Monoclonal antibodies as probes for functional domains in cAMP-dependent protein kinase II.

摘要: Abstract The antigenic regions of the type II regulatory subunit cAMP-dependent kinase from bovine heart have been correlated with previously established domain structure molecule. Immunoblotting both serum and monoclonal antibodies fragments generated by limited proteolysis or chemical cleavage R-subunit that major sites were confined to amino-terminal portion polypeptide chain (residues 1-145). Radioimmunoassays using two different antisera suggested one more high affinity antibody recognition further restricted residues 91-145. This includes hinge region which is particularly sensitive proteolysis, allowing be cleaved readily into a COOH-terminal retains cAMP-binding an NH2-terminal fragment appears site for interaction R-subunits in native dimer. Monoclonal recognized determinants on sides this characterized their specific localized. Accessibility holoenzyme contrast free R2 was compared. Although cAMP did tend slightly increase antibodies, all clearly exposed accessible holoenzyme. Furthermore, despite presumed close proximity between R- C-subunits, no case binding promote dissociation complex. fact would precipitate as well used ascertain importance amino acid C-subunits. domains isolated following chymotrypsin thermolysin. These differed only three at end. U these conjunction immunoadsorption chymotryptic fragment, contained Asp-Arg-Arg preceding autophosphorylation, capable forming stable complex C-subunit. In contrast, thermolytic those longer complexed C-subunit, indicating arginine not contribute specificity phosphorylation but also are essential component energetically stabilizing