The Journal of Cell Biology, Vol 97, 1062-1071, Copyright © 1983 by The Rockefeller University Press
Regulation of contraction and thick filament assembly-disassembly in glycerinated vertebrate smooth muscle cells
WZ Cande, PJ Tooth and J Kendrick-Jones
Isolated smooth muscle cells and cell fragments prepared by glycerination
and subsequent homogenization will contract to one-third their normal
length, provided Ca++ and ATP are present. Ca++- independent contraction
was obtained by preincubation in Ca++ and ATP gamma S, or by addition of
trypsin-treated myosin light chain kinase (MLCK) that no longer requires
Ca++ for activation. In the absence of Ca++, myosin was rapidly lost from
the cells upon addition of ATP. Glycerol-urea-PAGE gels showed that none of
this myosin is phosphorylated. The extent of myosin loss was ATP- and
pH-dependent and occurred under conditions similar to those previously
reported for the in vitro disassembly of gizzard myosin filaments.
Ca++-dependent contraction was restored to extracted cells by addition of
gizzard myosin under rigor conditions (i.e., no ATP), followed by addition
of MLCK, calmodulin, Ca++, and ATP. Function could also be restored by
adding all these proteins in relaxing conditions (i.e., in EGTA and ATP)
and then initiating contraction by Ca++ addition. Incubation with skeletal
myosin will restore contraction, but this was not Ca++- dependent unless
the cells were first incubated in troponin and tropomyosin. These results
strengthen the idea that contraction in glycerinated cells and presumably
also in intact cells is primarily thick filament regulated via MLCK, that
the myosin filaments are unstable in relaxing conditions, and that the
spatial information required for cell length change is present in the thin
filament- intermediate filament organization.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
