The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit.

Abstract

The time course of Mg ATP cleavage in chemically skinned muscle fibers of the rabbit was measured by a method in which Mg ATP cleavage was initiated by photolytic release of ATP from P3-1-(2-nitro)phenylethyl ATP (caged ATP) and terminated by rapid freezing 50 ms-8 s later. Up to 5 mM-ATP was released following a single 50 ns laser pulse at 347 nm. Mg ATP cleavage was measured at 19.degree. C in the presence and absence of Ca ions, for fibers near rest length and stretched beyond overlap of the myofilaments. At full overlap and in the absence of Ca (< 10-8 M) and nucleotide, the fibers developed rigor tension. Following the laser pulse the tension decreased to that of a relaxed fiber in 2 distinct phases. The 1st phase lasted about 40 ms and was followed by a 2nd phase during which tension decreased to 0 with an approximately exponential time course with a rate constant of 11 s-1. In the presence of 2 .times. 10-5 M-free Ca ions, the initial phase following the laser flash lasted .apprx. 13 ms, and was followed by an exponential rise of tension with a rate constant of 28 s-1. The active tension reached by the muscle fibers was 54 kN/m2. For fibers stretched beyond overlap, no change in tension was observed following the release of Mg ATP. Under all conditions the time course of Mg ATP cleavage was biphasic, and consisted of a rapid initial burst of ADP formation, complete within 50 ms, followed by a slower steady-state rate of Mg ATP cleavage. The number of molecules of Mg ATP cleaved during the burst was approximately equal to the number of myosin subfragment 1 heads for fibers at full myofilament overlap, and equal to 0.7 molecules per myosin subfragment 1 head for fibers stretched beyond overlap. At full overlap in the presence of Ca ions, the steady-state rate equalled 1.8 mol Mg ATP cleaved per mole myosin subfragment 1 head/s. In all other cases the steady-state rate of Mg ATP cleavage was at least 10-fold less. When fibers at full overlap were pre-incubated with 2 mM-ADP, the initial phase of the tension response was somewhat prolonged, but the burst of ADP formation was also complete within 50 ms. In the case of fibers stretched beyond overlap, the presence of ADP reduced the rate of the initial burst of ADP formation by at least a factor of 3. A low steady-state rate of Mg ATP cleavage for fibers stretched beyond overlap in the presence of Ca ions showed that other ATPases such as the Ca ion-dependent ATPase of the sarcoplasmic reticulum did not cleave Mg ATP significantly. This conclusion was supported by control experiments in which fibers had been treated with Triton X-100. During an isometric contraction most of the myosin heads are in the form of product intermediate states and this steady state is reached rapidly (< 50 ms). These properties are similar to those of the Mg-dependent actomyosin subfragment 1 ATPase. The difference in the amount of rapidly formed ADP in fibers relaxed from rigor compared to when ATP was released in fibers stretched beyond overlap can be attributed to cycling of cross-bridges in the former case because of protein co-operativity in the myofilament.