Pyruvate Reductase Research Articles

Transcriptional profile of ovine oocytes matured under lipopolysaccharide treatment in vitro

Lipopolysaccharide (LPS) derived from gram negative bacteria cell wall is known to cause ruminal acidosis and/or infectious diseases such as metritis and mastitis which has a significant negative impact on the reproductive performance. This study aimed to investigate the effect of LPS on oocyte maturation and subsequent development in vitro. Ovine cumulus oocyte complexes (COCs) were matured in a medium supplemented with 0 (control), 0.01, 0.1, 1 and 10 μg/mL LPS. Nuclear maturation, cleavage and blastocyst rate, mitochondrial membrane potential (ΔΨm), intracellular reactive oxygen species (ROS) content and changes to the transcript abundance were evaluated. In case of the maturation rate, the percentage of oocytes reaching the MII stage was lower following exposure to 10 μg/mL LPS in comparison to the control group (P < 0.05). Moreover, the blastocyst rate decreased in case of 1 and 10 μg/mL LPS when compared to the control group (P < 0.05). ROS overproduction accompanied by a decreased ΔΨm were recorded in LPS treated oocytes in comparison to the control group (P < 0.05). The 3′ tag digital gene expression profiling method revealed that 7887 genes were expressed while only seven genes exhibited changes in the transcript abundance following exposure to LPS. Tripartite motif containing 25 (TRIM25), Tripartite motif containing 26 (TRIM26), Zona Pellucida glycoprotein 3 (ZP3), Family with sequence similarity 50-member A (FAM50A), Glyoxalate and hydroxy pyruvate reductase (GRHPR), NADH ubiquinase oxireductase subunit A8 (NDUFA8) were down-regulated (P < 0.05), while only Centrin 3 (CETN3) was up-regulated (P < 0.05). Our findings show that LPS has undesirable effects on the maturation competence of ovine oocytes and subsequent embryo development. In addition, the transcriptomic profiling results may shed more light on the molecular mechanisms of LPS-induced infertility in ruminants.

An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera

Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (Vitis vinifera) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a V. vinifera aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer–specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in Arabidopsis thaliana. Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer–specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer–specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants.

Differential growth, nutrition, physiology, and gene expression in Melissa officinalis mediated by zinc oxide and elemental selenium nanoparticles.

Regarding the rapid progress in the production and consumption of nanobased products, this research considered the behavior of Melissa officinalis toward zinc oxide nanoparticles (nZnO), nanoelemental selenium (nSe), and bulk counterparts. Seedlings were irrigated with nutrient solution containing different doses of nZnO (0, 100, and 300 mg l-1) and/or nSe (0, 10, and 50 mg l-1). The supplements made changes in growth and morphological indexes in both shoot and roots. The mixed treatments of nSe10 and nZnO led to a drastic increase in biomass, activation of lateral buds, and stimulations in the development of lateral roots. However, the nSe50 reduced plants' growth (45.5%) and caused severe toxicity which was basically lower than the bulk. Furthermore, the nSe and nZnO improved K, Fe, and Zn concentrations in leaves and roots, except for seedlings exposed to nSe50 or BSe50. Moreover, the nSe and nZnO supplementations in a dose-dependent manner caused changes in leaf non-protein thiols (mean = 77%), leaf ascorbate content (mean = 65%), and soluble phenols in roots (mean = 28%) and leaves (mean = 61%). In addition, exposure to nZnO and/or nSe drastically induced the expression of rosmarinic acid synthase (RAS) and Hydroxy phenyl pyruvate reductase (HPPR) genes. Besides, the nSe, nZnO, or bulk counterparts influenced the activities of nitrate reductase in leaves and peroxidase in roots, depending on dose factor and compound form. The comparative physiological and molecular evidence on phytotoxicity and potential advantages of nSe, nZnO, and their bulk counterparts were served as a theoretical basis to be exploited in food, agricultural, and pharmaceutical industries.

