Kinetics of muscle contraction and actomyosin NTP hydrolysis from rabbit using a series of metal-nucleotide substrates.

Mechanical properties of skinned single fibres from rabbit psoas muscle have been correlated with biochemical steps in the cross-bridge cycle using a series of metal-nucleotide (Me.NTP) substrates (Mn(2+) or Ni(2+) substituted for Mg(2+); CTP or ITP for ATP) and inorganic phosphate. Measurements were made of the rate of force redevelopment following (1) slack tests in which force recovery followed a period of unloaded shortening, or (2) ramp shortening at low load terminated by a rapid restretch. The form and rate of force recovery were described as the sum of two exponential functions. Actomyosin-Subfragment 1 (acto-S1) Me.NTPase activity and Me.NDP release were monitored under the same conditions as the fibre experiments. Mn.ATP and Mg.CTP both supported contraction well and maintained good striation order. Relative to Mg.ATP, they increased the rates and Me.NTPase activity of cross-linked acto-S1 and the fast component of a double-exponential fit to force recovery by approximately 50% and 10-35%, respectively, while shortening velocity was moderately reduced (by 20-30%). Phosphate also increased the rate of the fast component of force recovery. In contrast to Mn(2+) and CTP, Ni.ATP and Mg.ITP did not support contraction well and caused striations to become disordered. The rates of force recovery and Me.NTPase activity were less than for Mg.ATP (by 40-80% and 50-85%, respectively), while shortening velocity was greatly reduced (by approximately 80%). Dissociation of ADP from acto-S1 was little affected by Ni(2+), suggesting that Ni.ADP dissociation does not account for the large reduction in shortening velocity. The different effects of Ni(2+) and Mn(2+) were also observed during brief activations elicited by photolytic release of ATP. These results confirm that at least one rate-limiting step is shared by acto-S1 ATPase activity and force development. Our results are consistent with a dual rate-limitation model in which the rate of force recovery is limited by both NTP cleavage and phosphate release, with their relative contributions and apparent rate constants influenced by an intervening rapid force-generating transition.