Characterization of myosin V binding to brain vesicles

Myosin II and V are important for the generation and segregation of subcell ular compartments. Ne observed that vesicular myosin II and V were associat ed with the protein scaffolding of a common subset of vesicles by density s edimentation, electron microscopy, and immunofluorescence. Solubilization o f either myosin II or V was caused by polyphosphates with the following eff icacy at 10 mM: for myosin II ATP-Mg2+ = ATP = AMP-PNP (5'-adenylyl imidodi phosphate) > pyrophosphate = tripolyphosphate much greater than tetrapolyph osphate = ADP > cAMP = Mg2+; and for myosin V pyrophosphate = tripolyphosph ate > ATP-Mg2+ = ATP = AMP-PNP much greater than ADP = tetrapolyphosphate > cAMP = Mg2+. Consequently, we suggest solubilization was not an effect of phosphorylation, hydrolysis, or disassociation of myosin from actin filamen ts. Scatchard analysis of myosin V binding to stripped dense vesicles showe d saturable binding with a K-m of 10 nM. Analysis of native vesicles indica tes that these sites are fully occupied. Together, these data show there ar e over 100 myosin Vs/vesicle (100-nm radius). Ne propose that polyphosphate anions bind to myosin Il and V and induce a conformational change that dis rupts binding to a receptor.