Identification of multiple isoforms of the cAMP-dependent protein kinase catalytic subunit in the bivalve mollusc Mytilus galloprovincialis.

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 Calpha-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.

Regulation of the futile cycle of fructose phosphate in sea mussel.

Carbohydrate metabolism in mussels shows two phases separated seasonally. During summer and linked to food supply, carbohydrates, mainly glycogen, are accumulated in the mantle tissue. During winter, mantle glycogen decreases concomitantly with an increase in triglyceride synthesis. In spring, after spawning, the animals go in to metabolic rest until the beginning of a new cycle. This cycle is regulated by the futile cycle of fructose phosphate that implicates PFK-1 and FBPase-1 activities. These enzymes and the bifunctional PFK-2/FBPase-2 that regulates the Fru-2,6-P2 levels, are seasonally modulated by covalent phosphorylation/dephosphorylation mechanisms, as a response to unknown factors. The futile cycle of the fructose phosphates also controls the transition from physiological aerobiosis to hypoxia. The process is independent of the phosphorylation state. In this sense, a pH decrease triggers a small Pasteur effect during the first 24 h of aerial exposure. Variations in the concentration of Fru-2,6-P2 and AMP are the sole factor responsible for this effect. Longer periods of hypoxia induce a metabolic depression characterized by a decrease in Fru-2,6-P2 which is hydrolyzed by drop in the pH. In this review, the authors speculate on the two regulation processes.

Measurement of adenosine 3',5'-cyclic monophosphate and guanosine 3', 5'-cyclic monophosphate in mussel (Mytilus galloprovincialis lmk.) by high-performance liquid chromatography with diode array detection.

Two fast and sensitive methods for the determination of cAMP and cGMP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. are described. Both methods use ion-pair high-performance liquid chromatography with diode array detection. The use of the diode array detector permitted the simultaneous detection of the absorbance at two different wavelengths and the obtaining of the UV absorption spectrum for each detected peak, confirming peak purity and identity. Method precision was good. The detection limit for both nucleotides was 2.5 pmol (signal-to-noise ratio = 4 at 254 nm). Previous to the injection onto the chromatograph, both nucleotides were purified by precipitation of the adenine and guanine 5'-ribonucleotides with ZnSO(4)-Na(2)CO(3). The supernatant obtained was subsequently passed over an anion-exchange column (AG l-X8 formate form resin). Early results seem to indicate that there is a seasonal variation in the contents of both cyclic nucleotides in mantle tissue. Such variation is probably related to the annual gametogenic cycle.

Purification of a novel isoform of the regulatory subunit of cAMP-dependent protein kinase from the bivalve mollusk Mytilus galloprovincialis.

Cytosolic extracts from the posterior adductor muscle of the bivalve mollusk Mytilus galloprovincialis contain significant amounts of both cGMP-binding and cGMP-stimulated protein kinase activities. However, photoaffinity labeling with 8-azido-[32P]cGMP revealed only a major cGMP-binding protein with an apparent molecular mass of 54 kDa (p54), lacking protein kinase activity itself. Instead, the purified and cGMP-free p54 protein has the ability to inhibit a mussel protein kinase homologous to the mammalian cAMP-dependent protein kinase (cAPK) catalytic subunit, the inhibition being relieved by cAMP or cGMP, which suggests that it can act as a regulatory subunit of cAPK. However, p54 failed to be recognized by a specific antibody against the regulatory subunit (type RII) previously isolated from mussel. Therefore, p54 must be a novel isoform of cAPK regulatory subunit that seems to have high affinity for both cGMP and cAMP.

In vivo phosphorylation of phosphofructokinase from the bivalve mollusk Mytilus galloprovincialis.

