Purification of a lipid-activated and Ca2+-independent protein kinase from the mantle tissue of Mytilus galloprovincialis Lmk.

A phospholipid-sensitive Ca²⁺-independent protein kinase (p105) was purified to homogeneity from mantle tissue of the mussel Mytilus galloprovincialis Lmk., employing consecutively DE-52 cellulose, Sephacryl S-200 and Biogel HTP chromatographies. The purified enzyme appeared as a single band on 10% SDS-PAGE, and had a molecular weight of 105 kDa.The positive Western blotting of the purified eluate for anti-human-PKC and PKC suggests that the enzyme from mussel mantle may be an ancestral nPKC isoform, with the kinetic properties of the enzyme very close to those of PKC isoform of vertebrates.Western blotting of samples from different steps of purification using specific mouse anti-p105, showed two protein bands in samples from the initial steps. However, only one band was detected in the Biogel-HTP eluate, the most purified fraction. The purification steps did not affect the presence of P-serine in p105. No P-tyrosine peptides were detected in any of the purification steps. These results open a new field of work on the study of several molecular processes related to energetic metabolism and reproduction in molluscs, whose regulation is associated with the activation of protein kinases.     

Nutrient storage cells isolation from mantle tissue of Mytilus galloprovincialis: glucose release and glycogen content

A method for obtaining isolated mantle nutrient storage cells and purifying vesicular (VC) and adipogranular (ADG) cells from mantle tissue of Mytilus galloprovincialis is reported. Tissue digestion is partly mechanical (stirring) and partly enzymatic (collagenase + dispase). Purification is carried out through continuous and discontinuous Percoll gradients. VC appears in fraction 3 (d = 1.05-1.08 g/ml) and ADG in fraction 2 (d = 1.09 g/ml). Intracellular glycogen and free-glucose content in September-April period is studied. When glycogen is detectable it is always accompanied by intracellular free-glucose pool in a concentration relationship glycogen/glucose 10:1. Furthermore, a glucose releasing activity elicited by the Ca2(+)-ionophore A23187 was found in isolated cells, which reproduce the former behaviour found with mantle tissue fragments in our laboratory.     

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.     

Ca2+ activates glycogenolysis in isolated mantle storage cells of Mytilus galloprovincialis Lmk.

Glycogenolytic activity (GA) in isolated mantle storage cells (MSC) from Mytilus galloprovincialis was studied, while glycogen and free-glucose content, as well as glucose released from cells were tested. In the period studied (November-December), the glucose releasing activity measured can be considered as an output of GA. In both, whole cells system (WCS) and crude cell-free system (CFS), a non-stimulated GA was detected. In WCS, dopamine and 5-hydroxytryptamine (5-HT) stimulated glycogenolysis, while epinephrine, norepinephrine and isoproterenol did not show any effect. Furthermore, mellitin and the Ca(2+)-ionophore, A23187, had a stimulating effect on the GA. In CFS, the absence of Ca2+ ions was a sufficient condition to depress GA. These and other findings suggest that: 1) GA in MSC may be stimulated by dopamine and 5-HT and not by adrenergic agonists; 2) cytosolyc Ca2+ signalling may have become an absolute requirement for activation of the glycogenolytic cascade in MSC; 3) a rapid high-affinity glucose transport may occur in these cells.     

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.     

Seasonal changes of nucleotides in mussel (Mytilus galloprovincialis) mantle tissue

Seasonal variations of nucleotides in Mytilus galloprovincialis mantle tissue were analyzed. Separation and quantification was achieved by reversed-phase high-performance liquid chromatography. Total nucleotides show a pronounced seasonal variation with maximum and minimum values in autumn and spring, respectively. Adenine nucleotides accounted for the major part in spring and summer, guanosine and cytidine nucleotides in winter; uridine nucleotides were relatively constant throughout the year. Their inverse variation suggests inter-conversion among them and the maintenance of the potential cell energy in winter by other triphosphate nucleotides different from ATP. These results reflect environmental and nutritional conditions, and also the reserves and gametogenic cycles taking place in M. galloprovincialis mantle tissue.     

Multilocus allozyme differentiation of populations of the mussel Mytilus galloprovincialis Lmk. from Moroccan coasts

Along the Moroccan coasts, the systematic status of Mytilus populations have been for a long time uncertain and confused, due to the use of unreliable morphometric criteria. In the present study, allozyme markers reveal the exclusive existence of M. galloprovincialis on Mediterranean and Atlantic coasts. Nei's genetic distances are low and reflect a high gene flow between Atlantic and Mediterranean populations. However, a significant multilocus discontinuity revealed by F-statistics separate southern Atlantic populations from Mediterranean and north Atlantic ones and could be explained by a gene flow breaking because of a larval dispersal decrease, due to a sea surface current direction change from Cap Ghir towards the Canaries archipelago, and probably by differential selection effects in these two geographic areas.     

Effect of temperature on the adenylate cyclase activity of Mytilus galloprovincialis mantle tissue.

1. The basal and NaF-stimulated adenylate cyclase activities of Mytilus galloprovincialis mantle tissue were studied at different temperatures. 2. There are no significant differences in the Km for ATP at 13 degrees C and 20 degrees C in both basal and NaF-stimulated conditions. 3. NaF increases the Vmax of the enzyme (5-fold) and decreases about 50% the Km for ATP at both temperatures assayed. 4. Activation energy of the enzyme reaction is 33.4 kJ/mol. K in basal conditions and 29.4 kJ/mol. K when NaF is present. The Q10, at saturating substrate concentrations, is approximately 1.5 and this value is constant in the temperature range studied, 10-30 degrees C. 5. The adenylate cyclase starts being inactivated from 30 degrees C. The enzyme shows greater sensitivity to denaturalization by temperature in NaF-stimulated than in basal conditions.     

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.     

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.     

A tentative mechanism of the ternary complex formation between phosphorylase kinase, glycogen phosphorylase b and glycogen

The kinetics of rabbit skeletal muscle phosphorylase kinase interaction with glycogen has been studied. At pH 6.8 the binding of phosphorylase kinase to glycogen proceeds only in the presence of Mg2+, whereas at pH 8.2 formation of the complex occurs even in the absence of Mg2+. On the other hand, the interaction of phosphorylase kinase with glycogen requires Ca2+ at both pH values. The initial rate of the complex formation is proportional to the enzyme and glycogen concentrations, suggesting the formation of the complex with stoichiometry 1:1 at the initial step of phosphorylase kinase binding by glycogen. According to the kinetic and sedimentation data, the substrate of the phosphorylase kinase reaction, glycogen phosphorylase b, favors the binding of phosphorylase kinase with glycogen. We suggest a model for the ordered binding of phosphorylase b and phosphorylase kinase to the glycogen particle that explains the increase in the tightness of phosphorylase kinase binding with glycogen in the presence of phosphorylase b.     

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.     

Interlineage Mytilus galloprovincialis Lmk. 1819 hybridization yields inconsistent genetic outcomes in the Southern hemisphere

A Southern hemisphere lineage of the blue mussel Mytilus galloprovincialis has been diverging in allopatry from Northern hemisphere conspecifics for 0.84–1.2 million years. Secondary contact between Southern and Northern hemisphere mussels in Chile, New Zealand and Australia provides an opportunity to better understand the extent and consequences of extensive range expansion. Non-native M. galloprovincialis and hybrids, as detected from RFLP assays of nuclear and mitochondrial DNA, are present in all three countries and significant cytonuclear disequilibria exist for native homozygotes in Chile and New Zealand, non-native homozygotes in Chile and non-native heterozygotes in New Zealand. Introductions into Australia are rare events given that no pure non-native mussels were detected. Immigration from one or both taxa into the hybrid zone may underlie disequilibria in New Zealand, whilst gender-directional crossing with limited ongoing hybridization contributes to disequilibria in Chile. Hybridization dynamics do not pose a threat to the Southern lineage in Chile and Australia, but in New Zealand, introgression, continued immigration and slight hybridization gender bias towards non-native maternal parents could lead to the regional extirpation of the native lineage.     

In vitro effects of LPS, IL-2, PDGF and CRF on haemocytes of Mytilus galloprovincialis Lmk

The cells in charge of the innate immune response in the marine mussel Mytilus galloprovincialis Lmk. are the haemocytes. These cells respond in different ways to agents such as lipopolysaccharide (LPS), interleukin-2 (IL-2), platelet-derived growth factor (PDGF) and corticotropin releasing factor (CRF). After stimulation of the haemocytes, the expression of molecules reactive with monoclonal antibodies raised to the alpha chain of the IL-2 receptor, present in their membrane, differed depending on the agent used. The same happened with regard to the levels of dopamine, adrenaline and noradrenaline released to the medium by the haemocytes. It should also be noted that no catecholamine release was detected and the level of expression of IL-2Ralpha showed no significant variation in cultured cells that had not been treated with inducers. These facts would indicate that most haemocytes were in the same starting condition at the moment that the stimulation was performed. Therefore, cultured haemocytes can be a highly reliable model in the study of the innate immune system.     

Evidence of selective mortality in favour of the Mytilus galloprovincialis Lmk phenotype in British mussel populations

Samples of mussels ( Mytilus ) were collected from 17 localities within hybrid zones of Mytilus edulis and Mytilus galloprovincialis in south-west and north-east England. The study of two polymorphic allozyme loci ( esterase-D and octopine dehydrogenase ), which are partially diagnostic for the two forms of mussel, reveal the existence of widespread length-dependent allele frequency variation. Larger mussels tend to have a higher frequency of alleles characteristically at high frequency in Mytilus galloprovincialis. Also at a given shell length galloprovincialis alleles have a higher frequency higher up the shore. Computer simulation is used to demonstrate that length-dependent variation may be generated not only by differential mortality but also by differential growth and in models including or excluding immigration. Evidence supports the hypothesis that selective mortality acting in favour of the galloprovincialis phenotype within hybrid populations in Britain is balanced by immigration of the more abundant Mytilus edulis.