AJP - Cell Physiology, Vol 262, Issue 6 C1437-C1445, Copyright © 1992 by American Physiological Society
Effects of myosin kinase inhibiting peptide on contractility and LC20 phosphorylation in skinned smooth muscle
J. D. Strauss, P. de Lanerolle and R. J. Paul
Department of Physiology and Biophysics, University of Cincinnati, Ohio 45267-0576.
A peptide inhibitor, myosin kinase inhibitor (MKI), of myosin light chain
kinase (MLCK) was tested for its effects on contractility and myosin light
chain phosphorylation in Triton X-100 skinned guinea pig taenia coli. MKI
is based on the amino acid sequence of the myosin light chain (residues
11-19 LC20) and is a competitive inhibitor [inhibitory constant (Ki)
congruent to 10 microM] of purified MLCK with respect to myosin light chain
(LC20). MKI inhibited unloaded shortening velocity (V(us)) and the
calcium-sensitive ATPase activity of the skinned fibers but had no
significant effect on steady-state isometric force or myosin light chain
phosphorylation, as measured by IEF-polyacrylamide gel electrophoresis
analysis. MKI had no significant effect on V(us) of thiophosphorylated
fibers in the absence of calcium. MKI inhibited MLCK activity in protein
extracts from taenia coli, as measured by radioactive phosphate
incorporation into LC20. Surprisingly, MKI also inhibited the phosphatase
activity of these same extracts. This peptide slowed the rate and extent of
relaxation of calcium-contracted fibers and elicited a contraction in
relaxed fibers. These results are consistent with the hypothesis that MKI
may be a phosphatase inhibitor as well as an inhibitor of MLCK. Our data
further suggest that the rate of phosphorylation-dephosphorylation turnover
may be important in regulating V(us) in smooth muscle.
