Fluorescence study of the high pressureâinduced denaturation of skeletal muscle actin
2002
Ikeuchi, Yoshihide | Suzuki, Atsusi | Oota, Takayoshi | Hagiwara, Kazuaki | Tatsumi, Ryuichi | Ito, Tatsumi | Balny, Claude
Ikkai & Ooi [Ikkai, T. & Ooi, T. (1966) Biochemistry5, 1551–1560] made a thorough study of the effect of pressure on Gââ£and Fâactins. 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 eATP 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 eATPâGâactin or eADP–Fâactin) followed firstâorder kinetics. The volume changes for the denaturation of Gâactin and Fâactin were estimated to be −72âmL·mol−1 and −67âmL·mol−1 in the presence of ATP, respectively. Changes in the intensity of fluorescence of Fâactin whilst under pressure suggested that eADP–Fâactin was initially depolymerized to eADP–Gâactin; subsequently there was quick exchange of the eADP for free eATP, and then polymerization occurred again with the liberation of phosphate from eATP bound to Gâactin in the presence of excess ATP. In the higher pressure range (>â250âMPa), the partial collapse of the threeâdimensional structure of actin, which had been depolymerized under pressure, proceeded immediately after release of the nucleotide, so that it lost the ability to exchange bound ADP with external free ATP and so was denatured irreversibly. An experiment monitoring eATP fluorescence also demonstrated that, in the absence of Mg2+âATP, the dissociation of actinâheavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.
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