Török K, Cowley D J, Brandmeier B D, Howell S, Aitken A, Trentham D R
School of Biological Sciences, Queen Mary and Westfield College, University of London, UK.
Biochemistry. 1998 Apr 28;37(17):6188-98. doi: 10.1021/bi972773e.
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), 0. 11-0.39 nM. The Ki 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-Lys75)-[6-(4-N, N-diethylamino-phenyl)-1,3,5-triazin-4-yl]-calmodulin (TA-calmodulin).1 Since, as here, Lys75 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-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-C1) at Lys75 (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK. The Km value for dansyl-calmodulin was equal to that of calmodulin, and that of TA-calmodulin was 3.5-fold greater. TA-calmodulin and Lys75-labeled dansyl-calmodulin thus distinguish between PDE and MLCK being agonists to the former and antagonists to the latter.
已探讨了钙调蛋白生物化学的多个方面，这些方面涉及其作为Ca2+调节介质的细胞生物学作用。从平滑肌肌球蛋白轻链激酶（MLCK）的氨基酸序列衍生而来的钙调蛋白结合肽被表征为钙调蛋白激活MLCK催化的平滑肌调节轻链（MLC）磷酸化的抑制剂。通过在离子强度0.15 M、pH 7.0和21℃下，在100μM CaCl2中通过连续荧光监测NADH的去除，在偶联酶测定中测量反应产物之一ADP的形成速率来测定MLCK活性。钙调蛋白的Km值为3.5 nM，该值比钙调蛋白对MLCK的Kd值大16 - 35倍[Török, K., 和Trentham D. R. (1994) Biochemistry 33, 12807 - 12820]。不同的Km和Kd值很可能与MLC磷酸化中的限速步骤有关，该步骤与MLCK的产物释放相关。抑制常数Ki的值如下：Ac - R - R - K - W - Q - K - T - G - H - A - V - R - A - I - G - R - L - CONH2（色氨酸肽），8.6（±1.4 sd）pM；色氨酸肽的Y4类似物（酪氨酸肽），7.3（±0.1）nM；以及A - R - R - K - W - Q - K - T - G - H - A - V - R - A - I - G - R - L - S - S（RS20样肽），0.11 - 0.39 nM。Ki值与通过动力学测定的肽对钙调蛋白的Kd值一致。Kd值的动力学测定需要使用荧光标记的钙调蛋白，2 - 氯 - (ε - 氨基 - Lys75)-[6 - (4 - N, N - 二乙氨基 - 苯基)-1,3,5 - 三嗪 - 4 - 基]-钙调蛋白（TA -钙调蛋白）。1由于，如此处所示，Lys75是钙调蛋白上用于引入荧光探针的便利标记位点，因此评估了Lys修饰的钙调蛋白的生物活性。TA -钙调蛋白和在Lys75处被1 - N, N - 二甲基氨基萘 - 5 - 磺酰氯（丹磺酰 - C1）选择性修饰的钙调蛋白（丹磺酰 - 钙调蛋白）被表征为环磷酸腺苷磷酸二酯酶（PDE）的激活剂和MLCK的抑制剂。丹磺酰 - 钙调蛋白的Km值与钙调蛋白的Km值相等，而TA -钙调蛋白的Km值大3.5倍。因此，TA -钙调蛋白和Lys75标记的丹磺酰 - 钙调蛋白区分了PDE和MLCK，前者是激动剂，后者是拮抗剂。
