“Refractory-like” State of Heavy-meromyosin Induced by the Reaction of Acto-heavy-meromyosin with Inosine Triphosphate¹

Abstract

Interactions of ITP with rabbit skeletal heavy-meromyosin (HMM) and with acto-heavy-meromyosin (acto-HMM) were studied by using transient as well as steady-state kinetics. (1) The results obtained from the studies of HMM-ITPase [EC 3. 6. 1. 3] in the steady state and of changes in the UV-absorption spectrum of HMM can be summarized by the following proposed reaction scheme (1):
M+ITPM_??_•ITP_??_M^IDP_P_??_M+IDP+P_i (1)
The kinetic parameters in the scheme were estimated to be as follows: K₁=40 μM, k₂=62.5 sec^-1, k^-2_??_k₂, and k₄=0.6 sec^-1. (2) The steady-state activity of acto-HMM-ITPase was found to consist of two different activities, and the following reaction scheme (2) is proposed to accomodate the findings:
AM+ITP_??_A+M^IDP_P_??_(2a)
M+ITP_??_AMP^IDP_P_??_(2b).
The two activities are thus characterized by different sets of kinetic parameters: we found K_m=0.9 μM and V_max=0.7 sec^-1 in one set, whereas K'_m=160 μM and V'_max=20-30 sec^-1 in the other. (3) The dissociation of acto-HMM, and reassociation of F-actin and HMM were studied by using the light-scattering technique. The results obtained indicate that ITP, when added to acto-HMM, induces a special state in HMM, and that the HMM in this special state, denoted by M (R), is free from IDP and Pi, and is nonetheless incapable of binding to F-actin. It is therefore proposed that the following reaction step should be added to the scheme (2a):
M^IDP_P_??_M(R)+IDP+P₁ M+IDP+P₁
The value of k₅ for the reaction in which M (R) returns to a normal state of HMM (denoted as M) was estimated to be 0.08 sec^-1.