Dependence of the Rate of Phosphate Dissociation from Actomyosin-ADP-Pi Upon the Alkali Light Chains in Skeletal Muscle Myosin

Abstract

The A1 light chain of rabbit skeletal myosin possesses a unique N-terminal extension of ∼40 amino acids which is lacking in A2. A C-terminal block of ∼140 amino acids is identical to both. The primary structures are completed by a small, non-identical peptide which is sandwiched between the above two sections of A1 and which is N-terminal in A2. The effect which A1 and A2 have on the product release step has been investigated by double-mixing stopped-flow in conjunction with a fluorescent phosphate binding protein (Buffer, 5mM MOPS, 2mM Mg acetate, pH 7). Rabbit skeletal myosin-S1 was chromatographically separated into A1 and A2 containing fractions. A slight excess of myosin-S1 over nucleotide was used in the first mix to ensure complete binding of the latter as well as a single turnover. After a 2 s delay to assure that ATP binding was complete, thin filaments (+Ca) were combined with the myosin-nucleotide mixture (M-ATP U M-ADP-Pi) in the second mix. The dominant component of the observed biphasic increase in fluorescence corresponds to Pi release from AM-ADP-Pi. Hyperbolic fits of the dependence of the rate upon the thin filament give maximum rates of: 80 s-1 (S1A1) and 139 s-1 (S1A2) and apparent binding constants of 10 μM (S1A1) and 72 μM (S1A2). The rates of phosphate dissociation are 4-5 more rapid than the steady state measurements of 20 s-1 (A1) and 36 s-1 (A2) and therefore cannot limit the rate of ATP hydrolysis. It is also evident that the N-terminal extension strengthens light chain affinity.