Pressure-relaxation studies of pyrene-labelled actin and myosin subfragment 1 from rabbit skeletal muscle. Evidence for two states of acto-subfragment 1.
We have used actin labelled at Cys-374 with N-(1-pyrenyl)iodoacetamide [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38] to monitor pressure-induced relaxations of acto-myosin subfragment 1. This label greatly increases the sensitivity of measurement of dissociated actin and reveals the presence of two relaxations. The experimental data can be fitted by a model in which actin binds subfragment 1 relatively weakly (K = 5.9 X 10(4) M-1) and then isomerizes to a more tightly bound complex (K = 1.7 X 10(7) M-1). This directly observed isomerization supports the model of Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell. Motil. 5, 351-361]. The rate of the isomerization is too high to be observed in the pressure-jump apparatus (less than 200 microseconds), but analysis of the amplitudes allows estimation of the equilibrium constant of the isomerization as 280 (20 degrees C, 0.1 M-KCl, pH 7). The equilibrium is sensitive to temperature, pressure, ionic strength and the presence of ethylene glycol. The pressure-sensitivity of the isomerization suggests a significant conformational change of the acto-myosin subfragment 1 complex.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives; D007536:Isomerism; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D011312:Pressure; D011817:Rabbits
D000199:Actins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; C029358:N-(1-pyrenyl)iodoacetamide; D009218:Myosins; D007460:Iodoacetamide