Phenyllactic Acid: A Potential Antimicrobial Compound in Lactic acid Bacteria

3-Phenyllactic acid (PhLA) is a broad spectrum antimicrobial compound active against bacteria and fungi. PhLA exists in two chiral isomers, L-PhLA and D-PhLA which show antimicrobial activity. D-PhLA shows more antimicrobial activity than L-PhLA and hence it is receiving great attention as food and feed additive in place antibiotics which control the microbial contamination and thereby increase the shelf life of food and food ingredients. This review summarizes the recent developments on the resources, detection and analysis and antimicrobial activity of PhLA. In addition, the article presents the recent studies of the key enzymes such as lactate dehydrogenases and pyruvate reductases involved in PhLA synthesis. The metabolic pathway and regulation of PLA synthesis in lactic acid bacteria (LAB) along with its high level production are also discussed.

Crystallization and structural analysis of 2-hydroxyacid dehydrogenase from Ketogulonicigenium vulgare

L-2-Hydroxyacid dehydrogenase (HDH) from Ketogulonicigenium vulgare Y25 was cloned and overexpressed in Escherichia coli. The protein was purified and crystallized by the sitting-drop vapour-diffusion method with polyethylene glycol 3350 as precipitant. The crystal structure of HDH was determined at 1.64Å resolution using the molecular replacement method with the crystal structure of hydroxyl (phenyl) pyruvate reductase from Coleus blumei Benth as the search model. The overall structure of HDH was similar to that of hydroxyl(phenyl)pyruvate reductase, consisting of two compact domains separated by a deep active cleft. The most significant structural divergence is located around the pocket gate comprising residues A210, T211 and R212, which is located on top of the catalytic triad.

Structure and substrate docking of a hydroxy(phenyl)pyruvate reductase from the higher plantColeus blumeiBenth.

Hydroxy(phenyl)pyruvate reductase [H(P)PR] belongs to the family of D-isomer-specific 2-hydroxyacid dehydrogenases and catalyzes the reduction of hydroxyphenylpyruvates as well as hydroxypyruvate and pyruvate to the corresponding lactates. Other non-aromatic substrates are also accepted. NADPH is the preferred cosubstrate. The crystal structure of the enzyme from Coleus blumei (Lamiaceae) has been determined at 1.47 A resolution. In addition to the apoenzyme, the structure of a complex with NADP(+) was determined at a resolution of 2.2 A. H(P)PR is a dimer with a molecular mass of 34 113 Da per subunit. The structure is similar to those of other members of the enzyme family and consists of two domains separated by a deep catalytic cleft. To gain insights into substrate binding, several compounds were docked into the cosubstrate complex structure using the program AutoDock. The results show two possible binding modes with similar docking energy. However, only binding mode A provides the necessary environment in the active centre for hydride and proton transfer during reduction, leading to the formation of the (R)-enantiomer of lactate and/or hydroxyphenyllactate.

Energy metabolism in the tropical abalone, Haliotis asinina Linné: Comparisons with temperate abalone species

The abalone, Haliotis asinina, is a large, highly active tropical abalone that feeds at night on shallow coral reefs where oxygen levels of the water may be low and the animals can be exposed to air. It is capable of more prolonged and rapid exercise than has been reported for temperate abalone. These unusual behaviours raised the question of whether H. asinina possesses enhanced capacities for aerobic or anaerobic metabolism. The blood oxygen transport system of H. asinina resembles that of temperate abalone in terms of a large hemolymph volume, similar hemocyanin concentrations, and in most hemocyanin oxygen binding properties; however, absence of a Root effect appears confined to hemocyanin from H. asinina and may assist oxygen uptake when hemolymph pH falls during exercise or environmental hypoxia. During exposure to air, H. asinina reduces oxygen uptake by at least 20-fold relative to animals at rest in aerated seawater, and there is no significant ATP production from anaerobic glycolysis or phosphagen hydrolysis in the foot or adductor muscles. This slowing of metabolism may contribute to survival at lower water oxygen levels than normally encountered by most temperate abalone. While crawling speeds of H. asinina in water are not exceptionally high, an aerobic expansibility of 5.5-fold at speeds less than 20% of maximum is more than 2.7-fold greater than reported for several temperate abalone. The high aerobic expansibility also supports the enhanced frequency and duration of flipping behaviour without recourse to the additional inputs from anaerobic glycolysis required by other abalone. Metabolic profiles of foot and adductor muscles of H. asinina are similar to those of other abalone. Common features are low activities of enzymes unique to aerobic ATP production, relatively high activities of arginine kinase, tauropine and d-lactate dehydrogenase as the predominant pyruvate reductases, and low intracellular pH buffering capacities. It is concluded that the exceptional abilities of H. asinina for prolonged and rapid exercise are supported by higher rates of aerobic metabolism rather than any enhanced capacity for anaerobic muscle work. It is unexpected, and instructive, that the exceptional aerobic expansibility is not apparent in obvious adjustments of the blood oxygen delivery system or muscle properties associated with aerobic ATP production. The absence of a hemocyanin Root effect, and the extent to which both aerobic and anaerobic metabolism can be reduced may be special features that assist prolonged exercise and survival of H. asinina when environmental oxygen becomes limiting.

