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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

Baculovirus expression of chicken nonmuscle heavy meromyosin II-B. Characterization of alternatively spliced isoforms.

We have expressed two truncated isoforms of chicken nonmuscle myosin II-B using the baculovirus expression system. One of the expressed heavy meromyosins (HMMexp) consists of two 150-kDa myosin heavy chains (MHCs), comprising amino acids 1-1231 as well as two pairs of 20-kDa and 17-kDa myosin light chains (MLCs) in a 1:1:1 molar ratio. The second HMMexp was identical except that it contained an insert of 10 amino acids (PESPKPVKHQ) at the 25-50-kDa domain boundary in the subfragment-1 region of the MHC. These 10 amino acids include a consensus sequence (SPK) for proline-directed kinases. Expressed HMMs were soluble at low ionic strength and bound to rabbit skeletal muscle actin in an ATP-dependent manner. These properties afforded a rapid purification of milligram quantities of expressed protein. Both isoforms were capable of moving actin filaments in an in vitro motility assay and manifested a greater than 20-fold activation of actin-activated MgATPase activity following phosphorylation of the 20-kDa MLC. HMMexp with the 10-amino acid insert was phosphorylated by Cdc2, Cdk5, and mitogen-activated protein kinase in vitro to 0.3-0.4 mol of PO4/mol of MHC. The site phosphorylated in the MHC was identified as the serine residue present in the 10-amino acid insert and its presence was confirmed in bovine brain MHCs. Characterization of the baculovirus expressed noninserted and inserted MHC isoforms with respect to actin-activated MgATPase activity and ability to translocate actin filaments in an in vitro motility assay produced the following average values following MLC phosphorylation: noninserted HMMexp, Vmax = 0.28 s-1, Km = 12.7 microM; translocation rate = 0.077 micron/s; inserted HMMexp, Vmax = 0.37 s-1, Km = 15.1 microM; translocation rate = 0.092 micron/s.[1]

References

  1. Baculovirus expression of chicken nonmuscle heavy meromyosin II-B. Characterization of alternatively spliced isoforms. Pato, M.D., Sellers, J.R., Preston, Y.A., Harvey, E.V., Adelstein, R.S. J. Biol. Chem. (1996) [Pubmed]
 
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