Mechanoenzymatic characterization of human myosin Vb.
There are three isoforms of class V myosin in mammals. While myosin Va has been studied well, little is known about the function of other myosin V isoforms (Vb and Vc) at a molecular level. Here we report the mechanoenzymatic function of human myosin Vb (HuM5B) for the first time. Electron microscopic observation showed that HuM5B has a double-headed structure with a long neck like myosin Va. V(max) and K(actin) of the actin-activated ATPase activity of HuM5B were 9.7 +/- 0.4 s(-)(1) and 8.5 +/- 0.1 microM, respectively. K(actin) and K(ATP) of the actin-activated ATPase activity were significantly higher than those of myosin Va. ADP markedly inhibited the ATPase activity. The rate of release of ADP from acto-HuM5B was 12.2 +/- 0.5 s(-)(1), which was comparable to the V(max) of the actin-activated ATPase activity. These results suggest that ADP release is the rate-limiting step for the actin-activated ATPase cycle; thus, HuM5B is a high duty ratio myosin. Consistently, the actin gliding velocity (0.22 +/- 0.03 microm/s) remained constant at a low motor density. The actin filament landing assay revealed that a single HuM5B molecule is sufficient to move the actin filament continuously, indicating that HuM5b is a processive motor.
Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655-0127, USA.
Shinya
S
Watanabe
Katsuhide
K
Mabuchi
Reiko
R
Ikebe
Mitsuo
M
Ikebe
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D018995:Myosin Heavy Chains; D024701:Myosin Type V; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013997:Time Factors
D000199:Actins; C505832:MYO5B protein, human; D000251:Adenosine Triphosphatases; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D009218:Myosins