Thermal constraints on glycolytic metabolism in the New Zealand abalone, Haliotis iris: the role of tauropine dehydrogenase

Black‐foot abalone, Haliotis iris, were sampled from two populations in warm northern waters, and from two in colder southern waters. Abalone muscle is characterised by high activity of the glycolytic pyruvate reductase enzyme, tauropine dehydrogenase (TDH). Adductor muscle TDH was profiled for thermostability and activity to test the hypothesis that the enzyme may show adaptation in titre or kinetic characteristics reflecting thermal habitat. Temperature dependency of the apparent Michaelis‐Menten constant of TDH for pyruvate (appKmpyr) suggested eurythermal enzyme behaviour below 20°C, and compromised function at the higher temperatures of northern populations occurring in the summer months. Thermostability profiles and enzyme activities suggest TDH expression does not differ significantly among populations (P > 0.05), indicating that this locus shows no compensation for temperature. The optimal temperature for efficient TDH function, estimated from Vmax./appKmpyr, is close to 20°C. The possible thermal constraints on glycolytic metabolism in H. iris are discussed.

Glycolytic Enzymes in the Tissues of Three Species of Scallop (Bivalvia: Pectinidae)

The maximal activities of 15 enzymes were compared in the striated and smooth adductor muscle, mantle muscle, foot, digestive gland, testis, and ovary of three species of scallops Chlamys nobilis, Pecten albicans, and Patinopecten yessoensis. The patterns of glycolytic enzyme activities in these three species of scallop were similar. However, the maximal activities of phosphofructokinase and malate dehydrogenase in the striated adductor muscle and octopine dehydrogenase in the striated and smooth adductor muscle differ significantly between species. All enzymes except for hexokinase were detected at the highest activity in the adductor muscle. In the striated muscle octopine dehydrogenase activity was highest among pyruvate reductases, followed by other opine dehydrogenases, D-lactate dehydrogenase. and L-lactate dehydrogenase. In these scallops phosphoenolpyruvate carboxykinase activity was very low compared with pyruvate kinase. Gonads and digestive gland showed fairly high activities of malate dehvdrosenase. asoartate aminotransferase. and alanine aminotransferase.

In vitro production (IVP) of domestic cat embryos using a modified IVP bovine system

Lipopolysaccharide (LPS) derived from gram negative bacteria cell wall is known to cause ruminal acidosis and/or infectious diseases such as metritis and mastitis which has a significant negative impact on the reproductive performance. This study aimed to investigate the effect of LPS on oocyte maturation and subsequent development in vitro. Ovine cumulus oocyte complexes (COCs) were matured in a medium supplemented with 0 (control), 0.01, 0.1, 1 and 10 μg/mL LPS. Nuclear maturation, cleavage and blastocyst rate, mitochondrial membrane potential (ΔΨm), intracellular reactive oxygen species (ROS) content and changes to the transcript abundance were evaluated. In case of the maturation rate, the percentage of oocytes reaching the MII stage was lower following exposure to 10 μg/mL LPS in comparison to the control group (P < 0.05). Moreover, the blastocyst rate decreased in case of 1 and 10 μg/mL LPS when compared to the control group (P < 0.05). ROS overproduction accompanied by a decreased ΔΨm were recorded in LPS treated oocytes in comparison to the control group (P < 0.05). The 3′ tag digital gene expression profiling method revealed that 7887 genes were expressed while only seven genes exhibited changes in the transcript abundance following exposure to LPS. Tripartite motif containing 25 (TRIM25), Tripartite motif containing 26 (TRIM26), Zona Pellucida glycoprotein 3 (ZP3), Family with sequence similarity 50-member A (FAM50A), Glyoxalate and hydroxy pyruvate reductase (GRHPR), NADH ubiquinase oxireductase subunit A8 (NDUFA8) were down-regulated (P < 0.05), while only Centrin 3 (CETN3) was up-regulated (P < 0.05). Our findings show that LPS has undesirable effects on the maturation competence of ovine oocytes and subsequent embryo development. In addition, the transcriptomic profiling results may shed more light on the molecular mechanisms of LPS-induced infertility in ruminants.

Effect of Wave Action on Muscle Composition, Metabolites and Growth Indices in the New Zealand Abalone, Paua (Haliotis iris), with Implications for Harvesting and Aquaculture

Abalone from exposed locations subject to heavy wave action may incur greater metabolic costs and structural stress in muscles used for attachment than do abalone in aquaculture or from sheltered sites. Because foot and adductor muscle texture and flavor affect economic value, we sought evidence of differences attributable to wave action among populations of the commercial New Zealand abalone, Haliotis iris. Evidence of environmentally induced changes were not found for condition indices relating shell and soft body proportions; muscle composition, such as total protein, collagen or water content; muscle biochemistry, such as intracellular pH buffering capacity, adenylate content, homarine or inositol monophosphate (IMP) content; blood hemocyanin concentration and muscle RNA content indicative of protein synthesis and somatic growth rates. The major difference in muscles of animals from exposed sites were higher activities of the glycolytic pyruvate reductase enzyme, tauropine dehydrogenase, and lower glycogen content. The differences imply that abalone exposed to heavy wave action routinely depend on bouts of anaerobic muscle work associated with attachment and locomotion but that the absence of wave action in aquacultural practice may not adversely affect muscle properties.

Plant lactate dehydrogenase: NADH kinetics and inhibition by ATP

Lactate dehydrogenase (LDH), isolated from either turnip ( Brassica rapa, Cruciferae) or from leek ( Allium porrum, Liliaceae) shows normal Michaelis-Menten kinetics with NAD + in the lactate dehydrogenase direction, but non-hyperbolic kinetics with NADH in the pyruvate reductase direction. These kinetics are not ‘sigmoidal’ and appear consistently as two intersecting straight lines in reciprocal plots, representing a sharp decline in the effectiveness of NADH as a substrate in its lower concentration range. The overall apparent affinity for NADH decreases with decreasing pH. Competitive inhibition by ATP, which is much stronger than that characteristic of mammalian LDH, also displays kinetics that seem to be biphasic, but the K i value does not vary significantly with pH in the physiological range. The counter-inhibitory effect of Mg 2+ ions is shown to be due to the formation of non-inhibitory MgATP complexes. The dissociation of this is strongly pH dependent and this results in most of the ATP being complexed, and therefore non-inhibitory at pH 7.4 (the normal pH in plant cytosol) but largely uncomplexed and inhibitory at pH 6.4 (probably a lower cytosolic pH limit). These factors, and particularly the last, combine to inhibit the enzyme more strongly as the pH drops (as a result of lactate-terminating anaerobic glycolysis) and may act as an effective negative feed-back mechanism in plant cells, allowing a pH-activated switchover to ethanol-terminating glycolysis while preventing ‘overshoot’ into harmful acidosis. © 1997 Elsevier Science Ltd. All rights reserved

Both d- and l-specific Lactate Dehydrogenases Co-exist in Individual Cephalopods

Abstract Both d -lactate-specific and l -lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l -specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.

Adjunctive nodule enzyme activities associated with tripartite symbiosis in Astragalus cicer L.

