Dephosphorylation Of Tonic And Phasic Smooth Muscle Myosin In The In Vitro Motility Assay Exhibits Different Kinetics
BIOPHYSICAL JOURNAL(2020)
Abstract
Tonic smooth muscle has a greater propensity to maintain force during dephosphorylation of its regulatory light chain compared to phasic smooth muscle. To elucidate the underlying mechanism, we injected myosin light chain phosphatase (MLCP) during regular in vitro motility assays (100% tonic or phasic smooth muscle myosin) and mixture assays (25% skeletal: 75% tonic or phasic). Tonic muscle myosin was purified from pig stomach fundus, phasic muscle myosin and MLCP were purified from avian gizzards, and skeletal muscle myosin was purified from chicken breast. In vitro motility assay chambers were devised with a top opening covered with a microporous membrane to allow diffusion of MLCP from injections during the assays. In the regular assay, we observed that the velocity of actin filaments started decreasing before the fraction of moving filaments (fmot) decreased. The time from the start of velocity decrease until near complete stop of the assay (<20% fmot) was 45% greater in tonic compared to phasic muscle myosin. In the mixture assay, we observed that the increase in velocity as a result of a reduction in the number of the slower, active smooth muscle myosin heads took 79% longer for the tonic-skeletal mixture compared to the phasic-skeletal mixture. These data could be explained by a slower rate of dephosphorylation of tonic muscle myosin, caused by either specificity of the MLCP purified from phasic muscle or inherent differences in dephosphorylation rates between tonic and phasic muscle myosin. Alternatively, reduced velocity could be caused by the formation of some slower detaching cross-bridges. These slowly detaching cross-bridges could result from dephosphorylation of myosin while attached to actin, as previously suggested in the latch-state hypothesis. More studies are underway to discriminate between these possibilities.
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Key words
phasic smooth muscle myosin,motility,kinetics,vitro
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