TIME-RESOLVED FLUORESCENCE DEPOLARIZATION MEASUREMENTS OF F-ACTIN BINDING TO MYOSIN SUBFRAGMENTS-1 BEARING DIFFERENT ALKALI LIGHT-CHAINS

Experiments were designed to test for functional differences which might shed light on the differences in actin-activated ATPase activities recently reported for myosin subfragments-1 bearing different light chains. By using the method of A. G. Weeds and R. S. Taylor (1975, Nature (London) 257, 54), two types of subfragment-1 (S-1) from myosin of rabbit fast skeletal muscle were prepared: (S-1)·A1 and (S-1)·A2 bearing, respectively, the alkali-1 and alkali-2 light chains. (In agreement with the findings of these investigators, actin enhanced the ATPase activity of (S-1)·A1 more than that of (S-1)·A2 at lower actin concentrations.) Through use of time-resolved fluorescence depolarization techniques, the affinity constants for the binding of the two types of S-1 to F-actin in the absence of ATP were found to be very similar: 3.4 ± 0.3 × 106 m-1 (N = 10) for (S-1)·A1 and 3.9 ± 0.2 × 106 m-1 (N = 7) for (S-1)·A2 of one preparation, and 6.4 ± 0.2 × 106 m-1 (N = 6) for (S-1)·A1 and 7.7 ± 0.5 × 106 m-1 (N = 12) for (S-1)·A2 of another preparation (pH 7.0, 25 °C, 0.28 m KCl, 1.5 mm MgCl2, 0.5 mm ethylene glycol bis (β-aminoethyl ether) N,N′-tetracetic acid, 10 mm imidazole, and 0.1 mm N-tris (hydroxymethyl) methyl-2-aminoethane sulfonate). The affinity constants for the two species of S-1 and actin also have a similar dependence on ionic strength and are not affected by addition of 0.6 mm CaCl2 to the above solution. The CaATPase (or the CaITPase) activities of the two species of S-1 show the same pH dependence. © 1979.