Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase.
We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).
Horowitz
J A
JA
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Voulalas
P
P
Wasco
W
W
MacLeod
J
J
Paupard
M C
MC
Orr
G A
GA
D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000032:analysis*; D002417:Cattle; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005407:Flagella / Q000032:analysis / Q000201:enzymology*; D047908:Intracellular Signaling Peptides and Proteins; D007525:Isoelectric Focusing; D008297:Male; D009206:Myocardium / Q000032:analysis; D010447:Peptide Hydrolases; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D013094:Spermatozoa / Q000201:enzymology*; D013737:Testis / Q000032:analysis
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C015482:protein kinase modulator; D011494:Protein Kinases; D010447:Peptide Hydrolases