A study of cAMP binding proteins on intact and disrupted sperm cells using 8-azidoadenosine 3',5'-cyclic monophosphate.
The photoaffinity probe (32P) 8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (32P) 8-N3 cAMP-binding proteins. The 8-N, cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells. Intact cells bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/10(8) cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP/mg protein and had a cAMP-PK activity of 2,206 units/10(8) cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.
D001386:Azides; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives*; D002373:Cyclic AMP Receptor Protein; D006801:Humans; D008297:Male; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism; D011953:Receptors, Cyclic AMP / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D014357:Trypsin
D001386:Azides; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D011953:Receptors, Cyclic AMP; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin