Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists
A. Sobieszek, Enzyme kinetic characterization of the smooth muscle myosin phosphorylating system: activation by calcium and calmodulin and possible inhibitory mechanisms of antagonists, BBA-MOL CEL, 1450(1), 1999, pp. 77-91
Citations number
31
Categorie Soggetti
Cell & Developmental Biology
Journal title
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
A native-like smooth muscle filamentous myosin system was characterized fro
m an enzyme kinetic point of view. The system contains endogenous myosin li
ght chain kinase (MLCKase) and calmodulin (CM) (A. Sobieszek, J. Muscle Res
. Cell Motil. 11 (1990) 114-124) and is, therefore, well suited for testing
the action of CM-antagonists or other inhibitory compounds. However, this
has not been done due to its complexity. The characterization of the system
includes: (1) derivation of a relationship for rate of myosin phosphorylat
ion in terms of total CM, free Ca2+ and total MLCKase concentrations, which
includes only three binding constants; and (2) derivation of relationships
between fractional inhibition rate (nu(i)/nu(o)) and total inhibitor conce
ntration (I-t) which cover most of the inhibitory mechanisms applicable to
the myosin system or to other CM-dependent enzymes. The three binding const
ants were subsequently evaluated from experimental data for filamentous myo
sin and for its isolated regulatory light chain (ReLC) using a non-linear r
egression software. They indicated differences in the interaction of myosin
filament with the active CM-MLCKase complex in comparison to that of the i
solated ReLC. The derived nu(i)/nu(o) versus I-t relationships, together wi
th the software, make it possible:to evaluate the inhibition constants and
binding stoichiometries of CM-antagonists and other compounds inhibiting my
osin phosphorylation. This approach was successfully applied to experimenta
l data on inhibition of MLCKase by amiloride, cadmium, or CM-binding peptid
e (M-12) for simple mechanisms. For more complex mechanisms, inhibition by
calmidozolium, trifluoperazine or melittin, the analysis showed that only c
almidozolium acted specifically at the CM level in a multiple-site activato
r-depletion mechanism. Melittin and trifluoperazine inhibited the phosphory
lation rate by a novel substrate-and-activator depletion mechanism, in whic
h additional inhibition of the substrate resulted in the removal of the inh
ibition at lower range of the antagonists' concentration. (C) 1999 Elsevier
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