Effect of ADP and ionic strength on the kinetic and motile properties of recombinant mouse myosin V.
Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and single-headed (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V(max) and a low K(ATPase). Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s(-1) under conditions where the V(max) was 3.3 s(-1), indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.
Wang
F
F
Laboratory of Molecular Cardiology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Chen
L
L
Arcucci
O
O
Harvey
E V
EV
Bowers
B
B
Xu
Y
Y
Hammer
J A
JA
Sellers
J R
JR
D000199:Actins / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000494:pharmacology*; D000818:Animals; D001921:Brain / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation; D005453:Fluorescence; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D015879:Myosin Subfragments / Q000737:chemistry / Q000648:ultrastructure; D024701:Myosin Type V; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000648:ultrastructure; D011721:Pyrenes; D011994:Recombinant Proteins / Q000737:chemistry
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D015879:Myosin Subfragments; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011721:Pyrenes; D011994:Recombinant Proteins; C078829:chicken brain myosin-V p190; D000244:Adenosine Diphosphate; C030984:pyrene; D017301:Ca(2+) Mg(2+)-ATPase; D024701:Myosin Type V; D002118:Calcium