CHICKEN SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASE IS ACETYLATED ON ITS NH2-TERMINAL METHIONINE

The reported cDNA structure of chicken smooth muscle myosin light chai n kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc . Natl. Acad. Sci USA, 87: 2284-2288, 1990). The calculated Mr is 107, 534 whereas the estimate by SDS-FAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possi bility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported cod ing region. The native smMLCK is NH2-terminally blocked. A CNBr peptid e derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-A la corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermol ytic peptide had a mass of approximately 797 by time of flight mass sp ectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfrag ment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753 . The difference in mass corresponds to the NH2-terminal Met being blo cked by acetylation. The results demonstrate that the NH2-terminal seq uence of smMLCK inferred from the reported cDNA sequence is correct an d that the proposed initiating Met is not removed, but modified by alp ha-NH2 acetylation of the translation product.