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A study of cAMP binding proteins on intact and disrupted sperm cells using 8‐azidoadenosine 3′,5′‐cyclic monophoshate
Author(s) -
Schoff Patrick K.,
Forrester Ian T.,
Haley Boyd E.,
Atherton Robert W.
Publication year - 1982
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240190102
Subject(s) - protein kinase a , endogeny , receptor , photoaffinity labeling , protein subunit , chemistry , sperm , biochemistry , cytosol , biology , microbiology and biotechnology , kinase , enzyme , gene , botany
The photoaffinity probe ( 32 P)8‐N 3 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 ( 12 P) 8‐N 3 cAMP‐binding proteins. The 8‐N 3 cAMP was also effective in stimulating endogenous cAMP‐PK activity in intact and disrupted sperm. A substantial amount of ( 32 P) 8‐N 3 cAMP binding to types I and II regulatory subunits and cAMP‐PK activity was detected on washed intact cells, intact cells. Intact cell bound 1.80 pmol of ( 32 P) 8‐N 3 cAMP/mg protein and had cAMP‐PK activity of 824 units/10 8 cells. Disrupted cells bound 3.95 pmol ( 32 P) 8‐N 3 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 ( 32 P) 8‐N 3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.