Purification and kinetic characterization of 6-phosphofructo-1-kinase from the liver of gilthead sea bream (Sparus aurata)

6-phosphofructo-1-kinase (PFK) was purified to homogeneity from liver of gilthead sea bream (Sparus aurata) and kinetic properties of the enzyme were determined. The native enzyme had an apparent molecular mass of 510 kDa and was composed of 86 kDa subunits, suggesting homohexameric structure. At pH 7, S. aurata liver PFK (PFKL) showed sigmoidal kinetics for fructose-6-phosphate (fru-6-P) and hyperbolic kinetics for ATP. Fructose-2,6-bisphosphate (fru-2,6-P2) converted saturation curves for fru-6-P to hyperbolic and activated PFKL synergistically with AMP. Fru-2,6-P2 counteracted the inhibition caused by ATP, ADP and citrate. Compared to the S. aurata muscle isozyme, PFKL had lower affinity for fru-6-P, higher cooperativity, hyperbolic kinetics in relation to ATP, increased susceptibility to inhibition by ATP, and was less affected by AMP, ADP and inhibition by 3-phosphoglycerate, phosphoenolpyruvate, 6-phosphogluconate or phosphocreatine. The effect of starvation-refeeding on PFKL expression was studied at the levels of enzyme activity and protein content in the liver of S. aurata. Our findings indicate that short-term recovery of PFKL activity after refeeding previously starved fish, may result from allosteric regulation by fru-2,6-P2, whereas combination of activation by fru-2,6-P2 and increase in protein content may determine the long-term recovery of the enzyme activity.     

Purification and kinetic properties of 6-phosphofructo-1-kinase from gilthead sea bream muscle

The kinetic properties of 6-phosphofructo-1-kinase (PFK) from skeletal muscle (PFKM) of gilthead sea bream (Sparus aurata) were studied, after 10,900-fold purification to homogeneity. The native enzyme had an apparent molecular mass of 662 kDa and is composed of 81 kDa subunits, suggesting a homooctameric structure. At physiological pH, S. aurata PFKM exhibited sigmoidal kinetics for the substrates, fructose-6-phosphate (fru-6-P) and ATP. Fructose-2,6-bisphosphate (fru-2,6-P(2)) converted the saturation curves for fru-6-P to hyperbolic, activated PFKM synergistically with other positive effectors of the enzyme such as AMP and ADP, and counteracted ATP and citrate inhibition. The fish enzyme showed differences regarding other animal PFKs: it is active as a homooctamer, and fru-2,6-P(2) and pH affected affinity for ATP. By monitoring incorporation of (32)P from ATP, we show that fish PFKM is a substrate for the cAMP-dependent protein kinase. The mechanism involved in PFKM activation by phosphorylation contrasts with previous observations in other species: it increased V(max) and did not affect affinity for fru-6-P. Unlike the mammalian muscle enzyme, our findings support that phosphorylation of PFKM may exert a major role during starvation in fish muscle.     

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene expression is regulated by diet composition and ration size in liver of gilthead sea bream, Sparus aurata

Modulation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P(2)ase) gene expression by diet composition and ration size was studied in the liver of gilthead sea bream, Sparus aurata. From five different types of diet supplied to fish, those with either high carbohydrate/low protein or high carbohydrate/low lipid content stimulated 6PF-2-K/Fru-2,6-P(2)ase expression at the levels of mRNA, immunodetectable protein and kinase activity as well as promoting higher fructose-2,6-bisphosphate (Fru-2,6-P(2)) values. The expression of the bifunctional enzyme and Fru-2,6-P(2) levels showed also direct dependence on the quantity of diet supplied. These findings demonstrate for the first time nutritional regulation of 6PF-2-K/Fru-2,6-P(2)ase at mRNA level by diet composition and ration size and suggest that the carnivorous fish S. aurata can adapt its metabolism, by stimulation of liver glycolysis, to partial substitution of protein by carbohydrate in the diet. In addition, the expression of 6PF-2-K/Fru-2,6-P(2)ase can be used as an indicator of nutritional condition.     

Molecular cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from liver of Sparus aurata: nutritional regulation of enzyme expression

A cDNA clone encoding full-length 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) was isolated and sequenced from a Sparus aurata liver cDNA library. The 2527 bp nucleotide sequence of the cDNA contains a 73 bp 5'-untranslated region (5'-UTR), an open reading frame that encodes a 469 amino acid protein and 1041 bp at the 3'-UTR. The deduced amino acid sequence is the first inferred 6PF-2-K/Fru-2, 6-P2ase in fish. The kinase and bisphosphatase domains, where the residues described as crucial for the mechanism of reaction of the bifunctional enzyme are located, present a high degree of homology with other liver isoenzymes. However, within the first 30 amino acids at the N-terminal regulatory domain of the fish enzyme a low homology is found. Nutritional regulation of the 6-phosphofructo-2-kinase activity, together with immunodetectable protein and mRNA levels of 6PF-2-K/Fru-2,6-P2ase, was observed after starvation and refeeding. In contrast to results previously described for rat liver, the decrease in immunodetectable protein and kinase activity caused by starvation was associated in the teleostean fish to a decrease in mRNA levels.     

