cAMP-binding proteins in epithelial cells cultured from human sweat glands.
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)
Pon
D J
DJ
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Wong
M
M
Riordan
J R
JR
Schimmer
B P
BP
D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D004439:Eccrine Glands / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism; D013545:Sweat Glands / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D046911:Macromolecular Substances; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases