Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.
Proteolysis by trypsin of gizzard myosin light chain kinase in the absence of Ca2+-calmodulin causes a biphasic effect on kinase activity. During the initial phase of proteolysis, Ca2+-calmodulin-dependent kinase activity is reduced over a thousand-fold. Further proteolysis, in the second phase, causes an increase in activity that is independent of Ca2+-calmodulin. Loss of activity is associated with the formation of a 64,000-dalton fragment. Calmodulin-independent activity is associated with the formation of a 61,000-dalton fragment. Procedures for the isolation of each fragment are outlined. Tryptic hydrolysis of the isolated 64,000-dalton peptide generates the 61,000-dalton peptide and increases calmodulin-independent activity. Km values for ATP and light chains for the native kinase and two fragments are the same, i.e. approximately 100 and 5 microM, respectively. Neither fragment binds to F-actin. Amino acid analyses of both fragments are given. Synthetic peptides corresponding to the calmodulin-binding regions of the smooth and skeletal muscle kinases are potent inhibitors of the 61,000-dalton fragment. These data demonstrate the existence of an inhibitory region that is suggested to be located between the active site and the calmodulin-binding site. Whether it is distinct from or at the N-terminal end of the calmodulin-binding site cannot be determined from these data.
Ikebe
M
M
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Stepinska
M
M
Kemp
B E
BE
Means
A R
AR
Hartshorne
D J
DJ
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000494:pharmacology*; D002645:Chickens; D005895:Gizzard, Avian / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin