| Popis: |
Caged-ATP (P3-1(2-nitrophenyl) ethyladenosine 5'-triphosphate) has been used to introduce millimolar concentrations of ATP rapidly into glycerinated muscle fibres, thus removing the limit imposed by diffusion to the time resolution of kinetic measurements. We have combined this technique with a rapid freezing method to arrest ATP hydrolysis abruptly. The work of Ferenczi et al. describes the time course of hydrolysis by fibres in the presence and absence of calcium, and for fibres stretched to a length where there is no myofilament overlap. Ferenczi measured the rate of the phosphate burst in muscle fibres. Here we show how these measurements can be extended to measure the equilibrium constant for the hydrolysis step and the rate of ATP release from the active site. Our initial results indicate that in chemically skinned psoas fibres of the rabbit at 12 degrees C, pH 7.1 and ionic strength of 200 mM, the equilibrium constant for the hydrolysis step is 6, and the rate of release of ATP from the active site is 13 s-1. |
