A study of cAMP binding proteins on intact and disrupted sperm cells using 8‐azidoadenosine 3′,5′‐cyclic monophoshate

Abstract

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.

Vaginal Fluid Adenosine 3',5'-Cyclic Monophosphate (cAMP) in the Rat: Interaction with Sperm cAMP-Dependent Protein Kinase Regulatory Subunits

“…3, bottom). Although their absence could be due to competition arising from endogenous CAMP, ovariectomized fluids that had the lowest levels of cAMP also did not photolabel, and cAMPdPK activity could not be detected [22] in these fluids, further indicating an absence of free catalytic subunit. These results suggest that large proteins (including the regulatory and catalytic subunits of the enzyme) as well as smaller molecules-e.g., CAMP-are not likely to be a product of damaged cells.…”

“…An implication of such data could be that this female genital tract cAMP is involved with the regulation of sperm function. Relatedly we have demonstrated with the use of [SZP]-8N3cAMP that both regulatory subunits RI (49K M, and RII (55K MJ of CAMP-dependent protein kinase (cAMPdPK) are readily accessible and trypsin-sensitive on washed human ejaculated sperm [22]. Similarly, these subunits are externally available on rat cauda sperm [l].…”

Phosphocreatine, an intracellular high-energy compound, is found in the extracellular fluid of the seminal vesicles in mice and rats.

“…Extracellular protein kinases ("ectokinases") have been found in rat, ram, and human sperm (34)(35)(36) …”

Modulation of intrasperm Ca2+: A possible maneuver for spermicidal activity

“…This ecto-CIK was not activated by Ca 21 , calmodulin, or phosphatidyl serine [Haldar and Majumder, 1986] and was inhibited by phosphatidyl inositol [Dey and Majumder, 1987a]. Similarly, evidences supporting the presence of ectokinases on human sperm surface have also been reported [Schoff et al, 1982;Pariset et al, 1983].…”