Biomedical subjects

J A Sleep

Publications and source records attributed to J A Sleep.

3 recordsLinked to original sources

PubMed1978

Effect of actin concentration on the intermediate oxygen exchange of myosin; relation to the refractory state and the mechanism of exchange.

The effect of actin concentration on the myosin catalyzed exchange of phosphate oxygens with water accompanying ATP hydrolysis has been investigated. The extent of exchange was found to extrapolate to zero at infinite actin concentration at 23 and 0 degrees C for myosin subfragments S1(A1) and S1(A2). This result is consistent with actin associating directly with the product of the hydrolysis step and is not readily consistent with refractory state schemes in which the entire flow goes via a dissociating pathway. The possibility of a refractory state in the form of a phosphorylated intermediate or a bound metaphosphate state with hydrolysis occurring in the transition to the refractory state merits consideration. A full analysis of the dependence of intermediate exchange on the rate constants of the acto-S1 scheme is given and the errors arising from other methods of analysis are discussed. The rate of oxygen exchange was measured as 10 s-1 (23 degrees C) a value comparable with but slightly lower than the rate of reversal of the ATP cleavage step.

Actins↗

PubMed1978

Actin mediated release of ATP from a myosin-ATP complex.

The apparent second-order rate constant, ka-2, of actin binding to a myosin-ATP state (M*.ATP) and releasing ATP to the medium has been determined by two methods. The first was the measurement of the amount of ATP released when actin was added to the intermediate state, M*.ATP; the second was the measurement of oxygen exchange between ATP and HOH. A quantitative treatment of ATP in equilibrium HOH exchange is given to allow extraction of elementary rate constants from the data. Agreement between the two methods was good and at low ionic strength and 23 degrees C, ka-2 is 6 X 10(5) M-1 s-1 which is about one-third the value of the apparent second-order rate constant, ka4, of actin binding to the myosin product state (M**.ADP.Pi). The determination of ka-2 allows a lower limit of 6 s-1 to be placed upon the first-order rate of ATP release from AM.ATP. This is to be compared with a value of less than or equal to 1.5 X 10(-4) s-1 for the equivalent steps of the myosin scheme; thus actin enhances the rate by a factor of 4 X 10(4) or more. A greater proportion of the bound ATP is released to the medium as ATP with increasing actin concentration. This reflects the contribution to rate limitation at saturating actin concentration of steps between myosin states dissociated from actin.

Actins↗

PubMed1976

Intermediate states of actomyosin adenosine triphosphatase.

The early kinetic steps of actomyosin subfragment 1 (acto-S1) adenosine triphosphatase have been investigated by simultaneous monitoring of fluorescence and light scattering and also by observation of the time course of the production of phosphate. The results show that fluorescence enhancement occurs after the dissociation of actomyosin and that the rate of enhancement is similar to the maximum rate of enhancement for S1 alone, under similar conditions of pH and temperature. The maximum rate of the phosphate burst for acto S1 is also approximately the same as that for S1 alone. The maximum rates for fluorescence enhancement or phosphate formation are reached at much lower adenosine triphosphate concentrations for acto-S1 than for S1. An extension of the actomyosin scheme is presented which accounts for these results.

Actins↗