Kinetics of calcium uptake by isolated sarcoplasmic reticulum vesicles using flash photolysis of caged adenosine 5'-triphosphate

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.     

Moderate resolution profile structure of the sarcoplasmic reticulum membrane under low temperature conditions for the transient trapping of E1 approximately P.

The calcium uptake reaction kinetics of isolated sarcoplasmic reticulum (SR) vesicles have previously been shown to be at least biphasic over a range of temperatures (26 to 10 degrees C) with a fast phase identified with the formation of E1 approximately P and calcium occlusion and a slow phase with Ca2+ translocation across the membrane and turnover of the Ca2+ ATPase ensemble. At "low" temperatures, namely 0 degrees C or lower, E1 approximately P formation is slowed and E1 approximately P is transiently trapped for at least several seconds, as indicated by the absence of the slow phase for 6 s or more. We now report that a reversible, temperature-induced structural transition occurs at about 2-3 degrees C for the isolated SR membrane. We have investigated the nature of this structural transition utilizing meridional and equatorial x-ray diffraction studies of the oriented SR membrane multilayers in the range of temperatures between 7.5 and -2 degrees C. The phase meridional (lamellar) diffraction has provided the profile structure for the SR membrane at the highest vs. lowest temperature at the same moderate resolution of 16-17 A while the equatorial diffraction has provided information on the average lipid chain packing in the SR membrane plane in the two cases. To identify the contribution of each membrane component in producing the differences between the profile structures at 7.5 and -2 degrees C, step-function models have been fitted to the moderate resolution electron density profiles. Lipid lateral phase separation may be responsible for inducing the structural change in the Ca2+ ATPase, thereby resulting in the slowing of E1 approximately P formation and the transient trapping of E1 approximately P at the "lower" temperatures.     

Effect of Mg2+ concentration on Ca2+ uptake kinetics and structure of the sarcoplasmic reticulum membrane.

Direct measurements of phosphorylation of the Ca2+ ATPase of the sarcoplasmic reticulum (SR) have shown that the lifetime of the first phosphorylated intermediate in the Ca2+ transport cycle, E1 approximately P, increases with decreasing [Mg2+] (Dupont, Y. 1980. Eur. J. Biochem. 109:231-238). Previous x-ray diffraction work (Pascolini, D., and J.K. Blasie. 1988. Biophys. J. 54:669-678) under high [Mg2+] conditions (25 mM) indicated that changes in the profile structure of the SR membrane could be responsible for the low-temperature transient trapping of E1 approximately P that occurs at temperatures below 2-3 degrees C, the upper characteristic temperature th for lipid lateral phase separation in the membrane. We now present results of our study of the Ca2+ uptake kinetics and of the structure of the SR membrane at low [Mg2+] (less than or equal to 100 microM). Our results show a slowing in the kinetics of both phases of the Ca2+ uptake process and an increase in the duration of the plateau of the fast phase before the onset of the slow phase, indicating an increase in the lifetime (transient trapping) of E1 approximately P. Calcium uptake kinetics at low [Mg2+] and moderately low temperature (approximately 0 degree C) are similar to those observed at much lower temperatures (approximately -10 degrees C) at high [Mg2+]. The temperature-induced structural changes that we observed at low [Mg2+] are much more pronounced than those found to occur at higher [Mg2+]. Also, at the lower [Mg2+] the upper characteristic temperature th for lipid lateral phase separation was found to be higher, at approximately 8-10 degrees C. Our studies indicate that both temperature and [Mg2+] affect the structure and the functionality (as measured by changes in the kinetics of Ca2+ uptake) of the SR membrane. Membrane lipid phase behavior and changes in the Ca2+ ATPase profile structure seem to be related, and we have found that structural changes are responsible for the slowing of the kinetics of the fast phase of Ca2+ uptake, and could also mediate the effect that [Mg2+] has on E1 approximately P lifetime.     

Time-resolved x-ray diffraction studies of the sarcoplasmic reticulum membrane during active transport.

X-ray and neutron diffraction studies of oriented multilayers of a highly purified fraction of isolated sarcoplasmic reticulum (SR) have previously provided the separate profile structures of the lipid bilayer and the Ca2+-ATPase molecule within the membrane profile to approximately 10-A resolution. These studies used biosynthetically deuterated SR phospholipids incorporated isomorphously into the isolated SR membranes via phospholipid transfer proteins. Time-resolved x-ray diffraction studies of these oriented SR membrane multilayers have detected significant changes in the membrane profile structure associated with phosphorylation of the Ca2+-ATPase within a single turnover of the Ca2+-transport cycle. These studies used the flash photolysis of caged ATP to effectively synchronize the ensemble of Ca2+-ATPase molecules in the multilayer, synchrotron x-radiation to provide 100-500-ms data collection times, and double-beam spectrophotometry to monitor the Ca2+-transport process directly in the oriented SR membrane multilayer.     

