Izaskun Ibarguren

Seasonal variations in glycolysis in Mytilus galloprovincialis Lmk.

1. 1. In the hepatopancreas and mantle of the sea mussel Mytilus galloprovincialis Lmk glycolysis shows seasonal variations. 2. 2. In both these tissues, glycolytic activity shows a maximum in winter and a minimum in summer. 3. 3. Smaller fluctuations also take place in the posterior adductor muscle. 4. 4. In all three tissues, Fru 2,6-P2 indicates the direction and strength of glycolysis, correlating well with other parameters such as energy charge or the phosphoenolpyruvate/pyruvate ratio. 5. 5. In the posterior adductor muscle, however, Fru 2,6-P2 does not correlate with the MAR/Keq ratio from phosphofructokinase owing to the synergic activation of the enzyme by both Fru 2,6-P2 and AMP.

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.

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. This conclusion is also supported by the following biochemical properties: (1) R(myt1) was proved to have interchain disulfide bonds stabilizing its dimeric structure; (2) it failed to be phosphorylated by the catalytic (C) subunit purified from mussel; (3) it has a higher pI value than that of the R(myt2) isoform; and (4) it showed cross-reactivity with mammalian anti-RIbeta antibody.

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. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.

Implication of adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, adenosine 5'-mono-, di-, and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in relation to the gametogenic cycle in the mussel mytilus galloprovincialis Lmk.

The changes in the contents of cyclic AMP, cyclic GMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occur in the mantle tissue of the mussel Mytilus galloprovincialis Lmk were analysed with regard to the annual gametogenic cycle. Throughout 2 years, the lowest contents of AMP, ADP and ATP were detected during late winter-spring, whereas the maximum appeared in the autumn months. During the second year, fructose-2,6-bisphosphate and cAMP showed a very similar behaviour. The levels of both compounds rose throughout the year until a maximum in September. Their behaviour was also similar to that observed during the first year, but displaced in time. Both in 1998 and in 1999, the highest level of cGMP was detected during the spring-summer months. The results obtained suggest that the glycolytic pathway, with regard to the breeding cycle, might be regulated by fructose-2,6-bisphosphate and cyclic AMP through the activation of 6-phosphofructo-1-kinase, which is the main regulating enzyme of the glycolysis in mantle of M. galloprovincialis.

DETERMINATION OF ADENINE NUCLEOTIDES IN MYTILUS GALLOPROVINCIALIS LMK. BY ION-PAIR HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH A DIODE ARRAY DETECTOR

A quick and sensitive method for the determination of AMP, ADP, and ATP levels in mantle tissue of the sea mussel Mytilus galloprovincialis Lmk. is described. The method uses ion-pair high-performance liquid chromatography with diode array detection, allowing obtaining of the UV absorption spectrum for each peak. Method precision was good. The detection limit for all three nucleotides was 20 pmol (much lower than the minimum concentrations in this tissue).

The role of fructose 2,6-bisphosphate in the regulation of marine mussels (Mytilus galloprovincialis and mytilus edulis) glycolysis/gluconeogenesis

Abstract 1. 1. Fru-2,6-P 2 plays an important modulating role in the glycolysis/gluconeogenesis of sessile marine molluscs, indicating by its content the change in both the flow direction and the glycogen reserves. 2. 2. PFK-1 is activated, while FBPase-1 is inhibited by Fru-2,6-P 2 . The phosphoester synergically enhances the effect caused by AMP on both enzymes. 3. 3. As in mammals, the different PFK-2 from marine molluscs are bifunctional enzymes with the hydrolytic FBPase-2 activity. The reversible covalent modification of PFK-2/FBPase-2 by phosphorylation/dephosphorylation displays different responses according to the source of the enzyme. 4. 4. A low temperature sea-water and the presence of glucose increase the concentration of Fru-2,6-P 2 parallel to the activation of the glycolytic pathway; whereas alanine and propionate, promoters of glyconeogenesis, reduce the Fru-2,6-P 2 content. 5. 5. The concentration of Fru-2,6-P 2 in mantle and hepatopancreas presents seasonal variations with peaks in winter and the low values in both spring and summer; these fluctuations are parallel to the feeding-breeding cycle. 6. 6. The variations in the concentration of Fru-2,6-P 2 during the physiological hypoxia or during anoxia are discussed, on the basis of processes of reversible phosphorylation/dephosphorylation of PFK2/FBPase-2.

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk. I: Purification and ion requirements

Abstract 1. 1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively. 2. 2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn 2+ , Zn 2+ and Mg 2+ . The K m for Mg 2+ is lower than the physiological concentration of cation in the tissue, whereas its K m for Mn 2+ and Zn 2+ is greater than the respective in vivo concentrations. 3. 3. The joint action of Mg 2+ and Zn 2+ increases the affinity of the enzyme for the substrate Fru-1,6-P 2 , though V max is reduced. 4. 4. Na + strongly inhibits the enzyme even at very low concentrations. K + has no effect whatsoever.

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.

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk.—III. Seasonal variation of enzymatic activity

1. 1. Fructose-1,6-bisphosphatase (FBPase-1) from the mantle and the posterior adductor muscle (PAM) of Mytilus galloprovincialis Lmk. was investigated over almost 2 years for changes in specific activity and the apparent Michaelis constant (Km) for Fru-1,6-P2 (purified enzyme preparations). 2. 2. The specific activity of the mantle changed, peaking in a period between August and November. 3. 3. The Km values for the substrate changed seasonally being low during the first part of the year (January–May) and high in the period between October and November. 4. 4. The changes in mantle FBPase-1 activity were discussed in relation to the effect of modulators and the possible seasonally covalent modification of the enzyme. 5. 5. No statistical changes were detected in posterior adductor muscle FBPase-1, which is in keeping with the glycolytic role suggested for the tissue.