Characterization of myosin V binding to brain vesicles

Myosin II and V are important for the generation and segregation of subcellular compartments. Ne observed that vesicular myosin II and V were associated with the protein scaffolding of a common subset of vesicles by density sedimentation, electron microscopy, and immunofluorescence. Solubilization of either myosin II or V was caused by polyphosphates with the following efficacy at 10 mM: for myosin II ATP-Mg2+ = ATP = AMP-PNP (5'-adenylyl imidodiphosphate) > pyrophosphate = tripolyphosphate much greater than tetrapolyphosphate = ADP > cAMP = Mg2+; and for myosin V pyrophosphate = tripolyphosphate > 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 filaments. Scatchard analysis of myosin V binding to stripped dense vesicles showed saturable binding with a K-m of 10 nM. Analysis of native vesicles indicates that these sites are fully occupied. Together, these data show there are 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 disrupts binding to a receptor.