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 mu M CaCl2 at ionic strength 0.15 M, pH 7.0 and 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 step in MLC phosphorylation being associated with product release from MLCK. The values of the inhibition constants, K-i, 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; 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), 0.11-0.39 nM. The K-i values were consistent with kinetically determined Kd values of the peptides to calmodulin. Kinetic determination of Kd values required the use of a fluorescently labeled calmodulin, 2-chloro-(epsilon-amino-Lys(75))-[6-(4-N,N-diethylamino-phenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin)(1). Since, as here, Lys(75) is a convenient labeling site on calmodulin for the introduction of fluorescent probes, the biological activity of the Lys-modified calmodulins was evaluated. TA-calmodulin and calmodulin selectively modified by 1-N,N-dimethylaminanaphthalene-5-sulfonyl chloride (dansyl-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)-labeled dansyl-calmodulin thus distinguish between PDE and MLCK being agonists to the farmer and antagonists to the latter.
