Filamin isoforms in molluscan smooth muscle.

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively.

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.

CK2-mediated phosphorylation of a type II regulatory subunit of cAMP-dependent protein kinase from the mollusk Mytilus galloprovincialis.

Two isoforms of regulatory (R) subunit of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), were identified so far in the sea mussel Mytilus galloprovincialis. Out of them, only R(myt2) was phosphorylated in vitro by casein kinase 2 (CK2) using GTP as phosphate donor. CK2 catalytic subunit (CK2alpha) itself was sufficient to phosphorylate R(myt2), but phosphorylation was enhanced by the presence of the regulatory subunit CK2beta.

Catalytic subunit of cAMP-dependent protein kinase from a catch muscle of the bivalve mollusk Mytilus galloprovincialis: purification, characterization, and phosphorylation of muscle proteins.

cAMP-dependent protein kinase (PKA) plays a crucial role in the release of the catch state of molluskan muscles, but the nature of the enzyme in such tissues is unknown. In this paper, we report the purification of the catalytic (C) subunit of PKA from the posterior adductor muscle (PAM) of the sea mussel Mytilus galloprovincialis. It is a monomeric protein with an apparent molecular mass of 40.

Isoforms of cAMP-dependent protein kinase in the bivalve mollusk Mytilus galloprovincialis: activation by cyclic nucleotides and effect of temperature.

Two different isoforms of cAMP-dependent protein kinase (PKA) have been partially purified from the posterior adductor muscle and the mantle tissue of the sea mussel Mytilus galloprovincialis. The holoenzymes contain as regulatory subunit (R) the previously identified isoforms Rmyt1 and Rmyt2, and were named PKAmyt1 and PKAmyt2, respectively. Both cAMP and cGMP can activate these PKA isoforms completely, although they exhibit a sensitivity approximately 100-fold higher for cAMP than for cGMP.

Characterization of a type I regulatory subunit of cAMP-dependent protein kinase from the bivalve mollusk Mytilus galloprovincialis.

Two isoforms of the regulatory subunit (R) of cAMP-dependent protein kinase (PKA), named R(myt1) and R(myt2), had been purified in our laboratory from two different tissues of the sea mussel Mytilus galloprovincialis. In this paper, we report the sequences of several peptides obtained from tryptic digestion of R(myt1). As a whole, these sequences showed high homology with regions of type I R subunits from invertebrate and also from mammalian sources, but homology with those of fungal and type II R subunits was much lower, which indicates that R(myt1) can be considered as a type I R isoform.

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.

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.

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.

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.

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.

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.

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.

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.

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].

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.