Inhibition of calmodulin-activated smooth-muscle myosin light-chain kinase by calmodulin-binding peptides and fluorescent (phosphodiesterase-activating) calmodulin derivatives

Biochemistry
Katalin TörökD R Trentham

Abstract

Aspects of the biochemistry of calmodulin have been addressed that bear on its cell biological role as a mediator of Ca2+ regulation. Calmodulin-binding peptides derived from the amino acid sequence of smooth-muscle myosin light-chain kinase (MLCK) were characterized as inhibitors of calmodulin activation of MLCK-catalyzed phosphorylation of the smooth-muscle regulatory light chain (MLC). MLCK activity was determined by measuring the rate of formation of one of the reaction products, ADP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0 and 21 degreesC. The Km value of calmodulin was 3.5 nM, a value 16-35-fold greater than the Kd value of calmodulin for MLCK [Török, K., and Trentham D. R. (1994) Biochemistry 33, 12807-12820]. The different Km and Kd values are most likely associated with the rate-limiting step in MLC phosphorylation being associated with product release from MLCK. The values of the inhibition constants, Ki, were the following: Ac-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide), 8.6 (+/-1. 4 sd) pM; Y4-analogue of Trp peptide (Tyr peptide), 7.3 (+/-0.1) nM; and A-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-S-S (RS20-like peptide),...Continue Reading

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Citations

Mar 5, 2008·Analytical and Bioanalytical Chemistry·Emre DikiciSylvia Daunert
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