-dependent phosphorylated intermediate (Mr 100,000) was detected which showed a low decomposition rate, consistent with it being the phosphorylated intermediate of the transport ATPase responsible for Ca2+ uptake. The Ca2+ uptake and the phosphorylated intermediate (E approximately P) displayed several properties that were different from those of the high-affinity Ca2+-ATPase previously observed in these membranes. Both Ca2+ uptake and E approximately P discriminated against ribonucleoside triphosphates other than ATP, whereas the ATPase split all the ribonucleoside triphosphates equally. Both Ca2+ uptake and E approximately P were sensitive to three different Hg-containing inhibitors, whereas the ATPase was inhibited much less. Ca2+ uptake required added Mg2+ (Km = 2.2 mM), whereas the ATPase required no added Mg2+. The maximum rate of Ca2+ uptake was about 400-fold less than that of ATP splitting; under different conditions, the decomposition rate of E approximately P was 1,000 times too slow to account for the ATPase activity observed. All of these features suggested that Ca2+ uptake was due to an enzyme of low activity, whose ATPase activity was not detected in the presence of the higher-specific-activity Ca2+-dependent ATPase.     

Evaluation of the cross-bridge-dependent change in the Ca2+ affinity of troponin C in aequorin-injected ferret ventricular muscles

Ca2+ affinity of cardiac troponin C (TnC) is regulated by the active cross-bridges (downstream-dependent mechanism). In the present study, we showed one of the methods to evaluate the downstream-dependent change in the Ca2+ affinity of TnC during contraction using the aequorin-injected ferret papillary muscle. For this purpose, the tension-dependent change in the extra-Ca2+ (a transient increase in the intracellular Ca2+ concentration ([Ca2+]i) in response to a quick length reduction) was measured under various conditions. We examined whether the regression line between the magnitude of tension reduction and the magnitude of the normalized extra-Ca2+ (the extra-Ca2+ was divided by [Ca2+]i immediately before length change) (the normalized extra-Ca2+-tension relation) in twitch contraction can be used for the estimation of the downstream-dependent change in the Ca2+ affinity of TnC. The normalized extra-Ca2+-tension relation became shallow by EMD 57033 (EMD) (one of the Ca2+ sensitizers) and by an increase in Ca2+ concentration in the solution ([Ca2+]o) in a concentration-dependent manner. However, 2,3-butanedione monoxime (BDM) (one of the desensitizers) antagonized the effects of EMD and higher [Ca2+]o in a concentration-dependent manner. These effects of EMD and BDM were also observed in the normalized extra-Ca2+-tension relation in tetanic contraction. The normalized extra-Ca2+-tension relation became steep by shortening the initial muscle length before contraction in tetanic contraction. Length-tension relation in twitch contraction was significantly shifted upward by higher [Ca2+]o and EMD, but BDM showed the opposite effects on them in a concentration-dependent manner. Thus, the downstream-dependent change in the Ca2+ affinity of TnC which physiologically functions in intact cardiac muscle can be evaluated using the normalized extra-Ca2+-tension relation.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase.

A rapid mixing technique was used to investigate the effects of Ca2+ ion on the kinetics of ATP hydrolysis by sarcoplasmic reticulum vesicles. "Basic" ATPase measured in the absence of Ca2+ showed an initial burst of inorganic phosphate production. Similarities in the transient state kinetic properties of basic and "extra" or Ca2+-dependent ATPase suggest that the two activities represent a single enzyme species. At low concentrations of Ca2+ (less than 10(-6) M) the time course of the partial reactions of extra ATPase appeared to fit a simple scheme in which the acid-stable, phosphorylated enzyme (E approximately P) breaks down directly to inorganic phosphate and free enzyme. A similar mechanism seemed to apply to moderate levels of ATP and high external concentrations of Ca2+ known to inhibit transport activity. In the intermediate range of Ca2+ concentrations inorganic phosphate production was resolved into two phases consisting of a fast initial rate (burst) and slow steady state. Acid-stable phosphorylated protein showed a transient decay which coincided with the appearance of the burst. This behavior is consistent with a scheme in which E approximately P breaks down to an acid-labile or noncovalent intermediate state (E-P). A slow secondary increase in phosphorylation followed the transient decay in E approximately P. This late phase of protein labeling was eliminated following pretreatment with Triton X-100, sodium oxalate, or diethyl ether which decrease or prevent the formation of a transport gradient. An analysis of the dependence of the steady state level of phosphorylation and rate of inorganic phosphate production on Ca2+ concentration indicated that the phosphorylation mechanism involves interaction of two Ca2+ ions with the enzymatic carrier. The pathway by which E approximately P breaks down, i.e. whether it goes to E + Pi or E-P, may depend on the extent to which these sites are occupied by Ca2+. The transport of Ca2+ is discussed in terms of a flip-flop mechanism in which E approximately P and E-P represent high and low affinity Ca2+ binding states occurring in separate halves of an enzyme dimer.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Regulation of molluscan actomyosin ATPase activity