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.     

Sucrose metabolism in developing endosperm of immature wheat (Triticum aestivum L.) grain

With a view to investigating the role of the enzyme pyrophosphate-fructose-6-phosphate-1-phosphotransferase (PFP) in sucrose breakdown in developing endosperm of wheat grain, the activity of PFP and related enzymes such as phosphofructokinase (PFK), fructose-6-bisphosphatase (FBPase), fructose-6-phosphate-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (F2, 6-P2ase) and the contents of the various intermediates of the pathway serving either the substrate or the effectors of these enzymes such as glu-6-P,glu-1-P,fru-6-P,fru-1,6-P2,DHAP,G3P, UDP-glucose, ADP-glucose, Pi,PPi and fru-2,6-P2 have been determined at 5 days intervals starting from day-5 after anthesis until day-40 after anthesis. These enzymes except PFK-2 had their peak activity at day-25 after anthesis. The activity of PFP was several fold higher than that of PFK at each stage of grain development. PFK-2 exhibited the lowest activity. The various intermediates again had their maximum concentration either at day-20 or day-25 after anthesis. Among hexose phosphates studied, glu-6-P was present in highest concentration at each stage of grain development. The level of Pi was much higher than those of PPi and fru-2,6-P2. Similarly, concentration of UDP-glucose was higher than that of ADP-glucose. Based on these results, it is proposed that the major role of the enzyme PFP in developing wheat grain is to provide PPi for sucrose breakdown via sucrose synthase.     

Increases in hepatic fructose-2,6-bisphosphate level and fructose-6-phosphate,2-kinase activity in rats with ventromedial lesions of the hypothalamus

To investigate altered fructose-2,6-bisphosphate (fructose-2,6-P2) metabolism, we measured fructose-2,6-P2 levels and fructose-6-phosphate,2-kinase (fructose-6-P,2-kinase) activities in various tissues, including liver, kidney, heart, and skeletal muscle, of ventromedial hypothalamus (VMH)-lesioned rats during feeding and starvation. The plasma insulin level was 6 times or more higher in these rats than in the controls. The fructose-2,6-P2 level in liver was much greater in VMH-lesioned rats than in the controls: 15.1 +/- 2.2 nmol/g tissue versus 7.7 +/- 0.7 in the fed state, 5.3 +/- 1.1 versus 1.6 +/- 0.4 in the starved state. In kidney, heart, and skeletal muscle, fructose-2,6-P2 levels were not different between the two animal groups. The activity of hepatic fructose-6-P,2-kinase remained high after 20 h of starvation in VMH-lesioned rats, whereas it was decreased markedly in the controls. The hepatic concentration of fructose-6-phosphate was also high in VMH-lesioned rats. Both fructose-6-P,2-kinase activity and fructose-6-phosphate concentration in the liver of starved VMH-lesioned rats were comparable to those of control rats in fed conditions. These results indicate that the alteration of fructose-2,6-P2 metabolism is characteristic of liver in VMH-lesioned rats, and that the increase in hepatic fructose-2,6-P2 may activate hepatic glycolysis not only during feeding but also during starvation, leading to the enhanced lipogenesis in these obese rats.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Content of 8-oxodG in chromosomal DNA of Sparus aurata fish as biomarker of oxidative stress and environmental pollution

The 8-oxodG content has been measured in chromosomal DNA of gilthead seabream (Sparus aurata) by HPLC-EC. Susceptibility of different tissues to oxidative DNA damage was studied by exposing fish to model pollutants. Cu(II), paraquat (PQ) and malathion failed to promote DNA oxidation in liver, while dieldrin significantly increased the 8-oxodG content in this organ, but not in gills or blood. After PQ exposure, fish liver showed high levels of glucose-6-P dehydrogenase (G-6PDH) and GSSG reductase activities. The increased antioxidant status and the lack of a specific transport system could explain the lack of susceptibility of liver to DNA oxidative damage induced by PQ. Increased levels of 8-oxodG were detected in the gills of PQ-exposed fish after 8 and 24 h. In contrast, after 48 h exposed fish contained lower 8-oxodG levels than controls. The existence of a PQ transport system in this O2-rich organ and the lack of a significant increase in antioxidant defenses would explain the sensitivity of gills to DNA damage promoted by PQ. Elimination of this soluble chemical and the putative induction of DNA-repair enzymes specific for oxidative damages could explain the drop of 8-oxodG levels at longer times. Fish exposed to moderate levels of urban and industrial pollution showed significantly high 8-oxodG content in hepatic DNA. We conclude that 8-oxodG determination in chromosomal DNA by HPLC-EC is a potentially useful biomarker of environmental pollution, although its response is still somewhat lower than that of other well-established biomarkers of oxidative stress.     

