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 epsilon ATP 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 epsilon ATP-G-actin or epsilon ADP-F-actin) followed first-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be -72 mL x mol(-1) and -67 mL x mol(-1) in the presence of ATP, respectively. Changes in the intensity of fluorescence of F-actin whilst under pressure suggested that epsilon ADP-F-actin was initially depolymerized to epsilon ADP-G-actin; subsequently there was quick exchange of the epsilon ADP for free epsilon ATP, and then polymerization occurred again with the liberation of phosphate from epsilon ATP 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, 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 epsilon ATP fluorescence also demonstrated that, in the absence of Mg(2+)-ATP, the dissociation of actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.
Ikeuchi
Yoshihide
Y
Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan. ikeuchiy@agr.kyushu-u.ac.jp
Suzuki
Atsusi
A
Oota
Takayoshi
T
Hagiwara
Kazuaki
K
Tatsumi
Ryuichi
R
Ito
Tatsumi
T
Balny
Claude
C
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018482:Muscle, Skeletal / Q000737:chemistry*; D015879:Myosin Subfragments / Q000378:metabolism; D011312:Pressure; D011489:Protein Denaturation; D011817:Rabbits; D013050:Spectrometry, Fluorescence
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate