Identification of morin and other compounds as inhibitors of the malic enzyme of Mucor circinelloides in vitro but not in vivo

Of 13 compounds tested, 11 inhibited malic enzyme activity in Mucor circinelloides, to some degree, at 5 mM. Four of these inhibitors (tartronic acid, morin, catechin and 2,5-dihydroxybenzoic acid) were studied further. Tartronic acid, morin and catechin were competitive inhibitors of malic enzyme (with respect to malate), with apparent K_i values of 0.04 mM, 5 μM and 0.6 mM, respectively. 2,5-Dihydroxybenzoic acid was a non-competitive inhibitor, with respect to malate, and had an apparent K_i value of 0.8 mM. Morin and tartronic acid did not inhibit any other NADPH-generating enzyme studied, although both inhibited malate dehydrogenase. The inhibitory actions of catechin and 2,5-dihydroxybenzoic acid were less specific. All four compounds inhibited malic enzyme, to some extent, when included in the culture medium. This inhibition was not as great as in vitro however and was insufficient to have an effect on lipid metabolism in M. circinelloides. This revised version was published online in July 2006 with corrections to the Cover Date.     

Isolation and biochemical characterization of grape malic enzyme

Malic enzyme (ME=L-malate: NADP oxidoreductase; E.C. 1.1.1.40) was extracted by Triton X-100-induced resolubilization of enzyme proteins which denaturize spontaneously upon homogenization of grape berry material. The purification procedure included fractionating with (NH₄)₂SO₄, preparative IEF, and Sephadex G-100 chromatography. ME was identified by TLC of the radioactive product after supplementing the assay mixture with [¹⁴C]malate. Cofactor dependence, pH-optimum and affinities for substrates and cosubstrates were determined. Enzymic pI was found to be 5.8, the Hill coefficients range from 1 to 3. In malate decarboxylating direction at pH 7.4, grape ME displayed positive cooperativity toward the substrate, the curve approaching normal Michaelis-Menten-kinetics at pH 7.0. Substituting Mn²⁺ for Mg²⁺ not only increased maximal turnover rates, but also enzymic affinity for malate. These features were considered indicative of the regulatory properties of the enzyme. Their relevance for grape malate metabolism and fruit ripening is discussed.Abbreviations EDTA ethylenediaminetetraacetic acid - IFF isoelectric focusing - MDH malate dehydrogenase - ME malic enzyme - OAA oxaloacetic acid - PAG polyacrylamide gel - TCA trichloroacetic acid - TLC thin layer chromatography     

Photosynthetic CO(2) Fixation Products and Activities of Enzymes Related to Photosynthesis in Bermudagrass and Other Plants

After a 5-second exposure of illuminated bermudagrass (Cynodon dactylon L. var. `Coastal') leaves to ¹⁴CO₂, 84% of the incorporated ¹⁴C was recovered as aspartate and malate. After transfer from ¹⁴CO₂-air to ¹²CO₂-air under continuous illumination, total radioactivity decreased in aspartate, increased in 3-phosphoglyceric acid and alanine, and remained relatively constant in malate. Carbon atom 1 of alanine was labeled predominantly, which was interpreted to indicate that alanine was derived from 3-phosphoglyceric acid. The activity of phosphoenolpyruvate carboxylase, alkaline pyrophosphatase, adenylate kinase, pyruvate-phosphate dikinase, and malic enzyme in bermudagrass leaf extracts was distinctly higher than those in fescue (Festuca arundinacea Schreb.), a reductive pentose phosphate cycle plant. Assays of malic enzyme activity indicated that the decarboxylation of malate was favored. Both malic enzyme and NADP⁺-specific malic dehydrogenase activity were low in bermudagrass compared to sugarcane (Saccharum officinarum L.). The activities of NAD⁺-specific malic dehydrogenase and acidic pyrophosphatase in leaf extracts were similar among the plant species examined, irrespective of the predominant cycle of photosynthesis. Ribulose-1, 5-diphosphate carboxylase in C₄-dicarboxylic acid cycle plant leaf extracts was about 60%, on a chlorophyll basis, of that in reductive pentose phosphate cycle plants.     

Changes in oxidative properties of Kalanchoe blossfeldiana leaf mitochondria during development of Crassulacean acid metabolism

Kalanchoe blossfeldiana plants grown under long days (16 h light) exhibit a C₃-type photosynthetic metabolism. Switching to short days (9 h light) leads to a gradual development of Crassulacean acid metabolism (CAM). Under the latter conditions, dark CO₂ fixation produces large amounts of malate. During the first hours of the day, malate is rapidly decarboxylated into pyruvate through the action of a cytosolic NADP⁺-or a mitochondrial NAD⁺-dependent malic enzyme. Mitochondria were isolated from leaves of plants grown under long days or after treatment by an increasing number of short days. Tricarboxylic acid cycle intermediates as well as exogenous NADH and NADPH were readily oxidized by mitochondria isolated from the two types of plants. Glycine, known to be oxidized by C₃-plant mitochondria, was still oxidized after CAM establishment. The experiments showed a marked parallelism in the increase of CAM level and the increase in substrate-oxidation capacity of the isolated mitochondria, particularly the capacity to oxidize malate in the presence of cyanide. These simultaneous variations in CAM level and in mitochondrial properties indicate that the mitochondrial NAD⁺-malic enzyme could account at least for a part of the oxidation of malate. The studies of whole-leaf respiration establish that mitochondria are implicated in malate degradation in vivo. Moreover, an increase in cyanide resistance of the leaf respiration has been observed during the first daylight hours, when malate was oxidized to pyruvate by cytosolic and mitochondrial malic enzymes.Abbreviations CAM Crassulacean acid metabolism - MDH malate dehydrogenase - ME malic enzyme     

Cyclic AMP-dependent and -independent protein kinases in HeLa cells

Malate saturation isotherms for the NAD⁺ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate^2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg²⁺ or malate^2? may bind first but the fact that the K_m's for both Mg²⁺ and malate^2? are smaller than the respective K_i's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD⁺ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved.     

Properties of NADP-malic enzyme from glumes of developing wheat grains

Properties of partially purified NADP-malic enzyme (EC 1.1.1.40) from glumes of developing wheat grains were examined. The pH optimum for enzyme activity was influenced by malate and shifted from 7.3 to 7.6 when the concentration of malate was increased from 2 to 10 mM. The K_m values, at pH 7.3, for various substrates were: malate, 0.76 mM; NADP, 20 μM and Mn²⁺, 0.06 mM. The requirement of Mn²⁺ cation for enzyme activity could be partially replaced by Mg²⁺ or Co²⁺. Mn²⁺ dependent enzyme activity was inhibited by Pb²⁺, Ni²⁺, Hg²⁺, Zn²⁺, Cd²⁺, Al³⁺ and Fe³⁺. During the reaction, substrate molecules (malate and NADP) reacted with enzyme sequentially. Activity of malic enzyme was inhibited by products of the reaction viz pyruvate, HCO₃^? and NADPH₂. At a limiting fixed concentration of NADP, these products induced a positive cooperative response to increasing concentrations of malate.     

The physiological role of malic enzyme in grape ripening

The high specificity of malic enzyme (ME; EC 1.1.1.40) from grape berries (Vitis vinifera L.) for the naturally occurring l-enantiomer of malic acid, its very selective C₄-decarboxylation, and certain allosteric properties, reported previously, favour the conjecture of a regulatory function of ME in fruit malic acid degradation. On the other hand, high ME activity was detected even during the acid-accumulating phase of berry development. Also, the in vitro reversibility of the reaction supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate, notably high CO₂/HCO ₃ ^- and NADPH/NADP ratios. However, a very limited incorporation of ¹⁴C into malate and the uniform labeling pattern of the dicarboxylic acid after administration of [U-¹⁴C] alanine to grape berries before and after the onset of ripening, indicate that the reverse reaction does not contribute essentially to grape malate synthesis. A regulatory mechanism mediating malic acid remetabolization on the basis of cosubstrate availability, comparable to the control of the hexose monophosphate shunt, is discussed.Abbreviation ME Malic enzyme (l-malate: NADP oxidoreductase)     

