2386 zyxwvutsrqpon Biochemistry zyxwvu 1982, zyxwvu 21, 2386-2391 Pederson, T., zyxwvutsrqp & Bhorjee, J. S. (1975) Biochemistry 14, Vertel, B. M., & Dorfman, A. (1978) Deu. Biol. 62, 1-12. von der Mark, H., & von der Mark, K. (1977) J. Cell zy Biol. Weintraub, H., & Groudine, M. (1976) Science (Washington, Weintraub, H., Larsen, A,, & Groudine, M. (1981) Cell Weisbrod, S., & Weintraub, H. (1979) Proc. Natl. Acad. Sci. Weisbrod, S., Groudine, M., & Weintraub, H. (1980) Cell 3238-3242. Perle, M. A., & Newman, S. A. (1 980) Proc. Natl. Acad. Sci. 73, 736-747. U.S.A. 77, 4828-2830. 210, 604-610. D.C.) 93, 848-858. Razin, A., & Riggs, A. D. (1980) Science (Washington, zyxwvu D.C.) Stalder, J., Groudine, M., Dodgson, J. B., Engel, J. D., & Weintraub, H. (1980a) Cell (Cambridge, Mass.) 19, 97 3-9 80. & Weintraub, H. (1980b) Cell (Cambridge, Mass.) 20, 45 1-460. (Cambridge, Mass.) 24, 333-444. Stalder, J., Larsen, A., Engel, J. D., Dolan, M., Groudine, M., U.S.A. 76, 631-635. (Cambridge, Mass.) 19, 289-301. Effects of pH, Ionic Strength, and Temperature on Activation by Calmodulin and Catalytic Activity of Myosin Light Chain Kinase? Donald K. Blumenthal and James T. Stull* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ABSTRACT: The reversible association of Ca~+.calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochem- istry 19, 560846141. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (>90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (<20% inhibition at zyxwvutsrq p = 0.22), and displayed a marked temperature dependence zyxwvutsrqp (Qlo N 2; E, = 10.4 kcal mol-'). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase Calmodulin is known to regulate a number of enzymes and cellular processes in a Ca2+-dependent manner [for reviews, see Wolff & Brostrom (1979), Klee et al. (1980), Cheung (1980), Means & Dedman (1980), and Wang & Waisman (1 979)]. Myosin light chain kinase is one of the several en- zymes whose activity is completely dependent on the presence of Ca2+ and calmodulin. This enzyme is responsible for catalyzing the phosphorylation of a specific subunit of myosin, known as the phosphorylatable or P light chain (Frearson & Perry, 1975). The enzyme and its substrate are present in nonmuscle as well as muscle tissue. The phosphorylation reaction may play an important role in the regulation of contraction in smooth and skeletal muscles (Stull, 1980; Stull et al., 1980), as well as in modulating cellular function in certain nonmuscle tissues (Adelstein, 1978; Salisbury et al., 1980). The details of the interaction of calmodulin with the many proteins it regulates are not well understood. Previous studies From the Department of Pharmacology and the Moss Heart Center, University of Texas Health Science Center at Dallas, Dallas, Texas 15235. Received September I, 1981. This work was supported by grants from the National Institutes of Health (HL 23990) and the Muscular Dystrophy Association of America. D.K.B. was supported as a post- doctoral trainee (HL-07360). activation by calmodulin had a narrower pH optimum (pH 6.0-7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (>70% inhibition at p = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 "C, activation was primarily entropically driven zy (AS" N 40 cal mol-' deg-'; AH" = -900 cal mol-'), but between 20 and 30 "C, enthalpic factors predominated in driving the activation process (M0 N 10 cal mol-' deg-'; AH" = -9980 cal mol-'). The apparent change in heat capacity (AC,) accompanying activation was estimated to be -910 cal mol-' deg-I. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interac- tions also make significant and probably obligatory contri- butions to the activation process. in our laboratory were concerned with determining the mechanism of activation of myosin light chain kinase (Blu- menthal & Stull, 1980). The purpose of this investigation was to extend our previous studies and to determine the effects of pH, temperature, various salts, and ionic strength on the ac- tivation and catalytic activity of myosin light chain kinase. From analysis of these results it is possible to obtain infor- mation regarding the factors that play important roles in the regulation of myosin light chain kinase activity. Because calmodulin is highly conserved throughout eukaryotic evolution (Jamieson et al., 1980), the general features of the interaction of calmodulin with myosin light chain kinase may be applicable to other calmodulin-dependent processes. Materials and Methods Chemicals were obtained from Sigma and were of analytical grade or better. [y-32P]ATP1 was prepared by the method ' Abbreviations: EGTA, ethylene glycol bis(&aminoethyl ether)- N,N,N',N'-tetraacetic acid; EDTA, (ethylenedinitri1o)tetraacetic acid; Mops, 4-morpholinepropanesulfonic acid; Hepps, 4-(2-hydroxyethyl)- 1 - piperazinepropanesulfonic acid; Mes, 2-(N-morpholino)ethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; ATP, adenosine 5'-tri- phosphate; NMR, nuclear magnetic resonance; SEM. standard error of the mean. 0006-2960/82/0421-2386$01.25/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 0 1982 American Chemical Society