Characterization of the motor activity of mammalian myosin VIIA.
Myosin VIIA was cloned from rat kidney, and the construct (M7IQ5) containing the motor domain, IQ domain, and the coiled-coil domain as well as the full-length myosin VIIA (M7full) was expressed. The M7IQ5 contained five calmodulins. Based upon native gel electrophoresis and gel filtration, it was found that M7IQ5 was single-headed, whereas M7full was two-headed, suggesting that the tail domain contributes to form the two-headed structure. M7IQ5 had Mg(2+)-ATPase activity that was markedly activated by actin with K(actin) of 33 microm and V(max) of 0.53 s(-1) head(-1). Myosin VIIA required an extremely high ATP concentration for ATPase activity, ATP-induced dissociation from actin, and in vitro actin-translocating activity. ADP markedly inhibited the actin-activated ATPase activity. ADP also significantly inhibited the ATP-induced dissociation of myosin VIIA from actin. Consistently, ADP decreased K(actin) of the actin-activated ATPase. ADP decreased the actin gliding velocity, although ADP did not stop the actin gliding even at high concentration. These results suggest that myosin VIIA has slow ATP binding or low affinity for ATP and relatively high affinity for ADP. The directionality of myosin VIIA was determined by using the polarity-marked dual fluorescence-labeled actin filaments. It was found that myosin VIIA is a plus-directed motor.
Inoue
Akira
A
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0127, USA.
Ikebe
Mitsuo
M
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004398:Dyneins; D020409:Molecular Motor Proteins; D008969:Molecular Sequence Data; D000081412:Myosin VIIa; D009218:Myosins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats
D000199:Actins; D020409:Molecular Motor Proteins; C000631457:Myo7a protein, rat; D000081412:Myosin VIIa; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins; D004398:Dyneins