Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin

Ikkai & Ooi [Ikkai, T. & Ooi, T. (1966) biochemistry 5, 1551-1560] made a thorough study of the effect of pressure on G- and F-actins. However, all of the measurements in their study were made after the release of pressure. In the present experiment in situ observations were attempted by using epsilonATP to obtain further detailed kinetic and thermodynamic information about the behaviour of actin under pressure. The dissociation rate constants of nucleotides from actin molecules (the decay curve of the intensity of fluorescence of epsilonATP-G-actin or epsilonADP-F-actin) followed first-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estirriated to be -72 mL.mol(-1) and -67 mL.mol(-1) in the presence of ATP, respectively. Changes in the intensity of fluorescence of F-actin whilst under pressure suggested that epsilonADP-F-actin was initially depolymerized to epsilonADP-G-actin; subsequently there was quick exchange of the epsilonADP for free epsilonATP, and then polymerization occurred again with the liberation of phosphate from epsilonATP bound to G-actin in the presence of excess ATP. In the higher pressure range (>250 MPa), the partial collapse of the three-dimensional structure of actin, which had been depolymerized under pressure, procceded immediately after release of the nucleotide, so that it lost die ability to exchange bound ADP with external free ATP and so was denatured irreversibly. An experiment monitoring epsilonATP fluorescence also demonstrated that, in the absence of Mg2+-ATP, the dissociation of actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.