Characterization of myosin V binding to brain vesicles

Kyle E. Miller, Michael Sheetz

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Myosin II and V are important for the generation and segregation of subcellular compartments. We 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 >> tetrapolyphosphate = ADP > cAMP = Mg2+; and for myosin V pyrophosphate = tripolyphosphate > ATP-Mg2+ = ATP = AMP-PNP >> 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). We propose that polyphosphate anions bind to myosin II and V and induce a conformational change that disrupts binding to a receptor.

Original languageEnglish (US)
Pages (from-to)2598-2606
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number4
DOIs
StatePublished - Jan 28 2000
Externally publishedYes

Fingerprint

Myosin Type V
Myosin Type II
Brain
Adenylyl Imidodiphosphate
Adenosine Triphosphate
Polyphosphates
Myosins
Adenosine Diphosphate
Phosphorylation
Actin Cytoskeleton
Sedimentation
Electron microscopy
Fluorescent Antibody Technique
Anions
Actins
Hydrolysis
Electron Microscopy

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Characterization of myosin V binding to brain vesicles. / Miller, Kyle E.; Sheetz, Michael.

In: Journal of Biological Chemistry, Vol. 275, No. 4, 28.01.2000, p. 2598-2606.

Research output: Contribution to journalArticle

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