The interaction of myosin and actin in many invertebrate muscles is mediated by the direct binding of Ca2+ to myosin, in contrast to modes of regulation in vertebrate skeletal and smooth muscles. Earlier work showed that the binding of skeletal muscle myosin subfragment 1 to the actin-troponin-tropomyosin complex in the presence of ATP is weakened by less than a factor of 2 by removal of Ca2+ although the maximum rate of ATP hydrolysis decreases by 96%. We have now studied the invertebrate type of regulation using heavy meromyosin (HMM) prepared from both the scallop Aequipecten irradians and the squid Loligo pealii. Binding of these HMMs to rabbit skeletal actin was determined by measuring the ATPase activity present in the supernatant after sedimenting acto-HMM in an ultracentrifuge. The HMM of both species bound to actin in the presence of ATP, even in the absence of Ca2+, although the binding constant in the absence of Ca2+ (4.3 X 10(3) M-1) was about 20% of that in the presence of Ca+ (2.2 X 10(4) M-1). Studies of the steady state ATPase activity of these HMMs as a function of actin concentration revealed that the major effect of removing Ca2+ was to decrease the maximum velocity, extrapolated to infinite actin concentration, by 80-85%. Furthermore, at high actin concentrations where most of the HMM was bound to actin, the rate of ATP hydrolysis remained inhibited in the absence of Ca+. Therefore, inhibition of the ATPase rate in the absence of Ca2+ cannot be due simply to an inhibition of the binding of HMM to actin; rather, Ca2+ must also directly alter the kinetics of ATP hydrolysis.     

Labeling the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum with 4-(bromomethyl)-6,7-dimethoxycoumarin: detection of conformational changes.

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with 4-(bromomethyl)-6,7-dimethoxycoumarin. It was shown that a single cysteine residue (Cys-344) was labeled on the ATPase, with a 25% reduction in steady-state ATPase activity and no reduction in the steady-state rate of hydrolysis of p-nitrophenyl phosphate. The fluorescence intensity of the labeled ATPase was sensitive to pH, consistent with an effect of protonation of a residue of pK 6.8. Fluorescence changes were observed on binding Mg2+, consistent with binding to a single site of Kd 4 mM. Comparable changes in fluorescence intensity were observed on binding ADP in the presence of Ca2+. Binding of AMP-PCP produced larger fluorescence changes, comparable to those observed on phosphorylation with ATP or acetyl phosphate. Phosphorylation with P(i) also resulted in fluorescence changes; the effect of pH on the fluorescence changes was greater than that on the level of phosphorylation measured directly using [32P]P(i). It is suggested that different conformational states of the phosphorylated ATPase are obtained at steady state in the presence of Ca2+ and ATP and at equilibrium in the presence of P(i) and absence of Ca2+.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. Dependence of an endogenous protein activator distinct from calmodulin

A Mg-dependent adenosine triphosphatase (ATPase) activated by submicromolar free Ca2+ was identified in detergent-dispersed rat liver plasma membranes after fractionation by concanavalin A-Ultrogel chromatography. Further resolution by DE-52 chromatography resulted in the separation of an activator from the enzyme. The activator, although sensitive to trypsin hydrolysis, was distinct from calmodulin for it was degraded by boiling for 2 min, and its action was not sensitive to trifluoperazine; in addition, calmodulin at concentrations ranging from 0.25 ng-25 micrograms/assay had no effect on enzyme activity. Ca2+ activation followed a cooperative mechanism (nH = 1.4), half-maximal activation occurring at 13 +/- 5 nM free Ca2+. ATP, ITP, GTP, CTP, UPT, and ADP displayed similar affinities for the enzyme; K0.5 for ATP was 21+/- 9 microM. However, the highest hydrolysis rate (20 mumol of Pi/mg of protein/10 min) was observed at 0.25 mM ATP. For all the substrates tested kinetic studies indicated that two interacting catalytic sites were involved. Half-maximal activity of the enzyme required less than 12 microM total Mg2+. This low requirement for Mg2+ of the high affinity (Ca2+-Mg2+)ATPase was probably the major kinetic difference between this activity and the nonspecific (Ca2+ or Mg2+)ATPase. In fact, definition of new assay conditions, i.e. a low ATP concentration (0.25 mM) and the absence of added Mg2+, allowed us to reveal the (Ca2+-Mg2+)ATPase activity in native rat liver plasma membranes. This enzyme belongs to the class of plasma membrane (Ca2+-Mg2+)ATPases dependent on submicromolar free Ca2+ probably responsible for extrusion of intracellular Ca2+.     

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.