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Paramecium Has Two Regulatory Subunits of Cyclic AMP‐Dependent Protein Kinase, One Unique to Cilia
Author(s) -
HOCHSTRASSER MARTIN,
CARLSON GAIL L.,
WALCZAK CLAIRE E.,
NELSON DAVID L.
Publication year - 1996
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1996.tb04000.x
Subject(s) - biology , protein subunit , autophosphorylation , biochemistry , protein kinase a , paramecium , fast protein liquid chromatography , enzyme , microbiology and biotechnology , size exclusion chromatography , affinity chromatography , kinase , gene
. The subunit composition and intracellular location of the two forms of cAMP‐dependent protein kinase of Paramecium cilia were determined using antibodies against the 40‐kDa catalytic (C) and 44‐kDa regulatory (R 44 ) subunits of the 70‐kDa cAMP‐dependent protein kinase purified from deciliated cell bodies. Both C and R 44 were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48‐kDa protein (R 48 ) weakly recognized by one of several monoclonal antibodies against R 44 , but not recognized by an anti‐R 44 polyclonal serum. Gel‐filtration chromatography of a soluble ciliary extract resolved a 220‐kDa form containing 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 conditions that allow autophosphorylation of R 44 The subunits of the large enzyme subsequently were purified to homogeneity by cAMP‐agarose chromatography. 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 results confirm that the ciliary cAMP‐dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell‐body enzyme, contains R 44 , whereas R 48 is the R subunit of the large enzyme.