Identification of goat sperm ecto-cyclic AMP independent protein kinase substrate localized on sperm outer surface.
We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.
Maiti
Arunima
A
Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700 032, India.
Mishra
K P
KP
Majumder
G C
GC
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002462:Cell Membrane / Q000378:metabolism; D006041:Goats; D008297:Male; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013081:Sperm Motility / Q000276:immunology / Q000502:physiology; D013094:Spermatozoa / Q000737:chemistry / Q000201:enzymology* / Q000648:ultrastructure; D013379:Substrate Specificity
D000906:Antibodies; D008565:Membrane Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases