Abstract
A synthetic heptadecapeptide corresponding to part of the NH2-terminal 17 residues of chicken gizzard myosin light chain (Mr = 20,000), Ser-Ser-Lys-Thr-Thr-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-(P)-Asn-Val-Phe-Ser-NH2, was readily phosphorylated by the myosin light chain kinase isolated from the same tissue. The synthetic peptide was phosphorylated stoichiometrically at serine 13, the same residue phosphorylated in the parent protein. The apparent Km and Vmax for peptide phosphorylation was 90 microM and 1.3 mumol min-1 mg-1 compared to 10 microM and 22 mumol min-1 mg-1, respectively, for the myosin light chain. The synthetic heptadecapeptide acted as a competitive inhibitor for myosin light chain phosphorylation with Ki approximately 600 microM. Acetylation of the heptadecapeptide alpha-amino group of serine 1 had little effect on Vmax (0.8 mumol min-1 mg-1) and increased the apparent Km 2-fold. The smooth muscle myosin light chain kinase did not phosphorylate the synthetic heptadecapeptide analog of the corresponding skeletal muscle myosin light chain (Mr = 18,500), nor did it phosphorylate synthetic peptide substrates specific for the cAMP-dependent protein kinase or phosphorylase b kinase. These findings support the idea that the myosin light chain kinase has particular protein substrate specificity requirements and that some of these are derived from the region of primary structure around the phosphorylation site in its native substrate.
Highlights
From the Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkuille, Victoria 3052,Australia
The syntheticpeptide was phosphorylatedstoichiometrically at serine 13, the same residue phosphorylatedin the parent protein.The apparent K, and V, for peptidephosphorylation was 90 PM and1.3pmolmin" mg"compared to 10 p~ and 22 pmoml in"mg", respectively, for the myosin light chain.The synthetic heptadecapeptide acted as a competitive inhibitor for light chains
In the present study we have examined whether chicken gizzard myosin light chain kinase can phosphorylate a heptadecapeptide corresponding to part of the region around the phosphorylation site in chickengizzardmyosin light chain [7]
Summary
Amino group of serine 1 had little effect on V,, (0.8 All materials were reagent grade unless otherwise indicated. [y-"'PpI pmol min" mg") andincreased the apparenKt, 2-fold. The enzyme showslittle or no activity toward histone, casein, phosphorylase b, phosphorylase b kinase, or bovine serum albumin ( 5 ) .myosin light chains are phosphorylated most readily by enzyme from the same tissue It from the pellet by homogenization in 2 volumes of 40 mM imidazole buffer, pH 7.2.4 mM EDTA, 5mM ATP, and 0.5 mM dithiothreitol or 15 mM 2-mercaptoethanol, and centrifuged at 9,OOO X g for 25 min. Assayed in a volume of 0.08 ml of40mM 4-(2-hydroxyethyl)-l-piper- Peptide Phosphorylation Site-When it was found that azineethanesulfonicacid buffer, pH 7.0,0.5 mM [y-32P]ATP(100-2000 cpm/pmol), 0.1 mM EGTA, 7 mM magnesium acetate, 0.55 mMCaClZ, 5 pgof calmodulin, 1 mg/ml of bovine serum albumin, myosin light serine was the site of phosphorylation in the heptadecapeptide, it was of interest to determine which of the 4 serine chains or peptide as indicated, and enzyme diluted in buffer containing residues present was esterified. This was consistent with the [32P]phosphatebeing esterified to one of the two serine residues in the carboxyl-terminal region of the peptide
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