Phosphorylation of .alpha..alpha.- and .beta..beta.-tropomyosin and synthetic peptide analogs

Publisher Website
Google Scholar
Abstract
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 .mu.M for .alpha..alpha.- and 265 .mu.M 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 .mu.M and 650 .mu.M, 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.degree. 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. At 25.degree. C, where tropomyosin exists as head-to-tail aggregates, two exponentials are needed to fit the time course data, indicating that phosphorylation of the two chains is ordered due to either nonidentical chain conformations or a negative cooperativity. In contrast, the time courses of phosphorylation for .beta..beta.-tropomyosin are biphasic at either 25 or 37.degree. C, indicative of a stronger polymerizability for the .beta..beta. isoform.