Location of the inhibitory region of smooth muscle myosin light chain kinase.
Proteolysis by trypsin of gizzard myosin light chain kinase (MLC kinase) in the absence of Ca2+-calmodulin produced a 64,000-dalton inactive fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment. This confirmed previous results (Ikebe, M., Stepinska, M., Kemp, B. E., Means, A. R., and Hartshorne, D. J. (1987) J. Biol. Chem. 262, 13828-13834). On the other hand, proteolysis of MLC kinase in the presence of Ca2+-calmodulin initially produced a 66,000-dalton Ca2+-calmodulin-dependent active fragment which was converted to a 61,000-dalton Ca2+-calmodulin-independent active fragment with further proteolysis. The amino acid sequences from the N terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton fragments were determined. The sequence was not found in the reported partial amino acid sequence of MLC kinase (C-terminal 60% of whole sequence) (Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381), and, therefore, the cleavage sites are in the remaining 40% N-terminal portion of the sequence of MLC kinase. The C terminus of these MLC kinase fragments was determined by employing the carboxypeptidases A, B, and Y digestion followed by the amino acid analysis of the released amino acids. As a result, it was concluded that the C terminus of the 66,000-dalton, 64,000-dalton, and 61,000-dalton MLC kinase fragments are arginine 522, lysine 490 and arginine 494, and lysine 473, respectively. These results show that the inhibitory domain is in the amino acid sequence of 474-490, and that the amino acid sequence 494-522 confers the calmodulin-dependent kinase activity.
Ikebe
M
M
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Maruta
S
S
Reardon
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism; D005895:Gizzard, Avian / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology; D014422:Turkeys
D002147:Calmodulin; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin