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

Abstract

SUMMARY1. The time course of magnesium adenosine triphosphate (Mg ATP) cleavage in chemically skinned muscle fibres 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)phenylethyladenosine 5'-triphosphate (caged ATP) and terminated by rapid freezing 50 ms to 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 0C in the presence and absence of calcium ions, for fibres near rest length and stretched beyond overlap of the myofilaments.2. At full overlap and in the absence of calcium (< 10-8 M) and nucleotide, the fibres developed rigor.tension. Following the laser pulse the tension decreased to that of a relaxed fibre in two distinct phases. The first phase lasted about 40 ms and was followed by a second phase during which tension decreased to zero with an approximately exponential time course with a rate constant of 11 s51. In the presence of 2 x 10-5 M-free calcium ions, the initial phase following the laser flash lasted approximately 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 fibres was 54 kN/m2. For fibres stretched beyond overlap, no change in tension was observed following the release of Mg ATP.3. 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 fibres at full myofilament overlap, and equal to 0-7 molecules per myosin subfragment 1 head for fibres stretched beyond overlap. At full overlap in the presence of calcium ions, the steady-state rate equalled 1'8 mol Mg ATP cleaved per mole myosin subfragment 1 head per second. In all other cases the steady-state rate of Mg ATP cleavage was at least 10-fold less.4. When fibres at full overlap were pre-incubated with 2 mM-ADP, the initial phase M. A. FERENCZI, E. HOMSHER AND D. R. TRENTHAMof the tension response was somewhat prolonged, but the burst of ADP formation was also complete within 50 ms. However, in the case of fibres stretched beyond overlap, the presence of ADP reduced the rate of the initial burst of ADP formation by at least a factor of 3. 5. A low steady-state rate of Mg ATP cleavage for fibres stretched beyond overlap in the presence of calcium ions showed that other ATPases such as the calcium ion-dependent ATPase of the sarcoplasmic reticulum did not cleave Mg ATP significantly. This conclusion was supported by control experiments in which fibres had been treated with Triton X-100.6. The results show that during an isometric contraction most of the myosin heads are in the form of product intermediate states and that this steady state is reached rapidly (<50 ms). These properties are similar to those of the Mg-dependent actomyosin subfra...

Nucleotide binding by myosin in glycerol‐extracted rabbit skeletal muscle fibres at temperatures between −35°C and 10°C

“…Marston [lo] found that this was the case in glycerol-extracted rabbit psoas fibres, as did Maruyama and Weber [9] and Sleep [ll] in myofibrils. The caged-ATP studied of Ferenczi et al [12] confirm the observation, and the oxygen exchange work of [I71 shows that the bound nucleotide is in rapid hydrolytic equilibrium. In the present experiments 160 -200 pM nucleotide was found in excess over the 100 pM in the bathing solution.…”

“…On protein denaturation the bound nucleotide is recovered as ADP, at temperatures down to near freezing [lo, 111. In similar fibres when a burst of caged ATP is released it is rapidly and entirely converted to ADP [12]. Thus it appears that within the contractile matrix the equilibrium of the hydrolysis of bound nucleotide may be shifted towards ADP.…”

Rapid relaxation of single frog skeletal muscle fibres following laser flash photolysis of the caged calcium chelator, diazo‐2

“…Single skinned fibres were mounted on aluminium T-clips [10] and suspended between stainless-steel hooks attached to a tension transducer (Akers) at one end and to a fixed mount at the other. The apparatus, previously described by Ferenczi et al [11] allows rapid changes of the solution which bathes the fibres. The laser photolysis technique and recording instrumentation was essentially the same as that described previously [11].…”

Skeletal muscle energetics with PNMR: personal views and historic perspectives

“…1) showed the immediate response of mitochondrial electron transport to muscle stimulation (18) and an early event in the cross-bridge cycle is ADP release (19). It was so fast that it was necessary to study it at 88 in pioneer experiments of Chance and Connelly (20).…”