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 aKapp o...

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Bibliographic Details
Published in:The Journal of biological chemistry 1999-09, Vol.274 (39), p.27448-27456
Main Authors: Trybus, Kathleen M., Krementsova, Elena, Freyzon, Yelena
Format: Article
Language:English
Subjects:
Actins - metabolism
Adenosine Diphosphate - metabolism
Adenosine Triphosphate - metabolism
Animals
Calcium - pharmacology
Calmodulin-Binding Proteins - chemistry
Calmodulin-Binding Proteins - metabolism
Cloning, Molecular
Dimerization
Egtazic Acid - pharmacology
Kinetics
Mice
Models, Chemical
Models, Molecular
Muscle, Skeletal - metabolism
Muscle, Smooth - metabolism
Myosin Light Chains - chemistry
Myosin Light Chains - metabolism
Myosin Type V
Myosins - metabolism
Nerve Tissue Proteins - chemistry
Nerve Tissue Proteins - metabolism
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
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Summary: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 aKapp of ∼1 μm (37 °C), and the velocity of actin movement was ∼0.3 μm/s (30 °C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to ∼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 ∼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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.274.39.27448