Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.

Update item information
Publication Type Journal Article
School or College College of Pharmacy; School of Medicine
Department Biomedical Informatics; Biochemistry; Pharmacology & Toxicology
Creator Blumenthal, Donald K.
Other Author Edelman, Arthur M.; Takio, Koji; Hansen, R. Scott; Walsh, Kenneth A.; Titani, Koiti; Krebs, Edwin G.
Title Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase.
Date 1995-09-15
Description Limited proteolysis has been utilized to study the structural organization of rabbit skeletal muscle myosin light chain kinase. The enzyme (Mr approximately 89,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) consists of an amino-terminal, protease-susceptible region of unidentified function and a carboxyl-terminal, protease-resistant region of Mr approximately 40,000 containing the catalytic and calmodulin-binding domains. Partial digestion with trypsin produced an intermediate 56,000-dalton fragment and a stable 38,000-dalton fragment, both of which were catalytically active and calmodulin-dependent. Chymotryptic digestion yielded three catalytically active fragments of about 37,000, 36,000, and 35,000 daltons. The Mr = 37,000 fragment was calmodulin-dependent with an apparent affinity equivalent to that of the native enzyme (approximately 1 nM). The 36,000-dalton fragment was also calmodulin-dependent but had a approximately 200-fold lower apparent affinity. The Mr = 35,000 fragment was calmodulin-independent. These three chymotryptic fragments, had identical amino termini. Nineteen residues were missing from the carboxyl terminus of the calmodulin-independent chymotryptic fragment whereas only 8 or 9 carboxyl-terminal residues were missing from the calmodulin-dependent tryptic fragments. These results suggest that the 11-residue sequence (IAVSAANRFKK) in the carboxyl-terminal region of myosin light chain kinase contributes directly to the binding of calmodulin. This conclusion is in accord with data (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, K., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3187-3191) that the carboxyl-terminal, 27-residue CNBr peptide of the native enzyme shows Ca2+-dependent, high affinity binding to calmodulin and that similar calmodulin-binding activity, although detectable in unfractionated CNBr digests of calmodulin-dependent enzyme forms, is much reduced in a CNBr digest of the calmodulin-independent, Mr = 35,000 chymotryptic fragment.
Type Text
Publisher American Society for Biochemistry and Molecular Biology (ASBMB)
Volume 260
Issue 20
First Page 11275
Last Page 11285
Subject Enzymology; Skeletal Muscle; Proteolysis, Peptide Fragments
Subject MESH Binding Sites; Calmodulin; Chymotrypsin; Electrophoresis; Endopeptidases; Kinetics; Molecular Weight; Myosin-Light-Chain Kinase; Peptide Fragments; Protein Kinases; Trypsin; Serine
Language eng
Bibliographic Citation Edelman AM, Takio K, Blumenthal DK, Hansen RS, Walsh KA, Titani K, Krebs EG. Characterization of the calmodulin-binding and catalytic domains in skeletal muscle myosin light chain kinase. J Biol Chem. 1985 Sep 15;260(20):11275-85. Retrieved September 15,2006 from
Rights Management Copyright © American Society for Biochemistry and Molecular Biology, J Biol Chem., 260, 11275-85, 1985.
Format Medium application/pdf
Identifier ir-main,405
ARK ark:/87278/s6tx3zzj
Setname ir_uspace
Date Created 2012-06-13
Date Modified 2021-05-06
ID 706550
Reference URL
Back to Search Results