Characterization of a cAMP-binding protein from the bivalve mollusc Mytilus galloprovincialis.
Three cAMP-binding proteins have been identified by photoaffinity labeling with 8-azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined 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 regulatory (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 agrees with a R2C2 stoichiometry for the mussel cAMP-dependent protein kinase holoenzyme. On the basis of the elution pattern from DEAE-cellulose chromatography and its ability to be phosphorylated by purified C subunit of cAMP-dependent protein kinase, the 54-kDa protein could be classified as a type II regulatory subunit. Furthermore, no mobility shift on SDS/PAGE upon phosphorylation/dephosphorylation of Mytilus protein was observed, a similar behaviour to that shown by the mammalian RII beta isoform. The 42-kDa and 37-kDa proteins, which were recognized by a specific antiserum against the 54-kDa protein and fail to be phosphorylated by Mytilus C subunit, are probably products generated by proteolysis of the 54-kDa protein, although they were shown even when inhibitors of the major types of proteases were used.
Cao
J
J
Departamento de Bioquímica e Bioloxía Molecular, Facultade de Veterinaria, Universidade de Santiago de Compostela, Spain.
Ramos-Martinez
J I
JI
Villamarín
J A
JA
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D049872:Bivalvia / Q000378:metabolism*; D002352:Carrier Proteins; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D011487:Protein Conformation
D000345:Affinity Labels; D001386:Azides; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases