J. Biol. Chem., Vol. 259, Issue 22, 14184-14189, Nov, 1984
Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB
JP Albanesi, H Fujisaki and ED Korn
The 130- and 125-kDa heavy chains of Acanthamoeba myosins IA and IB were
radioactively labeled at either the regulatory phosphorylation site or the
catalytic site and then subjected to controlled proteolysis by either
trypsin or chymotrypsin. The labeled and unlabeled peptides generated
during the course of proteolysis were identified by autoradiography and
Coomassie Blue staining after separation by electrophoresis on sodium
dodecyl sulfate-polyacrylamide gels. The relative positions of the
phosphorylation and active sites could be deduced. The catalytic site of
myosin IA is most probably within 38 kDa of one end of the 130-kDa heavy
chain, and the phosphorylation site, which can be no more than 40 kDa away
from the catalytic site, would then be between 38 and 78 kDa of that same
end of the heavy chain. Possibly, the phosphorylation site is further
restricted to the region between 38 and 64 kDa from the end of the heavy
chain. The catalytic and phosphorylation sites of myosin IB are both
contained within a segment of 62 kDa at one end of the 125-kDa heavy chain
and are within 40 kDa of each other. The phosphorylation site may be
restricted to a small segment between 60 and 62 kDa from one end of the
heavy chain which would limit the possible position of the catalytic site
to the region between 20 and 60 kDa of that end.
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