Phosphorylation of cAMP-dependent protein kinases in normal and abnormal human sperm.
The molecular characterization and distribution of cAMP-dependent protein kinases (cAPK) of normal and pathological (reduced progressive motility or altered morphology) human semen were investigated. Photoaffinity labeling with 8-N3-[32P]cAMP of human sperm cytosols revealed four specific cAMP-binding proteins (MW: 52 kD, 47 kD, 42 kD, and 37 kD) with the following quantitative distribution: 65% (47 kD), 30% (52 kD), and less than 5% (42/37 kD). In contrast, the seminal plasma of the respective semen samples incorporated the 8-azido cAMP photolabel predominantly into the 37 kD protein (greater than 50%). The cAMP-binding proteins with MW of 52 kD and 47 kD corresponded to the regulatory subunits R II and R I of the respective cAPK isoenzymes I and II. Neither qualitative nor quantitative differences could be detected in the distribution between seminal plasma and sperm as well as in the molecular properties of RI (47 kD), R II (52 kD), and of their proteolytic products (42 kD and 37 kD) between normal and pathological human semen. DEAE-cellulose chromatography revealed that type II isozyme was the predominant form (80% of the total cAMP-dependent protein kinase activity of sperm); the protein kinase isozyme pattern was similar in normal and pathological sperm. DEAE-cellulose chromatography in combination with photoaffinity labeling (8-N3-[32P]cAMP) resolved the kinase activity into type I and type II isoenzymes and into the corresponding subunits R I (47 kD) and R II (52 kD). The excessive amount of R I (47 kD) found by photoaffinity labeling in comparison to type I holoenzyme after DEAE-cellulose chromatography is due to a higher binding affinity of R I (5 nM) for the 8-N3[32P]cAMP as compared with that of R II (50 nM). In addition, endogenous phosphorylation of soluble sperm proteins revealed that the R II (52 kD) was present only as the phosphoform of R II.
D000293:Adolescent; D000328:Adult; D000345:Affinity Labels; D002352:Carrier Proteins / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002373:Cyclic AMP Receptor Protein; D003600:Cytosol / Q000201:enzymology; D006801:Humans; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism*; D012661:Semen / Q000201:enzymology; D013081:Sperm Motility; D013094:Spermatozoa / Q000002:abnormalities / Q000201:enzymology*
D000345:Affinity Labels; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D011494:Protein Kinases