Smooth muscle myosin is composed of homodimeric heavy chains.

C A Kelley,J R Sellers,P K Goldsmith,R S Adelstein

doi:10.1016/s0021-9258(18)45851-6

Abstract

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).

Highlights

Less is known aboutthestructureandfunction of the nonmuscle and smooth muscle myosin heavy chains (MHCs) isoforms
Two reports show that these two MHCs are isoforms generated by alternative mRNA splicing that differ in their carboxyl termini [21, 22]
It hasbeen demonstrated titatively removed from this mixture was completely that distinct vascular andintestinalsmooth muscle MHC

Summary

Smooth Muscle MyosinIs Composed of Homodimeric Heavy Chains*

Multigene family (I), and theirexpression is regulated developmentally, hormonally, in a tissue-specific manner, and by mechanicalstress [2,3,4,5,6,7]. To investigate the isoform composition of the MHCs in native myosin, antibodies specific for MHCZOw~ere generated and used to immunoprecipitatepurified bovine aortic smooth muscle myosin from a solution containing equal amounts of each isoform. Evidence for differential expression, together with our previousfindings that MHCzo, but not MHCzoo, is phosphorylatedby casein kinase I1 in intact cells [27], suggests that smooth muscle MHC isoforms function differently and may be differentially regulated. WeusedMHCzo4-specific antibodiestostudythe association of the two MHC isoforms by immunoprecipitation from a solution of purified bovine aortic smooth muscle myosin containing equal amounts of each isoform. The motility assay buffer contained 10mM MOPS (pH ing casein kinase I1 phosphorylation of bovine aortic smooth muscle 7.2), 20 mM KCl, 5 mMMgC12,0.1 M EGTA, 1 mM dithiothreitol, 1 myosin [27]: RRVIENADGSEEEVDAR (Fig. lC, single underline).

RESULTS

DISCUSSION

Velocity amls

Using an i n vitro motility assay we show that myosins