Phosphorylation of alpha alpha- and beta beta-tropomyosin and synthetic peptide analogues.
A tropomyosin kinase partially purified from chicken embryos was used to study the phosphorylation mechanism of alpha alpha- and beta beta-tropomyosin and synthetic peptides containing the site of phosphorylation at Ser-283 and corresponding to residues 264-284 of the tropomyosin isoforms. The apparent Km is 47 microM for alpha alpha- and 265 microM for beta beta-tropomyosin, whereas the Vmax values are similar. The alpha [264-284] and beta [264-284] peptides have apparent Km values of 500 microM and 650 microM, respectively, and Vmax values similar to that of the intact tropomyosin. This indicates that the conformation of the phosphorylation site at the COOH-terminal end of tropomyosin contributes significantly to the phosphorylation of the substrate. Furthermore, the marginal difference in the Km values of the alpha- and beta-peptide cannot account for the 5-fold difference in the Km of the native alpha alpha and beta beta isoforms, suggesting that the conformations of alpha alpha- and beta beta-tropomyosin at the phosphorylation sites are significantly different. Phosphorylation of beta-peptide analogues, each with a single substitution corresponding to the alpha sequence, indicates that His-276 and Ile-284 have negative influences on the phosphorylation of the beta-peptide, whereas Met-281 improves it. Direct analyses of the time courses of phosphorylation of alpha alpha-tropomyosin at 37 degrees C, where head-to-tail polymerization is minimized, show that a single exponential can fit the data satisfactorily. This indicates a random phosphorylation of two identical chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Watson
M H
MH
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Taneja
A K
AK
Hodges
R S
RS
Mak
A S
AS
D000818:Animals; D001665:Binding Sites; D002642:Chick Embryo; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism*
D010455:Peptides; D014335:Tropomyosin; D011494:Protein Kinases; C041311:tropomyosin kinase