Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine.
The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility 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 coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala 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 thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment 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 blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.
Faux
M C
MC
St. Vincent's Institute of Medical Research, Fitzroy Vic., Australia.
Mitchelhill
K I
KI
Katsis
F
F
Wettenhall
R E
RE
Kemp
B E
BE
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D016384:Consensus Sequence; D003488:Cyanogen Bromide; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D005895:Gizzard, Avian; D013058:Mass Spectrometry; D008715:Methionine / Q000032:analysis*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification
D018076:DNA, Complementary; D010446:Peptide Fragments; D008715:Methionine; D009219:Myosin-Light-Chain Kinase; D010450:Endopeptidases; D003488:Cyanogen Bromide