The phosphorylation state of phosphofructokinase from the mantle tissue of the facultative anaerobe mollusk Mytilus galloprovincialis was determined by a back-phosphorylation technique. The incubation of intact mantle tissue with 8-bromoadenosine 3':5'-cyclic monophosphate increased significantly the phosphate content of phosphofructokinase, which indicates that the enzyme can be phosphorylated in vivo by endogenous cAMP-dependent protein kinase. The phosphate content of mussel phosphofructokinase changes significantly during the year, in agreement with the kinetic data that show a more active enzyme form in earlier autumn. These results suggest that cAMP-dependent phosphorylation of phosphofructokinase can be partially responsible for the observed glycolytic changes associated with the annual gametogenic cycle that takes place in the mantle tissue of the mollusk. On the contrary, no differences were observed between aerobic and 24-h hypoxic mussels with regard to the phosphorylation state and the kinetic constants of phosphofructokinase. This result is inconsistent with the hypothesis that phosphorylation of phosphofructokinase is involved in the glycolytic depression that takes place during the long-term environmental hypoxia that the mollusk can undergo.

A method for the purification of cAMP-dependent protein kinase using immunoaffinity chromatography.

A rapid and efficient method for purifying cAMP-dependent protein kinase (PKA) holoenzyme based on immunoaffinity chromatography was developed. The affinity column was prepared by coupling a polyclonal antibody raised against the PKA regulatory subunit to NHS-activated Sepharose. The holoenzyme purified by this procedure from the bivalve mollusk Mytilus galloprovincialis was shown to be fully active as judged by (1) its cAMP-binding activity, (2) its cAMP-dependent protein kinase activity, and (3) its autophosphorylation ability. Moreover, together with both regulatory and catalytic subunits, which constitute the PKA holoenzyme, a protein with a molecular mass of approximately 200 kDa was copurified, and results from gel-filtration chromatography showed that it was associated with a fraction of PKA. Therefore, this immunoaffinity purification technique could also be useful to isolate such proteins as interact with PKA in vivo.

cAMP-dependent phosphorylation activates phosphofructokinase from mantle tissue of the mollusc Mytilus galloprovincialis. Identification of the phosphorylated site.

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.

Identification of RII-binding proteins in the mollusc Mytilus galloprovincialis.

Several proteins with M(r) > 70 kDa from various tissues of the sea mussel Mytilus galloprovincialis were specifically recognized in vitro by the regulatory subunit (type RII alpha) of cAMP-dependent protein kinase (cAPK) from porcine heart. However, none of these proteins interacted with the regulatory subunit of cAPK from the mollusc itself. The results suggest that, unlike mammalian RII, regulatory subunit from mussel lacks the specific residues responsible for interaction with R-binding proteins. Consequently, the identified molluscan RII alpha-binding proteins should play a distinct role from cAPK anchoring.

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.

Phosphofructokinase from mantle tissue of Mytilus galloprovincialis. Purification and effects of phosphorylation on the enzymatic activity.

Phosphofructokinase purified from mantle tissue of the sea mussel Mytilus galloprovincialis, was phosphorylated "in vitro" by the catalytic subunit of cyclic AMP-dependent protein kinase. The incorporation of phosphate gave rise to an activation of the enzyme by increasing its affinity for fructose-6-phosphate, by decreasing its sensitivity to the inhibition by ATP and by enhancing the effect of allosteric activators (5'-AMP and fructose-2,6-bisphosphate). In addition, the effects of phosphorylation on the catalytic activity are pH-dependent.

[Effect of hypoxia on glycolysis in the adductor muscle and hepatopancreas of the marine mussel Mytilus galloprovincialis Lmk]. / Efecto de la hipoxia sobre la glucolisis en músculo aductor y hepatopáncreas del mejillón marino Mytilus galloprovincialis Lmk.

Concentrations of glycolytic intermediates and adenine nucleotides have been estimated in adductor muscle and hepatopancreas from the sea mussel Mytilus galloprovincialis Lmk. after various periods of valve closure. Mass action ratios of enzyme steps involved in the metabolism of these components are compared with their equilibrium constants. This reveals hexokinase, phosphofructokinase, pyruvate kinase and fructose-1,6-bisphosphatase catalyze non-equilibrium reactions. The changes in the concentrations of the glycolytic intermediates and in the rate M.A.R./Keq during hypoxia suggest that the carbon flow after valve closure is first controlled by phophofructokinase, but later on the rate of transformation of phosphoenolyruvate regulates this flow.

New Granadan native populations: recent demographic research.

PIP: A survey of recent work on the demographic history of the colonial native populations in the area that is now Colombia is presented, with emphasis on the study of the Chibcha Indians. Information is included on published data, sources, issues, and future research questions.^ieng