KINETIC CHARACTERIZATION OF A CYTOPLASMIC MYOSIN MOTOR DOMAIN EXPRESSED IN DICTYOSTELIUM-DISCOIDEUM

A detailed kinetic study of the interaction of a recombinant myosin he ad fragment (MHF) of Dictyostelium discoideum with actin and adenine n ucleotides has been made by using a combination of rapid-reaction, equ ilibrium, and fluorescence methods. MHF is equivalent in size to a pro teolytic fragment of skeletal muscle myosin, subfragment 1 (S1), the s implest unit of myosin to retain enzymatic and functional activity. Th e results show that qualitatively the interactions of MHF with nucleot ides and actin are the same as those of S1. Both bind to rabbit actin with the same affinity, although differences in the rate constants of their interactions with nucleotides in the presence and absence of act in occur. The rate of ATP binding to MHF and the subsequent cleavage s tep are significantly slower than the corresponding rates with S1. The dissociation of a fluorescent analog of ADP from MHF was 5-fold faste r than from S1, while its rate of binding MHF was 3-fold slower, resul ting in a weaker association equilibrium constant. The ATP-induced iso merization of the actoMHF complex was 10-fold slower than for actoS1, but the binding affinities of ADP for actoMHF and actoS1 were indistin guishable. The results suggest a different degree of coupling between the nucleotide and actin binding sites of MHF and S1 which may be a co mmon feature of nonmuscle myosins. They also provide the basis for a s tudy of specifically modified myosins with which one can probe the sit es of interaction with nucleotides or actin, as well as functional mot ility.