Characterization and localization of cAMP-dependent protein kinases in rat caudal epididymal sperm.
We have studied the subcellular localization of type I and type II cAMP-dependent protein kinases in rat caudal epididymal sperm. Our studies indicated that a large fraction of the cAMP-binding activity (approximately 60%), as well as a significant portion of the catalytic activity (approximately 40%), remained associated with insoluble material after the plasma membrane was extracted with 1% Triton X-100. Only 15% of the cAMP-binding activity in sperm was membrane bound. To explore the possibility of a surface location for the membrane-associated cAMP-binding proteins, whole sperm were subjected to proteolysis. Sperm treated with alpha-chymotrypsin lost cAMP-binding activity in cytoplasmic, detergent-soluble, and detergent-resistant fractions. These results demonstrate that alpha-chymotrypsin had access to intracellular locations and to all of the cAMP-binding proteins in sperm. Photoaffinity labeling studies using 8-azido-[32P]cAMP suggest that the regulatory subunit (R) present in the membrane was predominantly RI, while the major species present in the cytoplasm and on detergent-resistant structures was RII. Detergent-resistant sperm structures could be depleted of endogenous catalytic activity by the addition of cAMP, and cAMP-dependent kinase activity could be subsequently regained by the addition of bovine heart catalytic subunits. Separation of head and tailpieces showed that the detergent-resistant regulatory subunits were almost exclusively located on the tailpiece. This finding is consistent with the involvement of cAMP-dependent protein kinases in sperm motility.
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D002470:Cell Survival; D002918:Chymotrypsin / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D004822:Epididymis; D008297:Male; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity; D013094:Spermatozoa / Q000166:cytology / Q000201:enzymology*; D013347:Subcellular Fractions / Q000378:metabolism
D001386:Azides; D000249:Adenosine Monophosphate; C021291:8-azidoadenosine 5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D002918:Chymotrypsin