Smooth muscle myosin is composed of homodimeric heavy chains.
Vertebrate smooth muscle myosin heavy chains (MHCs) exist as two isoforms with molecular masses of 204 and 200 kDa (MHC204 and MHC200) that are generated from a single gene by alternative splicing of mRNA (Nagai, R., Kuro-o, M., Babij, P., and Periasamy, M. (1989) J. Biol. Chem. 264, 9734-9737). A dimer of two MHCs associated with two pairs of myosin light chains forms a functional myosin molecule. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHC204 were generated and used to immunoprecipitate purified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. MHC204 quantitatively removed from this mixture was completely free of MHC200. Immunoprecipitation of the supernatant with an antiserum that recognizes both isoforms equally well revealed that only MHC200 remained. We conclude that only homodimers of MHC204 and MHC200 exist under these conditions. A method is described for the purification of enzymatically active MHC204 and myosin on a protein G-agarose high performance liquid chromatography column containing immobilized MHC204 antibodies. We show, using an in vitro motility assay, that the movement of actin filaments by myosin containing 204-kDa heavy chains (0.435 +/- 0.115 microns/s) was not significantly different from that of myosin containing 200-kDa heavy chains (0.361 +/- 0.078 microns/s) or from myosin containing equal amounts of each heavy chain isoform (0.347 +/- 0.082 microns/s).
Kelley
C A
CA
Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Sellers
J R
JR
Goldsmith
P K
PK
Adelstein
R S
RS
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D014422:Turkeys
D012333:RNA, Messenger; D009218:Myosins