Cicer milkvetch (Astragalus cicer L.) is a productive forage legume adapted to a wide range of soil types. It is a long‐lived, nonbloating rhizomatous perennial with tolerance for low soil fertility, unfavorable acid pH and salinity. It has much value for soil fertility improvement and soil erosion control. Objectives of this study were to determine soil fertility influencing growth, nodulation, cytosol reductases, and nitrogenase activity with effective mycorrhizal colonization. Significant increases in growth, nodulation, nitrogenase activity and seed yield resulted for mycorrhizal plants. Increases in nitrogenase levels were also congruent with increased nodulation for soil K amendments. Differences were not significant for N transformation enzyme activity levels for aspartate transaminase (AST), glutamate dehydrogenase (GDH), glutamine synthetase (GS), and glutamate‐oxoglutarate transaminase (GOGAT). However, highly significant differences were apparent for greater nonmycorrhizal nitrate reductase and cytosol nitrate N with an inverse higher cytosol pyruvate and thiosulfonate reductase (TSR) of mycorrhizal colonized plants. Definitive histological microscopy was highly correlated with both explicative enzyme cytochemistry and fluorescence parameters for nodule cytosol (reductase) enzyme transformations.

Factors Affecting Development of Peroxisomes and Glycolate Metabolism among Algae of Different Evolutionary Lines of the Prasinophyceae.

Leaf-type peroxisomes are not present in the primitive unicellular Prasinophycean line of algae but are present in the multicellular algae Mougeotia, Chara, and Nitella, which are in the one evolutionary line, Charophyceae, that led to higher plants. Processes related to glycolate metabolism that may have been modified or induced with the appearance of peroxisomes have been examined. The algal dissolved inorganic carbon-concentrating mechanism and alkalization of the medium during photosynthesis were not lost when peroxisomes appeared in the members of the Charophycean line of algae. Therefore, it is unlikely that lowering of the CO2 concentration in the environment was a major factor in the evolutionary appearance of peroxisomes. Multicellular Mougeotia, early members of the Charophycean line of algae, have peroxisomes, but they excrete excess glycolate into the medium. The cytosolic pyruvate reductase for D-lactate synthesis and the glycolate dehydrogenase activity almost disappeared when peroxisomal glycolate oxidase, which also oxidizes L-lactate, appeared. These biochemical changes do not indicate what caused the induction of leaf-type peroxisomes in this evolutionary line of algae. The oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and glycolate oxidase require about 200 to 400 [mu]M O2 for 0.5 Vmax. These high-O2-requiring steps in glycolate metabolism would have functioned faster with increasing atmospheric O2, which might have been the causative factor in the induction of peroxisomes.

A comparison of metabolic stress during air exposure in two species of New Zealand abalone, Haliotis iris and Haliotis australis: implications for the handling and shipping of live animals

Anaerobic metabolism in New Zealand blackfoot and yellowfoot abalone, Haliotis iris and Haliotis australis, is supported by specific pyruvate reductase enzymes and a moderate intracellular pH buffering capacity. Lactate and tauropine increased, while ATP and energy charge decreased in both species after 24 h air exposure. Responses were most marked in yellowfoot abalone, though allometric analysis revealed the simple explanation that the difference was due to size alone. Thus, small abalone accumulate higher metabolite concentrations and have a significantly lowered energetic status compared with larger specimens similarly exposed to air. These stress responses, which may ultimately affect both meat quality and survival during transport, are evaluated in the context of proposals to culture “cocktail” sized abalone.

Strategies of anaerobiosis in New Zealand infaunal bivalves: Adaptations to environmental and functional hypoxia

Abstract Under hypoxic environmental conditions, slowly burrowing bivalves such as the pipi (Paphies australis) and cockle (Austrovenus stutchburyi) support anaerobic energy production through the pyruvate reductase enzymes strombine dehydro‐genase and alanopine dehydrogenase. Rapidly digging surf clams such as the tuatua (P. sub‐triangulatum) and trough shell (Mactra discors) do not fare as well at low oxygen levels, but perform better under the functional anaerobiosis generated by burrowing, and energy production is supported by octopine dehydrogenase. Muscle buffering capacity is higher in rapidly burrowing species and is correlated with total pyruvate reductase activity. Anaerobic pathways in P. subtriangulatum led to both D‐lactate and octopine accumulation during environmental hypoxia, but only octopine accumulated during burrowing. Bivalves adapted to environmental hypoxia maintained high adenylate energy charge (AEC) under anaerobiosis, indicating a close matching of ATP production to consumption. AEC fell in P. subtriangulatum during environmental hypoxia. The significance of these findings is discussed within an ecological context, and in relation to the storage of live clams for the seafood industry.

