Formation and properties of smooth muscle myosin 20-kDa light chain-skeletal muscle myosin hybrids and photocrosslinking from the maleimidylbenzophenone-labeled light chain to the heavy chain.
Experimental conditions which permit the exchange of smooth muscle 20-kDa light chain into skeletal muscle myosin are described. The hybridization does not result in the regulation of actin-activated ATPase activity of the hybrid myosin by smooth light chain phosphorylation. Further, the KCl dependence of the Mg-ATPase activity of the hybrid was similar to that of skeletal muscle myosin. The dephosphorylation of the smooth light chain in the hybrid did not induce a conformational change in the hybrid from the 6 S to the 10 S state, thereby indicating that the conformational transition is dependent also on the nature of the heavy chain subunit. Exchange of the smooth light chain premodified at its Cys-108 by photolabile 4-(N-maleimido)benzophenone and photolysis resulted in crosslinking to the heavy chain subunit. Immunopeptide mapping using a monoclonal antibody against residues 1-23 at the N-terminus of the skeletal muscle myosin heavy chain identified the location of the photocrosslinking site to be beyond 92 kDa away from the N-terminus.
Rajasekharan
K N
KN
Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106.
Morita
J I
JI
Mayadevi
M
M
Ikebe
M
M
Burke
M
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D001577:Benzophenones / Q000494:pharmacology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008301:Maleimides; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010449:Peptide Mapping; D010782:Photolysis; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D055503:Protein Multimerization; D011817:Rabbits
D000199:Actins; D001577:Benzophenones; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D008301:Maleimides; D011189:Potassium Chloride; C046109:benzophenone-4-maleimide; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins