Modification of the interactions of myosin with actin and 5''-adenylyl imidodiphosphate by substitution of ethylene glycol for water.

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5'-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.     

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.     

Evidence for increased low force cross-bridge population in shortening skinned skeletal muscle fibers: implications for actomyosin kinetics.

The dynamic characteristics of the low force myosin cross-bridges were determined in fully calcium-activated skinned rabbit psoas muscle fibers shortening under constant loads (0.04-0.7 x full isometric tension Po). The shortening was interrupted at various times by a ramp stretch (duration, 10 ms; amplitude, up to 1.8% fiber length) and the resulting tension response was recorded. Except for the earlier period of velocity transients, the tension response showed nonlinear dependence on stretch amplitude; i.e., the magnitude of the tension response started to rise disproportionately as the stretch exceeded a critical amplitude, as in the presence of inorganic phosphate (Pi). This result, as well as the result of stiffness measurement, suggests that the low force cross-bridges similar to those observed in the presence of Pi (presumably A.M.ADP.Pi) are significantly populated during shortening. The critical amplitude of the shortening fibers was greater than that of isometrically contracting fibers, suggesting that the low force cross-bridges are more negatively strained during shortening. As the load was reduced from 0.3 to 0.04 P0, the shortening velocity increased more than twofold, but the amount of the negative strain stayed remarkably constant (approximately 3 nm). This This insensitiveness of the negative strain to velocity is best explained if the dissociation of the low force cross-bridges is accelerated approximately in proportion to velocity. Along with previous reports, the results suggest that the actomyosin ATPase cycle in muscle fibers has at least two key reaction steps in which rate constants are sensitively regulated by shortening velocity and that one of them is the dissociation of the low force A.M.ADP.Pi cross-bridges. This step may virtually limit the rate of actomyosin ATPase turnover and help increase efficiency in fibers shortening at high velocities.     

Two attached non-rigor crossbridge forms in insect flight muscle

We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.     

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.     

Effects of magnesium chloride on smooth muscle actomyosin adenosine-5'-triphosphatase activity, myosin conformation, and tension development in glycerinated smooth muscle fibers

The contractile system of smooth muscle exhibits distinctive responses to varying Mg2+ concentrations in that maximum adenosine-5'-triphosphatase (ATPase) activity of actomyosin requires relatively high concentrations of Mg2+ and also that tension in skinned smooth muscle fibers can be induced in the absence of Ca2+ by high Mg2+ concentrations. We have examined the effects of MgCl2 on actomyosin ATPase activity and on tension development in skinned gizzard fibers and suggest that the MgCl2-induced changes may be correlated to shifts in myosin conformation. At low concentrations of free Mg2+ (less than or equal to 1 mM) the actin-activated ATPase activity of phosphorylated turkey gizzard myosin is reduced and is increased as the Mg2+ concentration is raised. The increase in Mg2+ (over a range of 1-10 mM added MgCl2) induces the conversion of 10S phosphorylated myosin to the 6S form, and it was found that the proportion of myosin as 10S is inversely related to the level of actin-activated ATPase activity. Activation of the actin-activated ATPase activity also occurs with dephosphorylated myosin but at higher MgCl2 concentrations, between 10 and 40 mM added MgCl2. Viscosity and fluorescence measurements indicate that increasing Mg2+ levels over this concentration range favor the formation of the 6S conformation of dephosphorylated myosin, and it is proposed that the 10S to 6S transition is a prerequisite for the observed activation of ATPase activity. With glycerinated chicken gizzard fibers high MgCl2 concentrations (6-20 mM) promote tension in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers.

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca2+ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca2+ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechano-chemical cycle are Ca2+ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

Steady-state kinetic study on the inhibition of the adenosinetriphosphatase activity of dynein from Tetrahymena cilia by glycerol

The characteristics of glycerol-induced inhibition of the dynein ATPase extracted from Tetrahymena cilia were investigated. Fifty percent inhibition was observed at about 15% (v/v) glycerol with the 22S dynein Mg-ATPase. Ethylene glycol was equally inhibitory, while sucrose, a kind of polyol, was less effective. The glycerol-induced inhibition of the 22S dynein Mg-ATPase was not influenced by pH or by raising the ionic strength of the assay solution. An aqueous glycerol solution treated with anion or cation exchanger or charcoal was equally inhibitory to a non-treated solution. The inhibition was most likely to be due to glycerol or ethylene glycol itself, not to a contaminant. The inhibition of the 22S dynein Mg-ATPase was apparently noncompetitive: only the Vmax was reduced without a significant change in the apparent Km. The dynein ATPase is known to be inhibited potently by vanadate. Glycerol reduced the sensitivity of the dynein ATPase to the vanadate-induced inhibition. Glycerol exhibited a decelerating effect on the rate of the oxygen exchange between phosphate and water catalyzed by 22S dynein in the presence of ADP and Mg2+. If it is assumed that the rate constants of the ATP hydrolysis step are not affected by glycerol, it may be implied that the phosphate release from the E.ADP.P1 intermediate was decelerated by glycerol and that the deceleration of the phosphate release paralleled the reduction of the overall ATPase activity over a wide range of glycerol concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryoenzymic studies on an organized system: myofibrillar ATPases and shortening.

We have exploited cryoenzymology, first, to probe the product release steps of myofibrillar ATPase under relaxing conditions and, second, to define the conditions for studying the contractile process in slow motion. Cryoenzymology implies perturbation by temperature and by the antifreeze added to allow for work at subzero temperatures. Here, we studied myofibrillar shortening and ATPases by the rapid quench flow method over a wide temperature range (-15 to 30 degrees C) in two antifreezes, 40% ethylene glycol and 20% methanol. The choice of solvent and temperature was dictated by the purpose of the experiment. Ethylene glycol (40%) is suitable for investigating the kinetics of the products release steps which is difficult in water. In this cryosolvent, the myofibrillar ATPase is not activated by Ca2+ nor is there shortening, except under special conditions, i.e., Ca2+ plus strong rigor bridges [Stehle, R., Lionne, C., Travers, F., and Barman, T. (1998) J. Muscl. Res. Cell Motil. 19, 381-392]. By the use of the glycol, we show that at low Ca2+ the kinetics of the ADP release are much faster with myofibrils than with S1. On the other hand, the kinetics of the Pi release were very similar for the two materials. Therefore, we suggest that, upon Ca2+ activation, only the Pi release kinetics are accelerated. In 20% methanol, in the presence of Ca2+, myofibrils shortened at temperatures above -2 degrees C but not below. At a given temperature above -2 degrees C, both the shortening and ATPase rates were reduced by the methanol. The temperature dependences of the myofibrillar ATPases (+/-Ca2+) converged with a decrease in temperature: at 20 degrees C, Ca2+ activated 30-fold, but at -15 degrees C, only about 5-fold. We suggest that studies in methanol may open the way for an investigation of muscle contraction in slow motion and, further, to obtain thermodynamic information on the internal forces involved in the shortening process.     

Mechanochemical coupling in muscle: attempts to measure simultaneously shortening and ATPase rates in myofibrils.

We studied the ATPase of shortening myofibrils at 4 degrees C by the rapid flow quench method. The progress curve has three phases: a P(i) burst, a fast linear phase kF of duration tB, and a deceleration to a slow kS. We propose that kF is the ATPase of myofibrils shortening under zero external load; at tB shortening and ATPase rates are reduced by passive resistance. The total ATP consumed during the rapid shortening is ATPc. Our purpose was to obtain information on the myofibrillar shortening velocity from their ATPase progress curves. We tested tB as an indicator of shortening velocity by determining the effects of different probes upon it and the other ATPase parameters. The dependence of tB upon the initial sarcomere length was linear, giving a shortening velocity close to that of muscle fibres (Vo). The Km of ATP was larger for tB than for kF, as found with fibers for Vo and their ATPase. ADP and 2,3-butanedione monoxime, but not P(i), inhibited tB to the same extent as Vo. The delta H for tB and Vo were similar. ATPc was independent of the sarcomere length, implying that the more the myofibrils shorten, the less ATP expended per myosin head per micron shortened. We propose that tB can be used as an indicator for myofibrillar shortening velocities.     

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.     

Formation of ATP-insensitive weakly-binding crossbridges in single rabbit psoas fibers by treatment with phenylmaleimide or para-phenylenedimaleimide.

Chaen et al. (1986. J. Biol. Chem. 261:13632-13636) showed that treatment of relaxed single muscle fibers with para-phenylenedimaleimide (pPDM) results in inhibition of a fiber's ability to generate active force and a diminished ATPase activity. They postulated that the inhibition of force production was due to pPDM's ability to prevent crossbridges from participating in the normal ATP hydrolysis cycle. We find that the crossbridges produced by pPDM treatment of relaxed muscle cannot bind strongly to the actin filaments in rigor, but do bind weakly to the actin filaments in the presence and also absence of ATP. After pPDM treatment, fiber stiffness, as measured using ramp stretches of varying duration, is ATP-insensitive and identical to that of untreated relaxed fibers (both at high [165 mM] and low [40 mM] ionic strength). These results suggest that the pPDM-treated crossbridges, in both the presence and absence of ATP, are locked in a state that resembles the weakly-binding myosin ATP state of normal crossbridges. Their resemblance to the ATP-crossbridges of relaxed untreated fibers is quite strong; both bind to actin about equally tightly and have similar attachment and detachment rate constants. We also found that crossbridges are locked in a weakly-binding state after treatment with N-phenylmaleimide (NPM). In muscle fibers, this method of producing weakly-binding crossbridges appears preferable to pPDM treatment because, unlike treatment with pPDM, it does not increase the fiber's resting tension and stiffness and it does not disrupt the titin band seen on SDS-PAGE.     

