PARAMECIUM HAS 2 REGULATORY SUBUNITS OF CYCLIC-AMP-DEPENDENT PROTEIN-KINASE, ONE UNIQUE TO CILIA

The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined usi ng antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory ( R(44)) subunits of the 70-kDa cAMP-dependent protein kinase purified f rom deciliated cell bodies. Both C and R(44) were present in soluble a nd particulate fractions of cilia and deciliated cells. Crude cilia an d a soluble ciliary extract contained a 48-kDa protein (R(48)) weakly recognized by one of several monoclonal antibodies against R(44) but n ot recognized by an anti-R(44) polyclonal serum. Gel-filtration chroma tography of a soluble ciliary extract resolved a 220-kDa form containi ng C and R(48) and a 70-kDa form containing C and R(44). In the large enzyme, R(48) was the only protein to be autophosphorylated under cond itions that allow autophosphorylation of R(44). The subunits of the la rge enzyme subsequently were purified to homogeneity by cAMP-agarose c hromatography. Both C and R(48) were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These re sults confirm that the ciliary cAMP-dependent protein kinases have ind istinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R(44), whereas R(48) is t he R subunit of the large enzyme.