Cyclic AMP-dependent protein kinase in Paramecium tetraurelia. Its purification and the production of monoclonal antibodies against both subunits.
Two soluble cAMP-dependent protein kinases were purified from the cytoplasm of Paramecium tetraurelia. Both kinases consisted of a 40-kDa catalytic subunit and a 44-kDa regulatory subunit. The two forms of the enzyme were separated by anion-exchange chromatography. Affinity chromatography on cAMP-Sepharose separated the regulatory subunit (retained by the column) from the cAMP-independent catalytic subunit (not retained). Four classes of monoclonal antibodies were generated. One class was specific for the catalytic subunit of both cAMP-dependent protein kinases, and three classes recognized the regulatory subunit of both forms of the enzyme. Subunits of 40 and 44 kDa were detected on immunoblots of purified cilia and of crude cell extracts. In addition, one class of antibodies specific for the regulatory subunit detected a ciliary protein with a molecular mass of 48 kDa. The monoclonal antibodies did not recognize type I or type II cAMP-dependent protein kinase from rabbit muscle nor did they cross-react with proteins from several unicellular eucaryotes, with one exception: antibodies specific for the catalytic subunit recognized a 40-kDa protein of Tetrahymena pyriformis.
Hochstrasser
M
M
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
Nelson
D L
DL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000096:biosynthesis*; D000918:Antibody Specificity; D002384:Catalysis; D003429:Cross Reactions; D000242:Cyclic AMP / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology; D015151:Immunoblotting; D051379:Mice; D008807:Mice, Inbred BALB C; D010247:Paramecium / Q000201:enzymology*; D011494:Protein Kinases / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D013768:Tetrahymena / Q000032:analysis
D000911:Antibodies, Monoclonal; D000242:Cyclic AMP; D011494:Protein Kinases