Ca2+ dependence of tension and ADP production in segments of chemically skinned muscle fibers

Both ADP production and tension have been measured in segments of chemically skinned fibers contracting at different Ca²⁺ concentrations. Full mechanical activation occurred between pCa 7.00 and pCa 6.50. The total ATPase was due to both actomyosin and non-actomyosin ATPase. Actomyosin ATPase was observed at pCa 7.09 without accompanying tension. The Ca²⁺ dependence of tension was steeper than actomyosin ATPase. This finding implies some rate constants of the mechanochemical cycle are Ca²⁺ dependent. Non-actomyosin ATPase was measured in fibers stretched beyond overlap of the thick and thin filaments. Sarcoplasmic reticulum was isolated and sarcoplasmic reticulum activity was measured in vitro under the same conditions as the single-fiber experiments. Non-actomyosin ATPase in the single fibers was found to be small compared to maximally activated actomyosin ATPase but larger than the ATPase that could be attributed to sarcoplasmic reticulum activity.     

The effect of polyethylene glycol on the mechanics and ATPase activity of active muscle fibers

We have used polyethylene glycol (PEG) to perturb the actomyosin interaction in active skinned muscle fibers. PEG is known to potentiate protein-protein interactions, including the binding of myosin to actin. The addition of 5% w/v PEG (MW 300 or 4000) to active fibers increased fiber tension and decreased shortening velocity and ATPase activity, all by 25-40%. Variation in [ADP] or [ATP] showed that the addition of PEG had little effect on the dissociation of the cross-bridge at the end of the power stroke. Myosin complexed with ADP and the phosphate analog V(i) or AlF(4) binds weakly to actin and is an analog of a pre-power-stroke state. PEG substantially enhances binding of these states both in active fibers and in solution. Titration of force with increasing [P(i)] showed that PEG increased the free energy available to drive the power stroke by about the same amount as it increased the free energy available from the formation of the actomyosin bond. Thus PEG potentiates the binding of myosin to actin in active fibers, and it provides a method for enhancing populations of some states for structural or mechanical studies, particularly those of the normally weakly bound transient states that precede the power stroke.     

Stimulatory and inhibitory effects of dimethyl sulfoxide and ethylene glycol on ATPase activity and calcium transport of sarcoplasmic membranes.

1. The effect of dimethyl sulfoxide (Me2SO) and ethylene glycol on two different preparations of the sarcoplasmic reticulum, i.e. native membranes and membranes whose phospholipids were hydrolyzed by phospholipase A, were investigated using ATP and p-nitrophenylphosphate as substrates. 2. Me2SO and ethylene glycol inhibit both calcium-dependent ATP hydrolysis and ATP-supported calcium transport by native vesicles. 3. In contrast, calcium-dependent p-nitrophenylphosphatase activity as well as p-nitrophenyl-phosphate-supported calcium transport are activated by both agents at concentrations lower than 30% (v/v). 4. Me2SO strongly stimulates p-nitrophenylphosphate activity of vesicles treated with phospholipase A, but has relatively little effect on p-nitrophenylphosphatase activity of native vesicles. 5. Up to a concentration of approximately 40% Me2SO (v/v) the inhibiting effect on the calcium-dependent ATPase is fully reversible, but only partially reversible on calcium transport. 6. In the concentration range where Me2SO inhibits ATP hydrolysis and calcium transport, it does not affect ATP binding to the membranes nor calcium-dependent formation of phospho-protein. 7. The rate of dephosphorylation as well as the rate of Pi exchange between ATP and ADP are markedly reduced by the presence of 30% Me2SO (v/v). 8. While Me2SO inhibits passive calcium efflux, ethylene glycol produces a considerable activation. 9. ADP-dependent calcium efflux and ATP synthesis are activated by 15% Me2SO (v/v). Ethylene glycol reduces both activities. 10. The results suggest that the respective substrate-enzyme complexes are differently affected by the agents, resulting either in inhibition or stimulation     

Concentration-dependent inhibition of myosin ATPase by an indole metabolite of epinephrine

An indole metabolite of epinephrine (an isomer of adrenochrome) was shown to be a potent inhibitor (EC₅₀ of 1.50 μM to 1.85 μM) of myosin, actomyosin, and myofibrillar ATPase when assayed at or near physiologic ionic strength and pH. The inhibition of actomyosin ATPase by this epinephrine derivative was demonstrated to be competetive in nature. Complete inhibition of ATPase activity was never achieved under physiological conditions; maximum inhibition was 50% to 60%. It is concluded that the inhibitor reduced ATPase activity by reversibly attaching to sulfhydryl groups associated with ATPase activity. The reduction of ATPase activity by 50% may be explained by the known heterogeneity of the ATPase sites on myosin; only one-half of these sites may be sensitive or accessible to the inhibitor in the state of aggregation of myosin at physiologic ionic strength. The inhibitor was found to have no effect on hog cerebral cortex Na⁺,K⁺-activated ATPase, suggesting that it may be selective for contractile protein ATPase. These results further support the hypothesis proposed earlier from this laboratory that this inhibitory indole metabolite of epinephrine (which is formed only in smooth muscles relaxed by epinephrine) may be part of the mechanism by which epinephrine produces relaxation in certain smooth muscles.     

Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament

F-Actin was partially cross-linked to myosin subfragment-1 (S-1) at various molar ratios (r = S-1/actin) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked acto-S-1 ATPase showed so called "super-activation," Vx. S-1 was added further to the cross-linked acto-S-1 and the ATPase activity, Vy, was measured. Since the added S-1 can interact only with the bare actin protomers within the cross-linked actin filament, the difference, delta V = Vy - Vx - Vs (where Vs is the ATPase activity of the additional S-1 alone), can indicate the state of the bare actin protomers while the cross-linked acto-S-1 is hydrolyzing ATP. With increasing r, delta V decreased much more rapidly than delta Vo(1 - r) (where delta Vo is delta V at r = 0) and reached a minimum around r = 0.15. As r increased further, delta V approached the level of delta Vo(1 - r). When SH1/SH2-blocked S-1 was cross-linked to F-actin, delta V decreased according to delta Vo(1 - r). Therefore, the large reduction of delta V, observed when intact S-1 was cross-linked, was coupled to the high ATPase activity of the cross-linked acto-S-1. Combining these data with other kinetic data, we could deduce that structural distortion in a cross-linked actin induced by the ATPase reaction of the S-1 partner propagated over several bare actin protomers along the filament and reduced their affinity for the S-1-ADP-Pi complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of Escherichia coli F1-ATPase by lauryldimethylamine oxide and ethylene glycol: relationship of ATPase activity to the interaction of the epsilon and beta subunits

The stimulation of the ATPase activity of Escherichia coli F1-ATPase by the detergent lauryldimethylamine oxide (LDAO) and the relationship of this activation to removal of the inhibitory epsilon subunit were studied. The detergent caused a dramatic decrease in the affinity of epsilon-depleted enzyme for epsilon subunit, suggesting that release of epsilon is involved in LDAO activation. However, even in the absence of any epsilon subunit, the detergent caused a 140% increase in activity, indicating activation by effects independent of epsilon. In contrast, the addition of 30% ethylene glycol to the reaction buffer caused a modest inhibition of the ATPase activity of epsilon-depleted F1-ATPase but rendered the enzyme insensitive to inhibition by epsilon subunit. This solvent prevented the cross-linking of epsilon to beta by a water-soluble carbodiimide, although epsilon remained linkable to both beta and gamma by dithiobis(succinimidyl propionate). Thus, epsilon was not dissociated from F1-ATPase, but its intimate interaction with the beta subunit was altered. These results suggest that the inhibitory action of epsilon is expressed through its interaction with beta. Kinetic analysis revealed that LDAO activated hydrolysis at both the high- and low-affinity promotional sites, with little change in Km values. Ethylene glycol caused a substantial increase in Km at the low-affinity promotional site and made the enzyme resistant to inhibition by aurovertin D.     

An expanded latch-bridge model of protein kinase C-mediated smooth muscle contraction.

A thin-filament-regulated latch-bridge model of smooth muscle contraction is proposed to integrate thin-filament-based inhibition of actomyosin ATPase activity with myosin phosphorylation in the regulation of smooth muscle mechanics. The model included two latch-bridge cycles, one of which was identical to the four-state model as proposed by Hai and Murphy (Am J Physiol Cell Physiol 255: C86-C94, 1988), whereas the ultraslow cross-bridge cycle has lower cross-bridge cycling rates. The model-fitted phorbol ester induced slow contractions at constant myosin phosphorylation and predicted steeper dependence of force on myosin phosphorylation in phorbol ester-stimulated smooth muscle. By shifting cross bridges between the two latch-bridge cycles, the model predicts that a smooth muscle cell can either maintain force at extremely low-energy cost or change its contractile state rapidly, if necessary. Depending on the fraction of cross bridges engaged in the ultraslow latch-bridge cycle, the model predicted biphasic kinetics of smooth muscle mechanics and variable steady-state dependencies of force and shortening velocity on myosin phosphorylation. These results suggest that thin-filament-based regulatory proteins may function as tuners of actomyosin ATPase activity, thus allowing a smooth muscle cell to have two discrete cross-bridge cycles with different cross-bridge cycling rates.