Three CAMP-binding proteins have been identified by photoaffinity labeling with S-azido["?P]cAMP and purified from the mantle tissue of the sea mussel Mytilus ,eullo~,rovinc.ialis. Their molecular masses, determined by SDSPAGE, were 54, 42 and 37 kDa. The purified 54-kDa protein, which had two CAMPbinding sitedmonomer, 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 CAMPbinding protein and C subunit, estimated by analytical gel-filtration, was 220 kDa, a value which agrees with a RZCZ 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 11 regulatory subunit. Furthermore, no mobility shift on SDSlPAGE upon phosphorylation/dephosphoryIation of Myrilus protein was observed, a similar behaviour to that shown by the mammalian RIIP 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 M.ytiZu.s 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.Keyvords: CAMP-binding protein; CAMP-dependent protein kinase; regulatory subunit; protein phosphorylation ; Mytilus.For intertidal marine invertebrates, such as some bivalve molluscs, access to environmental oxygen is intermitent, so that these organisms are subjected to cyclic hypoxia periods. It is well described that, during hypoxia, a metabolic rate dcprcssion takes place, especially of glycolytic pathway [I]. One of the mechanisms involved in this biochemical adaptation seems to be the covalent modification (phosphorylation/dephosphorylation) of the key regulatory glycolytic enzymes : 6-phosphofructo-Ikinase, 6-phusphofructo-2-kinase and pyruvate kinase 12-41, However, despite the fact that certain neuroendocrine factors affecting the gycogen metabolism have been recently recognized in the sea mussel Mytilus edu1i.s [5, 61, their structure remains unknown and, so far, very little information is available about the mechanisms of intracellular signalling in these organisms.In our laboratory, we have proved that, in the sea mussel Mytilus ~~mllo~~r~~viiir~ialis, two key glycolytic enzymes, 6-phosphofructo-1 -kinase and 6-phosphofructo-2-kinase, are activated when phosphorylated in vitro by CAMP-dependent protein kinase [7, XI. On the other hand, it has also been reported that incubation of a dialyzed homogenate from mussels with cAMP C o t . r~. s~~o n d~~r i c~~ r o J. A. Villamarin,

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Phosphofructokinase from mantle tissue of the sea mussel Mytilus galloprovincialis was phosphorylated in vitro by a protein kinase isolated from the same tissue, homologous to mammalian cAMP‐dependent protein kinase; the maximal level of phosphorylation achieved was around 1 mol of Pi/mol of phosphofructokinase subunit. The covalent incorporation of phosphate leads to a notable increase in the enzyme activity assayed at near‐physiological concentrations of substrates and allosteric modulators and neutral pH. Tryptic digestion of labeled phosphofructokinase released a phosphopeptide whose sequence was Lys‐Asp‐Ser(P)‐Ile‐Trp‐Ile‐Gln‐Thr‐Gly‐Arg. This sequence showed high homology with the phosphopeptides from other invertebrates whose phosphofructokinase is also activated by cAMP‐dependent phosphorylation.

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Several isoforms of the cAMP‐dependent protein kinase catalytic subunit (C‐subunit) were separated from the posterior adductor muscle and the mantle tissues of the sea mussel Mytilus galloprovincialis by cation exchange chromatography, and identified by: (a) protein kinase activity; (b) antibody recognition; and (c) peptide mass fingerprinting. Some of the isozymes seemed to be tissue‐specific, and all them were phosphorylated at serine and threonine residues and showed slight but significant differences in their apparent molecular mass values, which ranged from 41.3 to 44.5 kDa. The results from the MS analysis suggest that at least some of the mussel C‐subunit isoforms arise as a result of alternative splicing events. Furthermore, several peptide sequences from mussel C‐subunits, determined by de novo sequencing, showed a high degree of homology with the mammalian Cα‐isoform, and contained some structural motifs that are essential for catalytic function. On the other hand, no significant differences were observed in the kinetic parameters of C‐subunit isoforms, determined by using synthetic peptides as substrate and inhibitor. However, the C‐subunit isoforms separated from the mantle tissue differed in their ability to phosphorylate in vitro some proteins present in a mantle extract.