Kinetic studies on the effects of ADP and ionic strength on the interaction between myosin subfragment-1 and actin: implications for load-sensitivityand regulation of the crossbridge cycle

The dynamics of the interaction of fast skeletal muscle myosin subfragment- 1 with pyrene-labelled actin were examined using both stopped-flow and pres sure relaxation methods. The data suggest a four-step model i.e.: [GRAPHICS] ADP weakens the acto-S1 affinity via a reduction in K-0, with no apparent e ffect on K-1 and no effect on K-2, whilst k(+2) and k(-2) are both markedly reduced. Increased ionic strength reduces both K-0 and k(+2) with no major effect on k(+1). Step 3 represents an extension to previous models and is ADP-dependent. The present work is discussed in relation to earlier studies which led to somewhat different conclusions (Taylor EW (1991) J Biol Chem 266: 294-302; Geeves MA (1989) Biochemistry 28: 5864-5871). It is likely th at the interaction proceeds via formation of a disordered complex stabilise d by ionic interactions (corresponding to step 0), followed by a disordered -to-ordered transition involving additional hydrophobic contacts (step 1) a fter which further contacts of both types are made coupled to internal conf ormational changes (steps 2 and 3). Step 3 could have a role in extending t he lifetime of force-generating crossbridges and limiting ATP turnover duri ng contraction against a load, and may be equivalent to a structural change observed in recent cryo-EM studies on the smooth muscle system (Whittaker M, Wilsonkubalek EM, Smith JE, Faust L, Milligan RA and Sweeney HL (1995) N ature 378: 748-751). Cooperative interactions between the two myosin heads also appear to have a role in this putative latch mechanism.