AJP - Cell Physiology, Vol 258, Issue 6 C1036-C1043, Copyright © 1990 by American Physiological Society
cAMP-binding proteins in epithelial cells cultured from human sweat glands
D. J. Pon, M. Wong, J. R. Riordan and B. P. Schimmer
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
This study characterized the various adenosine 3',5'-cyclic monophosphate
(cAMP)-binding proteins in epithelial cells cultured from human sweat
glands to identify potential pathways of cAMP action in this tissue. The
cAMP-binding proteins were identified by specific labeling with the
photoprobe 8-azido-[32P]-cAMP and visualized by autoradiography after
electrophoresis on polyacrylamide gels in the presence of sodium dodecyl
sulfate. Three cAMP-binding proteins, with molecular masses of 48, 52, and
54 kDa, were identified in subcellular fractions of the cultured sweat
glands. On the basis of their relative electrophoretic mobilities and
reactivity with specific antisera, the 48-kDa protein was identified as the
regulatory subunit of the type 1 cAMP-dependent protein isozyme, and the
54-kDa protein was identified as the regulatory subunit of the type 2
cAMP-dependent protein kinase isozyme. The 52-kDa cAMP-binding protein
appeared to be a distinct isoform of the regulatory subunit, possibly
related to the subclass associated with neural tissue. As determined from
the relative distributions of the photolabel, the 48-, 52-, and 54-kDa
binding proteins were present in the cell cytosol at a ratio of 1:2:1. The
48- and 52-kDa proteins bound the photoprobe with apparent dissociation
constants (Kd) of 20-40 nM, whereas the 54-kDa protein bound the photoprobe
with much lower affinity (Kd 230 nM). Only the 48- and 52-kDa proteins were
associated with cell membranes. They were present in the membrane fractions
in approximately equal amounts and bound the photoprobe with apparent Kd
values of greater than 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