Simultaneous internalization and binding of calcium during the initial phase of calcium uptake by the sarcoplasmic reticulum Ca pump.

The kinetics of Ca2+ transport mediated by the sarcoplasmic reticulum (SR) Ca-ATPase were investigated by rapid kinetic techniques that either measure the disappearance of Ca2+ from the medium [stopped-flow photometry of Ca2+ indicators or rapid filtration (method 1)] or directly detect the changes in the accessibility of Ca2+ to the exterior of the membrane, i.e., occlusion of Ca2+ within the Ca pump and Ca2+ transport into the lumen of SR vesicles [EGTA quench (method 2)]. SR vesicles were preincubated in micromolar Ca2+ to form the E.2Cacyt intermediate of the Ca-ATPase, and then Ca2+ transport was initiated by addition of ATP. It was found that Ca2+ uptake measured by method 1 began with no lag phase, in spite of the prediction of kinetic models of the Ca-ATPase. Instead, the time course of Ca2+ uptake was found to have two components: a fast and a slow phase, similar to that obtained using method 2, although the rate constant of the fast phase determined by method 1 was considerably lower than that measured by method 2. The fast phase of Ca2+ uptake measured by method 1 was not influenced by either Ca2+ ionophore or detergent treatment, whereas the slow phase was diminished.(ABSTRACT TRUNCATED AT 250 WORDS)     

A direct analysis of lamellar x-ray diffraction from hydrated oriented multilayers of fully functional sarcoplasmic reticulum.

The profile structure of functional sarcoplasmic reticulum (SR) membranes was investigated by X-ray diffraction methods to a resolution of 10 A. The lamellar diffraction data from hydrated oriented multilayers of SR vesicles showed monotonically increasing widths for higher order lamellar reflections, indicative of simple lattice disorder within the multilayer. A generalized Patterson function analysis, previously developed for treating lamellar diffraction from lattice-disordered multilayers, was used to identify the autocorrelation function of the unit cell electron density profile. Subsequent deconvolution of this autocorrelation function provided the most probable unit cell electron density profile of the SR vesicle membrane pair. The resulting single membrane profile possesses marked asymmetry, suggesting that a major portion of the Ca++ -ATPase resides on the exterior of the vesicle. The electron density profile also suggests that the Ca++-dependent ATPase penetrates into the lipid hydrocarbon core of the SR membrane. Under conditions suitable for X-ray analysis, SR vesicles prepared as partially dehydrated oriented multilayers are shown to conserve most of their ATP-induced Ca++ uptake functionality, as monitored spectrophotometrically with the Ca++ indicator arsenazo III. This has been verified both in resuspensions of SR after centrifugation and slow partial dehydration, and directly in SR multilayers in a partially dehydrated state (20-30 percent water). Therefore, the profile structure of the SR membrane that we have determined may closely resemble that found in vivo.     

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.     

Guanosine triphosphate utilization by canine cardiac muscle sarcoplasmic reticulum

We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Phosphorylation of the calcium adenosinetriphosphatase of sarcoplasmic reticulum: rate-limiting conformational change followed by rapid phosphoryl transfer

The calcium adenosinetriphosphatase of sarcoplasmic reticulum, preincubated with Ca2+ on the vesicle exterior (cE X Ca2), reacts with 0.3-0.5 mM Mg X ATP to form covalent phosphoenzyme (E approximately P X Ca2) with an observed rate constant of 220 s-1 (pH 7.0, 25 degrees C, 100 mM KCl, 5 mM MgSO4, 23 microM free external Ca2+, intact SR vesicles passively loaded with 20 mM Ca2+). If the phosphoryl-transfer step were rate-limiting, with kf = 220 s-1, the approach to equilibrium in the presence of ADP, to give 50% EP and kf = kr, would follow kobsd = kf + kr = 440 s-1. The reaction of cE X Ca2 with 0.8-1.2 mM ATP plus 0.25 mM ADP proceeds to 50% completion with kobsd = 270 s-1. This result shows that phosphoryl transfer from bound ATP to the enzyme is not the rate-limiting step for phosphoenzyme formation from cE X Ca2. The result is consistent with a rate-limiting conformational change of the cE X Ca2 X ATP intermediate followed by rapid (greater than or equal to 1000 s-1) phosphoryl transfer. Calcium dissociates from cE X Ca2 X ATP with kobsd = 80 s-1 and ATP dissociates with kobsd = 120 s-1 when cE X Ca2 X ATP is formed by the addition of ATP to cE X Ca2. However, when E X Ca2 X ATP is formed in the reverse direction, from the reaction of E approximately P X Ca2 and ADP, Ca2+ dissociates with kobsd = 45 s-1 and ATP dissociates with kobsd = 35 s-1. This shows that different E X Ca2 X ATP intermediates are generated in the forward and reverse directions, which are interconverted by a conformational change.     

Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump.

The giant-patch technique was used to study the Na+,K(+)-ATPase in excised patches from rat or guinea pig ventricular myocytes. Na+,K(+)-pump currents showed a saturable ATP dependence with aK(m) of approximately 150 microM at 24 degrees C. The pump current can be completely abolished by ortho-vanadate. Dissociation of vanadate from the enzyme in the absence of extracellular Na+ was slow, with a Koff of 3.10(-4) S-1 (K1 approximately 0.5 microM, at 24 degrees C). Stationary currents were markedly dependent on intracellular pH, with a maximum at pH 7.9. Temperature-dependence measurements of the stationary pump current yielded an activation energy of approximately 100 kJ mol-1. Partial reactions in the transport cycle were investigated by generating ATP concentration jumps through photolytic release of ATP from caged ATP at pH 7.4 and 6.3. Transient outward currents were obtained at pH 6.3 with a fast rising phase followed by a slower decay to a stationary current. It was concluded that the fast rate constant of approximately 200 s-1 at 24 degrees C (pH 6.3) reflects a step rate-limiting the electrogenic Na+ release. Simulating the data with a simple three-state model enabled us to estimate the turnover rate under saturating substrate concentrations, yielding rates (at pH 7.4) of approximately 60 s-1 and 200 s-1 at 24 degrees C and 36 degrees C, respectively.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

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)     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells.

The Ca2+ binding kinetics of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer, which determine the time course of Ca2+ changes after photolysis of DM-nitrophen, were studied in bovine chromaffin cells. The in vivo Ca2+ association rate constants of fura-2, DM-nitrophen, and the endogenous Ca2+ buffer were measured to be 5.17 x 10(8) M-1 s-1, 3.5 x 10(7) M-1 s-1, and 1.07 x 10(8) M-1 s-1, respectively. The endogenous Ca2+ buffer appeared to have a low affinity for Ca2+ with a dissociation constant around 100 microM. A fast Ca2+ uptake mechanism was also found to play a dominant role in the clearance of Ca2+ after flashes at high intracellular free Ca2+ concentrations ([Ca2+]), causing a fast [Ca2+]i decay within seconds. This Ca2+ clearance was identified as mitochondrial Ca2+ uptake. Its uptake kinetics were studied by analyzing the Ca2+ decay at high [Ca2+]i after flash photolysis of DM-nitrophen. The capacity of the mitochondrial uptake corresponds to a total cytosolic Ca2+ load of approximately 1 mM.     

Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).     

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Abnormal rapid Ca2+ release from sarcoplasmic reticulum of malignant hyperthermia susceptible pigs.

Using the rapid filtration technique to investigate Ca2+ movements across the sarcoplasmic reticulum (SR) membrane, we compare the initial phases of Ca2+ release and Ca2+ uptake in malignant hyperthermia susceptible (MHS) and normal (N) pig SR vesicles. Ca2+ release is measured from passively loaded SR vesicles. MHS SR vesicles present a 2-fold increase in the initial rate of calcium release induced by 0.3 microM Ca2+ (20.1 +/- 2.1 vs. 6.3 +/- 2.6 nmol mg-1 s-1). Maximal Ca2+ release is obtained with 3 microM Ca2+. At this optimal concentration, rate of Ca2+ efflux in absence of ATP is 55 and 25 nmol mg-1 s-1 for MHS and N SR, respectively. Ca(2+)-induced Ca2+ release is inhibited by Mg2+ in a dose-dependent manner for both MHS and N pig SR vesicles (K1/2 = 0.2 mM). Caffeine (5 mM) and halothane (0.01% v/v) increase the Ca2+ sensitivity of Ca(2+)-induced Ca2+ release. ATP (5 mM) strongly enhances the rate of Ca2+ efflux (to about 20-40-fold in both MHS and N pig SR vesicles). Furthermore, both types of vesicles do not differ in their high-affinity site for ryanodine (Kd = 12 nM and Bmax = 6 pmol/mg), lipid content, ATPase activity and initial rate of Ca2+ uptake (0.948 +/- 0.034 vs. 0.835 +/- 0.130 mumol mg-1 min-1 for MHS and N SR, respectively). Our results show that MH syndrome is associated to a higher rate of Ca2+ release in the earliest phase of the calcium efflux.