NUCLEOTIDE AND ACTIN-BINDING PROPERTIES OF THE ISOLATED MOTOR DOMAIN FROM DICTYOSTELIUM-DISCOIDEUM MYOSIN

Nucleotide and actin binding properties of the truncated myosin head ( S1dC) from Dictyostelium myosin II were studied in solution using rabb it skeletal myosin subfragment 1 as a reference material. S1dC and sub fragment 1 had similar affinities for ADP analogues, epsilon ADP and T NP-ADP. The complexes of epsilon ADP and BeFx or A1F(4)(-) were less s table with S1dC than with subfragment 1. Stem-Volmer constants for acr ylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.A1F( 4)(-) and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. Th e corresponding values for subfragment 1 were 2.6, 1.5 and 1.1M(-1). T he environment of the nucleotide binding site was probed by using a hy drophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca2+-ATPase (K-i = 1.6 mu M). The binding of nucleotides to subf ragment 1 enhanced PPBA fluorescence and caused blue shifts in the wav elength of its maximum emission in the order: ATP approximate to ADP.A 1F(4)(-) approximate to ADP.BeFx > ATP gamma S > ADP > PPi. In the cas e of S1dC, the effects of different nucleotides were smaller and indis tinguishable from each other. S1dC bound actin tighter than S1 (K-d = 7 nM and 60 nM, respectively). The actin activated MgATPase activity o f S1dC varied between preparations, and the V-max and K-m values range d between 3 and 7 s(-1) and 60 and 190 mu M, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inact ivations at 35 degrees C. These results show that the nucleotide and a ctin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and t he communication between the nucleotide and actin binding sites in the se proteins.