ISOLATION AND CHARACTERIZATION OF 3 DICTYOSTELIUM MYOSIN-I ISOZYMES

Using ion exchange chromatography and an ATP-dependent actin precipita tion step, we have isolated three myosin-I isozymes that, together, ac count for most of the K+EDTA-ATPase activity recovered from extracts o f Dictyostelium. The two major myosin-I isozymes, present in approxima tely equal amounts, had apparent molecular masses of 125 kDa on SDS ge ls and have been identified by amino acid sequence analysis as the pro ducts of the Dictyostelium myosin-IB (DMIB) and myosin-ID (DMID) genes . DMIB, with a specific K+EDTA-ATPase activity 10-fold higher than DMI D, was responsible for most of the activity in cell extracts. The thir d isozyme, present in low amounts, had an apparent molecular mass of 1 37 kDa on SDS gels and is too large to be the product of any of the kn own myosin-I genes. DMIB eluted from DE53 cellulose columns as two dis tinct peaks (II and III). Addition of the phosphatase inhibitor okadai c acid to the extraction buffer increased the fraction of DMIB recover ed from growth phase cells in peak III from 35 to 70%. DMIB isolated f rom peak III, but not from peak II, displayed a significant level of a ctin-activated MgATPase activity. These results indicate that peak III represents a phosphorylated, actin-activatable form of DMIB.