Establishment of dominance relationships in gilthead sea bream Sparus aurata juveniles during feeding: effects on feeding behaviour, feed utilization and fish health

In gilthead sea bream Sparus aurata held in groups of two, five or 10 fish, social hierarchies were observed. Subordinate S. aurata were characterized by elevation of basal levels of plasma cortisol, together with a reduced immunological potential. Subordinate fish also showed lower feed intake, feed utilization and lower growth. Fatty acid composition was also affected by social status, with a lower content of saturated acids, oleic and eicosapentaenoic, in muscle and liver of fish considered as subordinate. Results show that social hierarchy acts as a stressor in S . aurata .     

Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata)

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.     

Regulation of Carbon Partitioning in Source and Sink Leaf Parts in Sweet Pepper (Capsicum annuum L.) Plants : Role of Fructose 2,6-Bisphosphate

Area expansion rate, partitioning of photosynthetically fixed carbon, and levels of fructose 2,6-bisphosphate (fru-2,6-P₂) were determined in individual parts of developing leaves of sweet pepper (Capsicum annuum L.). The base was rapidly expanding and allocated less carbon to sucrose synthesis in comparison to the leaf tip, where expansion had almost stopped. The change in leaf expansion rate and carbon partitioning happened gradually. During day time levels of fru-2,6-P₂ were consistently higher in the leaf base than in the leaf tip. Leaf expansion rate and carbon partitioning were closely related to day time levels of fru-2,6-P₂, suggesting that fru-2,6-P₂ is an important factor in adjustment of metabolism during sink-to-source transition of leaf tissue. The levels of fru-2,6-P₂ changed markedly after a dark-to-light transition in the leaf base, but not in the leaf tip, suggesting that regulatory systems based on fru-2,6-P₂ are different in sink and source leaf tissue. During the period upon dark-to-light transition the variations in level of fru-2,6-P₂ did not show a close correlation to changes in the carbon partitioning, until the metabolism had reached a steady state.     

Chronic toxicity of ammonia to juvenile gilthead seabream Sparus aurata and related histopathological effects

Effect of 20 days exposure of juvenile gilthead seabream ( Sparus aurata ) to elevated levels of ammonia on growth and survival was examined in a continuous flow system. Suppressed growth and reduced survival were observed at concentrations of 8.2 and 13 mg l⁻¹ total ammonia-N (0.5 and 0.7 mg l⁻¹ un-ionized ammonia-N, respectively) and higher. The maximum acceptable toxic concentration (MATC) for growth was between 4.8 to 8.2 mg l⁻¹ total ammonia-N (0.3 and 0.5 mg l⁻¹ un-ionized ammonia-N, respectively). Fish exposed to high ammonia levels (13 mg l⁻¹ total ammonia-N, 0.7 mg l⁻¹ un-ionized ammonia-N) displayed clear signs of liver pathology. Existing evidence suggests that S. aurata is less sensitive to ammonia than other reported marine and freshwater fish.
Under certain conditions ammonia concentration in the intensive fish ponds in Eilat may exceed the no observed effect concentration for S. aurata .     

Lipid and fatty acid composition of female gilthead seabream during their reproductive cycle: effects of a diet lacking n-3 HUFA

In female sea bream Sparus aurata fed a control diet (C), ovarian levels of neutral lipids (NL) and polar lipids (PL) remained constant between November and March, while a decrease in NL content was observed in liver and muscle. In the same period, liver PL content increased, while no changes were observed in muscle. Between March and June ovarian NL and PL showed a strong decrease, while NL remained constant in liver and muscle. When fish were fed a diet lacking in n-3 highly unsaturated fatty acids, n-3 HUFA (D), the pattern observed was similar to that found in the fish fed diet C, with the exception of liver NL, which increased between March and June. In general, the changes in fatty acid content, in both groups of fish, were highly influenced by the diet given to the broodstock, although these effects were greater on ovarian NL and PL than on liver and muscle lipids. Despite the fact that gilthead seabream females continue feeding during the spawning season, they probably make use of their liver and muscle reserves during the gonadal maturation process. Furthermore, the fatty acid composition of the broodstock diet was reflected in the body composition, especially in the ovaries.     

