INHIBITION OF CALMODULIN-ACTIVATED SMOOTH-MUSCLE MYOSIN LIGHT-CHAIN KINASE BY CALMODULIN-BINDING PEPTIDES AND FLUORESCENT (PHOSPHODIESTERASE-ACTIVATING) CALMODULIN DERIVATIVES

Aspects of the biochemistry of calmodulin have been addressed that bea r on its cell biological role as a mediator of Ca2+ regulation. Calmod ulin-binding peptides derived from the amino acid sequence of smooth-m uscle myosin light-chain kinase (MLCK) were characterized as inhibitor s of calmodulin activation of MLCK-catalyzed phosphorylation of the sm ooth-muscle regulatory light chain (MLC). MLCK activity was determined by measuring the rate of formation of one of the reaction products, A DP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 mu M CaCl2 at ionic strength 0.15 M, pH 7.0 an d 21 degrees C. The K-m value of calmodulin was 3.5 nM, a value 16-35- fold greater than the K-d value of calmodulin for MLCK [Torok, K., and Trentham D. R. (1994) Biochemistry 33, 12807-12820]. The different K- m and K-d values are most likely associated with the rate-limiting ste p in MLC phosphorylation being associated with product release from ML CK. The values of the inhibition constants, K-i, were the following: A c-R-R-K-W-Q-K-T-G-H-A-V-R-A-I-G-R-L-CONH2 (Trp peptide), 8.6 (+/-1.4 s d) pM; Y-4-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), 0.11-0.3 9 nM. The K-i values were consistent with kinetically determined Kd va lues of the peptides to calmodulin. Kinetic determination of Kd values required the use of a fluorescently labeled calmodulin, iethylamino-p henyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin)(1). Since, as he re, Lys(75) is a convenient labeling site on calmodulin for the introd uction of fluorescent probes, the biological activity of the Lys-modif ied calmodulins was evaluated. TA-calmodulin and calmodulin selectivel y modified by 1-N,N-dimethylaminanaphthalene-5-sulfonyl chloride (dans yl-C1) at Lys(75) (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK. The K-m value for dansyl-calmodulin was equal to that of calmodulin, and that of TA-calmodulin was 3.5-fold greater. TA-calmodulin and Lys(75)-labe led dansyl-calmodulin thus distinguish between PDE and MLCK being agon ists to the farmer and antagonists to the latter.