Characterization of the motor and enzymatic properties of smooth muscle long S1 and short HMM: role of the two-headed structure on the activity and regulation of the myosin motor.
Truncated mutants of smooth muscle myosin containing various lengths of the S2 portion were expressed in Sf9 cells and purified. Truncated myosin having a heavy chain molecular mass of 128 kDa and larger formed a stable dimer, while 108 kDa myosin remained a monomer. On the other hand, 114 and 110 kDa myosins existed as both monomer and dimer. The enzymatic activity and also the in vitro actin sliding activity of these mutant myosins were measured, and the following findings were obtained. (1) Both the actin sliding activity and the actin-activated ATPase activity showed phosphorylation dependence when myosin forms a dimer while the monomeric form was phosphorylation-independent. This indicates that the interaction between the two heads is operating and critical for the regulation. (2) The actin sliding velocity of the dimer form was twice as large as that of the monomer form, while the actin-activated ATPase activity of the two forms was identical, suggesting that the mechano-chemical efficiency is affected by the interaction between the two heads. (3) The depression of the Mg(2+)-ATPase activity of myosin at low ionic strength, characteristic of the 6S-10S transition of smooth muscle myosin, is abolished with the monomer form, suggesting that the association of the two heads is critical for the 6S-10S transition.
Sata
M
M
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655-0127, USA.
Matsuura
M
M
Ikebe
M
M
D000199:Actins / Q000378:metabolism / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D009130:Muscle, Smooth / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins