Magnesium ion dependent adenosine triphosphatase activity of heavy meromyosin as a function of temperature between +20 and -15.degree.C

Abstract

The hydrolysis of Mg2+-adenosine 5'-triphosphate (ATP) by heavy meromyosin has been studied between +20 and -15 degrees C, especially in the low-temperature range, in a medium containing 30% (v/v) ethylene glycol by fluorometric, spectrophotometric, and potentiometric measurements. The time course of the fluorescence changes of the enzyme during the reaction depends markedly on the temperature in consequence of large differences between the activation energies of the various steps. The observed kinetics have been analyzed according to the simplified scheme of Bagshaw & Trentham [Bagshaw, C. R., & Trentham, D. R. (1974) Biochem. J. 141, 331-349]. The following results have been obtained. (1) The rate-limiting step of the reaction changes in this temperature range; at 20 degrees C M**.ADP.Pi is the predominant steady-state complex, and M*.ADP predominates at -15 degrees C, with a half-life of approximately 10 min. (2) As expected, on the basis that it is the dissociation of the M*.ADP complex which becomes rate limiting at low temperature, one observes, in the pre-steady-state below 0 degrees C, both a proton burst and a lag phase in ADP release. (3) At low temperature, the equilibrium M*.ATP in equilibrium M**.ADP.Pi is displaced to the left All the kinetic data obtained in this study are compatible with a simple pathway for the Mg2+-ATP hydrolysis by myosin and with sequential release of the reaction products.

Cryoenzymological Studies on Myosin Subfragment 1

“…Further studies on the reaction pathway of myosin by rapid reaction techniques at low temperatures should clarify the effect of temperature on the equilibria of the various intermediates. Fluorescence studies at low temperature have lead to similar conclusions to our work [33].…”

Effect of the filamentous structure of myosin on the actomyosin ATPase activity

“…Heavy meromyosin and actin were prepared and characterized as described [12, 131. Actin was dialyzed before use against a 5 mM Tris/HCl buffer containing 6 mM KC1 and 3.6 mM MgC12; its reduced viscosity [q] was equal to 9.8 f 0.95 dl/g at 25 "C in this solution.…”

“…Purity of myosin was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate; the patterns showed a very intense heavychain band and three light-chain bands in the expected staining ratios. Solutions of minifilaments were kept at 4°C and used between three days after their preparation.Heavy meromyosin and actin were prepared and characterized as described [12, 131. Actin was dialyzed before use against a 5 mM Tris/HCl buffer containing 6 mM KC1 and 3.6 mM MgC12; its reduced viscosity [q] was equal to 9.8 f 0.95 dl/g at 25 "C in this solution.…”

Conformational transitions in the myosin head induced by temperature, nucleotide and actin

“…The nucleotide-promoted specific cleavages (steps 3 and 4 in the digestion scheme) seem to reflect the changes in myosin heavy-chain conformation upon nucleotide binding that have been detected by a variety of other techniques [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][49][50][51]]. Here we show that the same conformational change is promoted by lowering the temperature.…”

“…That the binding of ATP is also a three-step process [49] has been shown unambigously by ATP-chase experiments [50] and by using 1,P-ethenoadenosinetriphosphate, a fluorescent analogue of ATP [51]. These and other studies have shown that the equilibrium constant for the transition between the two forms of binary nucleotide-myosin complexes varies with temperature, ionic strength and pH [28,29,32,33, 35-37, 52, 531. The results have been interpreted as indicating that the myosin head itself exists in two conformational states in equilibrium which depends on ambient factors and is perturbed by the binding of nucleotides [36, 37, 52, 541.…”