Kinetic studies on the effects of ADP and ionic strength on the interaction between myosin subfragment-1 and actin: implications for load-sensitivity and 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 pressure relaxation methods. The data suggest a four-step model i.e.: A + M.(N)(K0)<-->A approximately M.(N)(K1)<-->A - M.(N)(K2)<-->A.M.(N)(K3)<-->A.M.(N)#. ADP weakens the acto-S1 affinity via a reduction in Ko, with no apparent effect on K1 and no effect on K2, whilst k(+2) and k(-2) are both markedly reduced. Increased ionic strength reduces both K0 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 that the interaction proceeds via formation of a disordered complex stabilised by ionic interactions (corresponding to step 0), followed by a disordered-to-ordered transition involving additional hydrophobic contacts (step 1) after which further contacts of both types are made coupled to internal conformational changes (steps 2 and 3). Step 3 could have a role in extending the lifetime of force-generating crossbridges and limiting ATP turnover during 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) Nature 378: 748-751). Cooperative interactions between the two myosin heads also appear to have a role in this putative latch mechanism.
Conibear
P B
PB
Department of Biochemistry, University of Leicester, UK. pbc1@le.ac.uk
D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D007700:Kinetics; D018482:Muscle, Skeletal / Q000737:chemistry / Q000187:drug effects; D015879:Myosin Subfragments / Q000737:chemistry*; D009994:Osmolar Concentration; D011817:Rabbits
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate