CHARACTERIZATION OF A CAMP-BINDING PROTEIN FROM THE BIVALVE MOLLUSK MYTILUS-GALLOPROVINCIALIS

Three cAMP-binding proteins have been identified by photoaffinity labe ling with 8-azido[P-32]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determi ned by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two cAMP-binding sites/monomer, was judged to be a regulator y (R) subunit of cAMP-dependent protein kinase since it re-associated with and inhibited purified catalytic (C) subunit of this enzyme from mussel, in the absence but not in the presence of cAMP. The molecular mass of the complex between Mytilus cAMP-binding protein and C subunit , estimated by analytical gel-filtration, was 220 kDa, a value which a grees with a R(2)C(2) stoichiometry for the mussel cAMP-dependent prot ein kinase holoenzyme. On the basis of the elution pattern from DEAE-c ellulose chromatography and its ability to be phosphorylated by purifi ed C subunit of cAMP-dependent protein kinase, the 54-kDa protein coul d be classified as a type II regulatory subunit. Furthermore, no mobil ity shift on SDS/PAGE upon phosphorylation/dephosphorylation of Mytilu s protein was observed, a similar behaviour to that shown by the mamma lian RII beta isoform. The 42-kDa and 37-kDa proteins, which were reco gnized by a specific antiserum against the 54-kDa protein and fail to be phosphorylated by Mytilus C subunit, are probably products generate d by proteolysis of the 54-kDa protein, although they were shown even when inhibitors of the major types of proteases were used.