Myosin assembly critical for the enzyme activity of smooth muscle myosin phosphatase: effects of MgATP, ionic strength, and Mg(2+).
We suggested that an assembled form of phosphorylated myosin (P-myosin) might exhibit higher affinity for smooth muscle myosin phosphatase (SMMP) than dissociated P-myosin on the basis of the effect of MgATP [Sato and Ogawa (1999) J. Biochem. 126, 787-797]. To further deepen our understanding, we examined the SMMP activity and P-myosin assembly with various ionic strengths and Mg(2+) concentrations, with and without MgATP, all of which are well known to be critical for myosin assembly. The structure of myosin molecules was directly observed by electron microscopy using a rotary shadowing procedure, which was found to be consistent with the sedimentation assay. We found that the SMMP activity was always high when P-myosin was assembled. MgATP, which disassembled P-myosin mostly into a folded conformation, in contrast, decreased the enzyme activity. We also found that glycerol had a dissociating action on P-myosin, primarily dissociating it into an extended conformation, resulting in reduced SMMP activity, and that increases in the ionic strength and Mg(2+) (>5 mM) inhibited SMMP. These results indicate that myosin assembly is essential for SMMP activity.
Sato
O
O
Department of Pharmacology, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Ogawa
Y
Y
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology*; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000201:enzymology; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure*; D009994:Osmolar Concentration; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D013270:Stomach / Q000201:enzymology; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D009218:Myosins; D008274:Magnesium