Implication of PKC isozymes in the release of biogenic amines by mussel hemocytes: effect of PDGF, IL-2, and LPS.

The innate immune system of marine mussels (Mytilus galloprovincialis) is operated by phagocytic cells termed hemocytes. Lipopolysaccharide (LPS), interleukin-2 (IL-2), or platelet-derived growth factor (PDGF) increase biogenic amine synthesis in these cells, and the enzymes Ca(2+)-independent protein kinase C (PKC) (p105/108) and Ca(2+)-dependent PKC (p60) are involved in these processes. Stimulation by PDGF induces a down-regulation process affecting the form p108 of the Ca(2+)-independent PKC. In addition, PDGF produces the increase of expression of p60 in the membrane fraction. IL-2 induces the disappearance of p108 from the membrane but does not affect the presence of p60 in cytosol or membrane. For its part, LPS activates exclusively p60 by a down-regulation mechanism. The ensemble of results suggests that each agonist starts a pathway that implicates the PKC isoenzymes that mediate the regulation of the activities dopa decarboxylase, dopamine beta-hydroxilase, and phenyletanolamine N-methyltranferase, which lead to different actions related to biogenic amine synthesis.

The Ca2+-independent PKC (p105) mediates the PMA-activation of marine mussel hemocytes and the Ca2+-dependent PKC (p60) does not intervene.

Previous works revealed the presence of a Ca(2+)-dependent protein kinase (p60) and a Ca(2+)-independent protein kinase (p105) in the mantle tissue from the sea mussel Mytilus galloprovincialis Lmk. The expression of both isoforms shows a balance between cytosolic and membrane fractions in mantle, gills, and hepatopancreas, whereas, in hemocytes, their expression is mainly cytosolic, as happens in muscle tissues with p60 alone. Both enzymatic forms contain phosphorylated serines, and no phosphorylation was detected in tyrosines. Only the form p105 mediates the PMA-induced activation of the hemocytes of M. galloprovincialis, and it does so by a process of down-regulation. The form p60 does not respond to the presence of the phorbol ester, suggesting structural differences related to the binding sites of the diacylglycerol.

Purification and characterization of a new Ca2+-dependent protein kinase C in mussel (Mytilus galloprovincialis Lmk.) mantle.

An enzyme that can be included into the so-called conventional PKCs has been purified to homogeneity from the mantle tissue of the sea mussel Mytilus galloprovincialis. This enzyme has a molecular weight of 60 kDa, which is DAG-dependent, PS-activated, and Ca2+-dependent. It was separated from a Ca2+-independent PKC (p105) (Mercado et al., Mol Cell Biochem 233:99-105, 2002) by means of an ionic exchange chromatography on DE-52 cellulose. The molecular weights and kinetic properties of both the enzymes are different. The protein p60 is broadly distributed among the tissues, which suggests that it may carry out specific functions, different from those performed by p105.

Effect of thermal stress on protein expression in the mussel Mytilus galloprovincialis Lmk.

The exposure of organisms to stressing agents may affect the level and pattern of protein expression. Certain proteins with an important role in protein homeostasis and in the tolerance to stress, known as stress proteins, are especially affected. Different tissues and cells show a range of sensitivities to stress, depending on the habitat to which organisms have adapted. The response of different tissues and cells from the mussel Mytilus galloprovincialis Lmk. to heat shock has been studied in this work using different exposure times and temperatures. During the assays, protein expression was observed to vary depending on the tissue studied, the temperature or the exposure time used. But maybe the most prominent thing is the different response obtained from the cultured haemocytes and those freshly obtained from stressed mussels, which makes us think that the extraction procedure is the main cause of the response of non-cultured cells, although the haemolymph may contain components that modulate haemocyte response.

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.

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk.

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 receptor (IL-2R) subunits. Mussel IL-2R alpha and IL-2R beta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL-2R gamma is smaller than that in vertebrates. Both lipopolysaccharide and IL-2 induce IL-2R alpha expression, and such induction depends on the concentration of both agonists.

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.

The 28S fraction of rRNA in molluscs displays electrophoretic behaviour different from that of mammal cells.

Electrophoresis on agarose/formaldehyde gels of rRNA in molluscs display a pattern of bands which could suggest a RNase action due to incorrect manipulation of the samples. This study shows that the disappearance of the band corresponding to the 28S fraction is due to the denaturing conditions used when electrophoresis is carried out and not to RNases action.

Identification of protein kinase C isoforms in marine mussels.

