Physical, enzymatic, and contractile properties of brain myosin with anti-brain myosin Fab fragment bound on its tail.
An antibody obtained by immunizing a rabbit with purified bovine brain myosin was found to react with the tail portion of the myosin heavy chain. An Fab fragment obtained by limited papain digestion of the antibody was allowed to bind to brain myosin, and the complex of the Fab fragment and brain myosin (Fab-myosin) was isolated. On examination of the rotary-shadowed Fab-myosin by electron microscopy, most of the Fab fragment was located on the middle to C-terminal regions of the tails of the myosin molecules. The solubility of Fab-myosin in low salt solutions was higher than that of control brain myosin. Fab-myosin was found to form small irregular aggregates in low salt solutions instead of regular bipolar filaments, and the relative population of the monomeric form of myosin molecules observed for the Fab-myosin was much larger than that observed for the control myosin. The actin-activated Mg2+-ATPase activity of Fab-myosin was stimulated two- to threefold by phosphorylation of the light chains with myosin light chain kinase, as observed for the control brain myosin. Furthermore, the levels of the ATPase activity of the phosphorylated and dephosphorylated Fab-myosins were similar to those of the phosphorylated and dephosphorylated control myosins, respectively. The superprecipitation activity of Fab-myosin was also highly dependent on phosphorylation of the light chains. Although control brain myosin formed a large superprecipitate network which contracted to a dense particle, Fab-myosin generated only numerous tiny superprecipitates under the same conditions. From these results it was deduced that a regular filamentous state of brain myosin was not prerequisite for its actin-activated Mg2+-ATPase and superprecipitation activities but was indispensable for the formation of a large and well contractible superprecipitate.
Matsumura
S
S
Department of Biochemistry, Saga Medical School.
Ohmori
K
K
Chiba
T
T
Kumon
A
A
D000818:Animals; D000917:Antibody Formation; D000918:Antibody Specificity; D001923:Brain Chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007140:Immunoglobulin Fab Fragments / Q000032:analysis*; D008854:Microscopy, Electron; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology; D010206:Papain; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D012995:Solubility
D007140:Immunoglobulin Fab Fragments; D011189:Potassium Chloride; D009219:Myosin-Light-Chain Kinase; D010206:Papain; D009218:Myosins