Kinetic characterization of a monomeric unconventional myosin V construct.
An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulin-binding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 +/- 1.6 s(-1) with a K(app) of approximately 1 microM (37 degrees C), and the velocity of actin movement was approximately 0.3 micrometer/s (30 degrees C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to approximately 70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s(-1)), and ATP hydrolysis occurs at approximately 200 s(-1). The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.
Trybus
K M
KM
Department of Physiology and Molecular Biophysics, University of Vermont, Burlington, Vermont 05405, USA. trybus@salus.uvm.edu
Krementsova
E
E
Freyzon
Y
Y
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D019281:Dimerization; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D024701:Myosin Type V; D009218:Myosins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D002148:Calmodulin-Binding Proteins; D018994:Myosin Light Chains; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; C078829:chicken brain myosin-V p190; D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D024701:Myosin Type V; D009218:Myosins; D002118:Calcium