An unusual fructose 1,6-bisphosphate-activated lactate dehydrogenase from the ascidian Pyura stolonifera

Lactate dehydrogenase (LDH) was purified from the siphon muscle of the intertidal ascidian Pyura stolonifera. The enzyme is unique among chordate LDHs but resembles some bacterial and platyhelminth LDHs in being activated by fructose 1,6-bisphosphate (FBP). Concentrations of FBP in the range 5μM to 0.5 mM increase Vmax of the pyruvate reductase reaction by 130% to 210%, and decrease Km pyruvate 5 to 11 fold and Km NADH 2.5 to 5 fold. The enzyme is also activated by inorganic phosphate, but requires a 50 fold higher concentration to attain the maximum activation achieved by 0.5 mM FBP. Of a range of metabolites tested, including other glycolytic sugar phosphates, only FBP and inorganic phosphate activated the enzyme. FBP activation was not observed with 16 representative vertebrate LDH homotetramers, but did occur to a limited extent with LDH from an echinoderm. LDH was the only pyruvate reductase enzyme detected in P. stolonifera siphon muscle, and its activity was much greater than that of phosphorylase or phosphofructokinase. The LDH reaction is utilized by P. Stolonifera during prolonged siphon closure on exposure to air when lactate, but not succinate, accumulates in the siphon muscle. While the ascidian enzyme provides the first example of a FBP activated LDH from a chordate, it remains to be determined if this unusual property has any role in metabolic regulation.

Lactate dehydrogenase, alanopine dehydrogenase and octopine dehydrogenase from the heart of Concholepas concholepas (Gastropoda: Muricidae)

Alanopine dehydrogenase, octopine dehydrogenase and lactate dehydrogenase activities were detected in the heart of C. concholepas. As determined by gel filtration on Sephadex G-100, the molecular weights ( M r) were 85,000, 42,000 and 52,000 for lactate dehydrogenase, alanopine dehydrogenase and octopine dehydrogenase, respectively. Substrate inhibition of the opine dehydrogenases was observed at high concentrations of both pyruvate and the corresponding amino acid (alanine or arginine). Moderate substrate inhibition of lactate dehydrogenase by pyruvate was observed; K m values for lactate and NAD + were unusually high. Alanoonine dehydrogenase was very specific for alanine and glycine was not a substrate for this enzyme. In addition to arginine, lysine was also a substrate for octopine dehydrogenase. Possible functions of the pyruvate reductases are discussed in connection with adaptation to anoxia and other regulatory processes in the heart of C. concholepas.

Are cell redox or lactate dehydrogenase kinetics responsible for the absence of gluconeogenesis from lactate in sea raven, hepatocytes?

Previous studies have reported very low rates of gluconeogenesis from lactate in sea raven (Hemitripterus americanus) hepatocytes compared to other teleosts studied. This study examines whether hepatic cell redox or lactate dehydrogenase (LDH) characteristics may explain this observation. Sea raven hepatic optimal LDH activities (pyruvate reductase direction) were more than 40 times less compared with rainbow trout liver values (40 vs 1914 μmol·min(-1)·g(-1) protein). The Km(lactate) was 9.24 and 0.86 mM for sea raven and trout hepatic LDH, but the Km(pyruvate) was similar between the two species (0.11 and 0.21 mM, respectively). These results suggested that sea raven liver LDH did not favour lactate use and was more indicative of the mammalian M-isozyme. Gel electrophoresis showed a predominant intermediate isozyme, with a small amount of the M-type LDH. Phosphoenolpyruvate carboxykinase (PEPCK) was localized to the mitochondrial compartment, while there was no apparent mitochondrial glutamate-oxaloacetate transaminase (GOT) activity. No in vitro lactate flux to glucose was found in untreated, 10 mM ethanol-treated, or 3 mM NH4Cl-treated sea raven hepatocytes, although CO2 production from lactate was decreased by ethanol and increased by NH4Cl. These results provide evidence that cell redox does not limit gluconeogenesis from lactate, while low activities and the kinetic characteristics of LDH may partially explain the low lactate gluconeogenesis reported in sea raven hepatocytes.