Abstract
The molecular and biochemical properties of myosin light chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin light chain kinase resulted in a decrease in Vmax with no significant change in Km values for light chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin light chain kinase increased the Km values for light chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin light chain kinases is involved in light chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin light chain kinases share structural elements necessary for regulation.
Highlights
The molecular and biochemical properties of myosindynamic and CD spectroscopic studies of the native rabbit light chain kinases from chicken skeletal and smooth skeletal muscle MLCK and selected proteolytic fragments muscle were investigated by recombinant DNA tech- have shown that theenzyme exists as anasymmetric aminoniques
A cDNA encoding the full-length chicken skeletal muscle MLCK was constructed from two partial cDNAs (Fig. 1A)
The translational start site of the chicken skeletal muscle MLCK is encoded by nucleotides 187-189
Summary
The molecular and biochemical properties of myosindynamic and CD spectroscopic studies of the native rabbit light chain kinases from chicken skeletal and smooth skeletal muscle MLCK and selected proteolytic fragments muscle were investigated by recombinant DNA tech- have shown that theenzyme exists as anasymmetric aminoniques. MLCKs from skeletal and smooth muscles molecules, lacking bothamino- and carboxyl-terminal secan be distinguished on the basis of molecular mass (Nunnally quences which exhibit Ca2+/calmodulin-independentkinase and Stull, 1984;Edelman et al, 1987),antigenicity (Kammet activity (Foyt et al, 1985; Ikebe et al, 1987; Kennelly et al, al., 1987), and catalytic specificity for synthetic peptide sub- 1987; Mayr and Heilmeyer, 1983). Amino-terminal tail region plays a role in catalysis We used This construction results in a smooth muscle product except for the skeletal/smooth muscle chimeric molecules and site-specific two most amino-terminal residues, G and P,from the skeletal muscle mutants to examine regions inside and outside the catalytic enzyme.
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