Proteins from fish eggs that protect DNA from acid precipitation and inhibit DNA synthesis

We partially characterized proteins that inhibit DNA acid precipitation from various fish eggs (Sparus aurata, Dicentrarchus labrax, Mugil cephalus and Zeus faber). The active proteins were purified by acetone fractionation. The activity was found to be heat resistant. Of bivalent cations tested only Co(2+) and Cu(2+) exerted a profound promoting effect in the activity from all fish. The protein fraction from Sparus aurata inhibited DNA synthesis in PCR performed by different DNA polymerases. The possible role of DNA protective proteins in fish egg physiology is discussed.     

Lack of consistency between the trophic interrelationships of five sparid species in two adjacent central Mediterranean coastal lagoons

Both interspecific and intraspecific inter-lagoon differences, between the central Mediterranean coastal lakes of Fogliano and Caprolace, in the trophic patterns of the examined species Diplodus annularis, Diplodus puntazza, Diplodus sargus, Lithognathus mormyrus and Sparus aurata were revealed by means of correspondence analysis. The most evident inter-lagoon qualitative and quantitative diet changes were observed in S. aurata . Feeding similarities among species were synthesized for each lake by means of multidimensional scaling. Trophic interrelationships among sparid fish were different in the two lagoons, as a direct consequence of diet variations in each species. Such differences are interpreted as possibly reflecting differences in the overall structure of the benthic communities. Different feeding models could be identified, each corresponding to a peculiar adaptive strategy for the consumption of lagoon benthic resources. The use of benthophagous fish feeding habits as a tool for the monitoring of the structure and the dynamics of coastal lagoon communities is discussed.     

Molecular cloning of hepatic glucose-6-phosphatase catalytic subunit from gilthead sea bream (Sparus aurata): response of its mRNA levels and glucokinase expression to refeeding and diet composition

To examine the relationship between structure and function of glucose-6-phosphatase (G6Pase) in fish, we undertook molecular cloning and modulation of G6Pase expression by starvation and refeeding on diets with different nutrient composition in the liver of the carnivorous fish, Sparus aurata. A cDNA encoding the full-length G6Pase catalytic subunit from the liver of S. aurata was isolated. This cDNA encodes a 350-amino acid protein, with low homology to the mammalian G6Pase, although it contains most of the key residues required for catalysis. Based on hydrophobicity and membrane structure prediction, we propose a model containing nine-transmembrane regions for S. aurata G6Pase. Northern blots showed that refeeding after a prolonged starvation rapidly reverses the glucose/glucose-6-phosphate substrate cycle flux in the fish liver through decreased G6Pase expression and strong glucokinase (GK) induction. The effect of refeeding different diets on G6Pase and GK expression, indicated that hepatic intermediary metabolism of fish fed diets with low protein/high carbohydrate diets is impelled towards utilization of dietary carbohydrates, by means of modulation of GK mRNA levels rather than G6Pase expression. These findings challenge the role attributed to dysregulation of G6Pase or GK expression in the low ability of carnivorous fish to metabolise glucose.     

The effect of dietary intake of vitamins C and E on the stress response of gilthead seabream (Sparus aurata L.)

High dietary doses of the antioxidant vitamins C and E were administered to gilthead seabream (Sparus aurata L.) in an attempt to reduce the stress response in specimens exposed to a multiple stress situation. Fish were fed four different diets for 6 weeks: a commercial feed containing 0.1g vitamin C and 0.1g vitamin E kg(-1) acted as control diet, while experimental diets consisted of the same feed supplemented with 3g vitamin C kg(-1), 1.2g vitamin E kg(-1) or both 3g vitamin C and 1.2g vitamin E kg(-1). After 2, 4 and 6 weeks fish were exposed to stressors typical of aquacultural practices, and serum cortisol levels, complement activity (measured by the alternative pathway), blood glucose level and respiratory burst activity of head-kidney leucocytes were evaluated. The results showed that all stress-induced increases in blood glucose concentration were lower in fish fed the vitamin C and/or E-supplemented diet than in fish fed the control diet after 2 weeks of treatment, although no other differences were found at the rest of the times. Cortisol levels increased in stressed fish and did not suffer depletion as a consequence of administering vitamins C and/or E as a supplement. The natural haemolytic complement activity was not affected by the stressors but enhanced in specimens fed vitamin-supplemented diets at week 6. The respiratory burst activity was depressed by the stressors in fish fed the control diet, although only after 6 weeks of treatment were the differences statistically significant. These results suggest that vitamins C and E are involved in the hypothalamic-sympathetic-chromaffin cell axis and also interfere in tertiary stress responses such as immunodepression, where they protect the leucocyte functions.