Abstract
The COOH-terminal residue in peptide analogs of the phosphorylation site sequence in smooth muscle myosin light chains, Lys11-Lys12-Arg13-Ala-Ala-Arg16-Ala-Thr-Ser19 -(P)Asn20-Val21-Phe22-Ala23, were shown to have a strong influence on the kinetics of peptide phosphorylation. The peptides 11-19, 11-20, 11-21, 11-22, and 11-23 were all phosphorylated by the myosin light chain kinase with similar apparent Km values in the range 11-17 microM. The Vmax varied 40-fold, with the peptides 11-19, 11-20, 11-21, 11-22, and 11-23 having Vmax values of 0.035, 0.045, 0.32, 1.74, and 1.43 mumol X min-1 X mg-1 respectively. These results indicated that Ala23 was not essential whereas Phe22 and Val21 had a strong influence on the Vmax of peptide phosphorylation. This series of peptides competitively inhibited myosin light chain phosphorylation with Ki values similar to their respective Km values. Peptide 11-19 had a Ki value of approximately 10 microM and a Vmax less than 0.1% of the value with myosin light chains and is therefore an effective inhibitor of the smooth muscle myosin kinase.
Highlights
Peptide Synthesis and Purification-The synthetic peptides were synthesized as the COOH-terminal amidfeorm bythe Merrified solidphase synthesis procedure [7]
The COOH-terminalresidue in peptide analogs of the phosphorylationsite sequence in smoothmusclemyosin the t-butyloxycarbonyl group in the a-aminoposition and benzyhydrylamine resin were obtained from the Protein Research Foundation (Osaka, Japan)
Protein KinaseAssay-Myosin kinase was assayed in a volume of 0.08 ml of 40 mM Hepes' buffer, pH 7.0, 5 mM magnesium acetate, Alaz3was not essential whereasPheZzand Val" had a 0.50 mM [y-32P]ATP(100-2000 cpm/pmol), 0.55 mM CaC12, 5 pg of strong influence on the V, of peptide phosphoryla- calmodulin, 1 mg/ml bovine serum albumin, 0.1% (w/v) Tween 80, tion
Summary
Vol 261, No 1, Issue of January 5, pp. 25-27,1986 0 1986 by The American Society of Biological Chemists, Inc. Protein KinaseAssay-Myosin kinase was assayed in a volume of 0.08 ml of 40 mM Hepes' buffer, pH 7.0, 5 mM magnesium acetate, Alaz3was not essential whereasPheZzand Val" had a 0.50 mM [y-32P]ATP(100-2000 cpm/pmol), 0.55 mM CaC12, 5 pg of strong influence on the V,,, of peptide phosphoryla- calmodulin, 1 mg/ml bovine serum albumin, 0.1% (w/v) Tween 80, tion. Under these conditions the synthetic contraction [1] This enzyme is responsible for phosphoryl- peptide is not retained on the filter paper andoes not contribute to ating Ser" in smooth muscle myosin light chains [2, 3].
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