AJP - Cell Physiology, Vol 252, Issue 3 C277-C284, Copyright © 1987 by American Physiological Society
Identification of high-affinity calmodulin-binding proteins in rat liver
R. M. Hanley, J. R. Dedman and S. Shenolikar
The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was utilized to identify CaM binding or "acceptor" proteins or
CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and
45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM
binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by
a variety of chromatographic steps, including affinity chromatography on
CaM Sepharose. CAP-60 possessed a native molecular size of 400,000,
indicating it to be the CaM-binding "subunit" of a larger oligomeric
complex. In contrast, CAP-45 was monomeric as judged by gel filtration.
Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent
with known CaM-dependent enzymes reported in the literature.
Two-dimensional peptide mapping provided convincing evidence that CAP-60
and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+
(CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic
nucleotide phosphodiesterase. The relative abundance and high affinity for
CaM could suggest that these novel target proteins, CAP-60 and CAP-45,
represent a dominant pathway for CaM action in the mammalian liver.
