Functional chicken gizzard heavy meromyosin expression in and purification from baculovirus-infected insect cells.
The heavy chain and the essential and the regulatory light chains of chicken gizzard heavy meromyosin (HMM) were coexpressed in Spodoptera frugiperda (fall armyworm) cells infected with a mixture of two recombinant Autographa californica baculoviruses. Soluble HMM consisting of the heavy chain and the two types of light chains was obtained. The recombinant HMM was purified from the virus-infected cells and characterized. The regulatory light chain of the isolated recombinant HMM was phosphorylated by myosin light chain kinase in the presence of calmodulin in a Ca(2+)-dependent manner. The ATPase of the recombinant HMM was activated by rabbit skeletal muscle actin when myosin light chain kinase, calmodulin, and Ca2+ were present in the reaction medium. Chicken gizzard tropomyosin enhanced the actin-activated ATPase activity. The recombinant HMM decorated actin filaments, displaying the characteristic arrowhead pattern along the filaments. This report on a functional recombinant double-headed smooth muscle myosin fragment opens the way to detailed studies on the molecule.
Department of Structural Analysis, National Cardiovascular Center Research Institute, Osaka, Japan.
H
H
Onishi
K
K
Maéda
Y
Y
Maéda
A
A
Inoue
K
K
Fujiwara
D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D005822:Genetic Vectors; D005895:Gizzard, Avian; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry; D015879:Myosin Subfragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D018411:Spodoptera; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D002147:Calmodulin; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D014335:Tropomyosin; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium