Temperature dependence of nucleotide association and kinetic characterization of myo1b.
Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 degrees C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide-inaccessible state to a nucleotide-accessible state (k(alpha)). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3-fold greater than other characterized myosins (e.g., myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (Vmax and K(ATPase)). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to k(alpha) suggesting that a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.
Lewis
John H
JH
The Pennsylvania Muscle Institute and Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6085, USA.
Lin
Tianming
T
Hokanson
David E
DE
Ostap
E Michael
EM
D008841:Actin Cytoskeleton / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D006868:Hydrolysis; D007700:Kinetics; D024461:Myosin Type I / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
C104297:Myo1b protein, rat; D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024461:Myosin Type I