The data presented here confirm and provide further experimental evidence that rabbit skeletal-muscle myosin subfragment-1 (S-1) binds to the postulated actin-(338-348) hydrophobic segment [Kabsch, Mannherz, Suck, Pai and Holmes (1990) Nature (London) 347, 37-44] with high affinity in the absence and presence of MgATP. The apparent dissociation constant of the S-1 interaction (5.5 x 10(-7) M) with the actin-(338-348) peptide was of the same order of magnitude as that of the actin-(18-28) binding site (2 x 10(-6) M). In similar conditions, fragmented (27 kDa-50 kDa-20 kDa) S-1 also bound to the peptide. Antibodies directed to the vicinal sequence 348-358 were rapidly eliminated from actin by S-1 interaction and weakened S-1 binding to monomeric or filamentous actin. The antigenic site (348-358) is located very close to the C-terminal S-1-binding site (360-369) and encompasses some residues (Leu-349 and Phe-352) included in the hydrophobic S-1-binding region [Schröder, Manstein, Jahn, Holden, Rayment, Holmes and Spudich (1993) Nature (London) 364, 171-174]. It was observed that anti-[actin-(348-358)] antibodies were also unable to decrease actomyosin ATPase activity, in contrast with previous results obtained with anti-[actin-(18-28)] antibodies [Adams and Reisler (1993) Biochemistry 32, 5051-5056]. The hydrophobic actin-(338-348) peptide used in considerable excess was unable to perturb acto-S-1 and S-1 activities in contrast with results obtained with the N-terminal actin peptide [Kôgler, Moir, Trayer and Ruegg (1991) FEBS Lett. 294, 31-34].
Studies on the subunit interactions of skeletal muscle myosin subfragment 1. Evidence for subunit exchange between isozymes under physiological ionic strength and temperature.
