Localization of cellular regulatory proteins using postembedding immunogold labeling.
Cyclic AMP-dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP-binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RII) of differing molecular weight, primary structure, and cyclic AMP-binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK subunits in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP-binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK subunits in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.
National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
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Jungmann
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D004702:Endocrine Glands / Q000378:metabolism*; D005088:Exocrine Glands / Q000378:metabolism*; D005260:Female; D006046:Gold; D006107:Granulosa Cells / Q000378:metabolism* / Q000648:ultrastructure; D007120:Immunochemistry / Q000379:methods*; D008854:Microscopy, Electron; D010306:Parotid Gland / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D006046:Gold; D011494:Protein Kinases