In this paper we have examined the distribution of some isoforms of protein kinase C in different tissues from the sea mussel Mytilus galloprovincialis Lmk.. By immunoblot analysis, we have detected the presence of at least three PKC isoforms, all preferably associated with the cellular cytosolic fraction. The Ca(2+)-independent form PKC delta was separated from the Ca(2+)-dependent forms (PKC alpha and beta) by means of an ionic change chromatography on DE-52. A comparative study was carried out on the phosphorylatable non-artificial substrates. The M.B.P. protein proved to be the best substrate, while the worst was HIIIS histone which, however, is frequently used in evaluation assays of the activity.

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.

The modulation of the oxidative phase of the pentose phosphate pathway in mouse liver.

The glucose-6-phosphate dehydrogenase from mouse liver is fully inhibited in vitro by physiological concentrations of NADPH. This suggests that the oxidative phase of the pentose phosphate pathway requires some deinhibitory system. In order to investigate regulation of the pentose phosphate pathway, various parameters (intermediate concentrations, mass-action ratios of reactions, etc.) were measured in liver from control mice and from meal-fed mice. Assays were also carried out to detect any molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH. The liver of meal-fed mice show greater glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities. They also had greater concentrations of several metabolic intermediates and triglycerides than the control animals (P < 0.001). These results prove that the diet increases the flow of the pentose phosphate pathway in a lipogenic sense. The glutathione reductase does not change with the diet, suggesting that this enzyme does not participate in the modulating process. Unlike rat liver, no molecules causing the reverse of glucose-6-phosphate dehydrogenase inhibition by NADPH were detected. These data suggest that the increase of flow of the pentose phosphate pathway during lipogenesis is obtained by an increase in enzyme synthesis.

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.

Purification, characterization and kinetic mechanism of glucose-6-phosphate dehydrogenase from mouse liver.

1. Glucose-6-phosphate dehydrogenase (G6PDH EC 1.1.1.49) from mouse liver has been purified 1100-fold by extraction, ion-exchange chromatography on DE-52, absorption chromatography on Bio-Gel HTP and gel filtration through sepharose 6 HR 10/30. The purified enzyme showed a single band in silver stained SDS-PAGE. 2. The native and subunit molecular weight were 117 and 31 kDa respectively. 3. The kinetic studies and the patterns obtained from the inhibition by-products suggest that the enzyme follows an ordered sequential kinetic mechanism. 4. The reduced Km values for the substrates favour the operativity of the enzyme. The "fine control" of the enzymatic activity was exerted by the NADPH, whose Ki is several fold lower than the in vivo concentration.

Regulation of fructose-2,6-bisphosphate content in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. Regulation of fructose-2,6-bisphosphatase activity.

Fructose-2,6-bisphosphatase (FBPase-2) from the mantle tissue of the mussel Mytilus galloprovincialis shows a hyperbolic kinetic with a Km value (0.40 mM) for its substrate, that suggest that the "in vivo" Fru-2,6-P2 concentration is not a limiting factor for activity. The enzyme possesses an optimum pH for activity between 6 and 7 units, similar to the reached in mussel mantle during physiological hypoxia. The modulation of activity by the pH, and in addition, the positive effect of ATP are in keeping with the little decrease in concentration of the Fru-2,6-P2 that occurs during the first hour of hypoxia due to the valve closure.

Phosphorylation-activated 6-phosphofructo-2-kinase from mantle tissue of marine mussels.

PKF-2 from mussel mantle was phosphorylated by cAMP-dependent protein kinase. The phosphorylation does not change the enzyme activity at neutral pH values, but at acid pH the activity of the phosphorylated form is higher than the native PFK-2. With respect to the native enzyme, the activation consisted of a reduction in the Km for Fru-6-P and a decrease in the inhibitory effect of PEP. These results are in keeping with the stabilized concentration of Fru-2,6-P2 found in the mussel mantle during the physiological hypoxia caused by the closure of the valves.

Amino-acids imitate the EDTA activation on the fructose-1,6-bisphosphatase of mantle tissue from the sea mussel (Mytilus galloprovincialis Lmk).

In the absence of AMP and Fru-2,6-P2, several amino-acids such as histidine, lysine, alanine, aspartic acid, and other molecules, as reduced glutathione or citrate, activate FBPase-1 from Mytilus galloprovincialis mantle. AMP decreases Vmax and Km for Fru-1,6-P2 both in the absence and in the presence of activators; but the addition of Fru-2,6-P2 decreases the affinity of the enzyme by its substrate. Na+ acts as a inhibitor reducing both Vmax and Km. The Km value is lower than the physiological level of Fru-1,6-P2, suggesting that the enzyme is operative but its activity is very reduced.

[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.