In vitro production of enzymatically active myosin heavy chain.
In order to initiate studies on the structural and functional relationships of the myosin heavy chain, we constructed a full-length complementary DNA encoding the isoform that is found in the fast white muscle of the embryonic chicken. The complementary DNA contained 108 basepairs of its 3'-untranslated region and was preceded by a leader sequence derived from the alfalfa mosaic virus. Similarly, a complementary DNA encoding 963 amino acids which encompass the subfragment-1 of myosin and part of the subfragment-2 was also constructed. Each was inserted into the expression vector pMT2 and transiently transfected into COS-1 cells. Both constructs directed the expression of the respective proteins, each of which was immunogenic. The full-length and subfragment-1 proteins interacted with actin and demonstrated high levels of a K(+)-activated, EDTA-resistant ATPase activity, which is characteristic of myosin.
Rindt
H
H
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, OH 45267.
Bauer
B J
BJ
Robbins
J
J
D000818:Animals; D002460:Cell Line; D002642:Chick Embryo; D003001:Cloning, Molecular; D004247:DNA; D015870:Gene Expression; D066298:In Vitro Techniques; D009218:Myosins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis
D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D009218:Myosins