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO₂ via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. -Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.These data were presented in part at the meeting of the American Society for Neurochemistry in Richmond, Virginia, March 1993     

Purification and Characterization of NAD Malic Enzyme from Leaves of Eleusine coracana and Panicum dichotomiflorum

NAD malic enzyme (EC 1.1.1.39), which is involved in C₄ photosynthesis, was purified to electrophoretic homogeneity from leaves of Eleusine coracana and to near homogeneity from leaves of Panicum dichotomiflorum. The enzyme from each C₄ species was found to have only one type of subunit by SDS polyacrylamide gel electrophoresis. The M_r of subunits of the enzme from E. coracana and P. dichotommiflorum was 63 and 61 kilodaltons, respectively. The native Mr of the enzyme from each species was determined by gel filtration to be about 500 kilodaltons, indicating that the NAD malic enzyme from C₄ species is an octamer of identical subunits. The purified NAD malic enzyme from each C₄ species showed similar kinetic properties with respect to concentrations of malate and NAD; each had a requirement for Mn²⁺ and activation by fructose- 1,6-bisphosphate (FBP) or CoA. A cooperativity with respect to Mn²⁺ was apparent with both enzymes. The activator (FBP) did not change the Hill value but greatly decreased K_0.5 (the concentration giving half-maximal activity) for Mn²⁺. The enzyme from E. coracana showed a very low level of activity when NADP was used as substrate, but this activity was also stimulated by FBP. Significant differences between the enzymes from E. coracana and P. dichotomiflorum were observed in their responses to the activators and their immunochemical properties. The enzyme from E. coracana was largely dependent on the activators FBP or CoA, regardless of concentration of Mn²⁺. In contrast, the enzyme from P. dichotomiflorum showed significant activity in the absence of the activator, especially at high concentrations of Mn²⁺. Both immunodiffusion and immunoprecipitation, using antiserum raised against the purified NAD malic enzyme from E. coracana, revealed partial antigenic differences between the enzymes from E. coracana and P. dichotomiflorum. The activity of the NAD malic enzyme from Amaranthus edulis, a typical NAD malic enzyme type C₄ dicot, was not inhibited by the antiserum raised against the NAD malic enzyme from E. coracana.     

Enzymes of nitrogen assimilation in maize roots

The intracellular distribution of enzymes involved in the Crassulacean acid metabolism (CAM) has been studied in Bryophyllum calycinum Salisb. and Crassula lycopodioides Lam. After separation of cell organelles by isopycnic centrifugation, enzymes of the Crassulacean acid metabolism were found in the following cell fractions: Phosphoenolpyruvate carboxylase in the chloroplasts; NAD-dependent malate dehydrogenase in the mitochondria and in the supernatant; NADP-dependent malate dehydrogenase and phosphoenolpyruvate carboxykinase in the chloroplasts; NADP-dependent malic enzyme in the supernatant and to a minor extent in the chloroplasts; NAD-dependent malic enzyme in the supernatant and to some degree in the mitochondria; and pyruvate; orthophosphate dikinase in the chloroplasts. The activity of the NAD-dependent malate dehydrogenase was due to three isoenzymes separated by (NH₄)₂SO₄ gradient solubilization. These isoenzymes represented 17, 78, and 5% of the activity recovered, respectively, in the order of elution. The isoenzyme eluting first was associated with the mitochondria and the second isoenzyme was of cytosolic origin, while the intracellular location of the third isoenzyme was probably the peroxisome. Based on these findings, the metabolic path of Crassulacean acid metabolism within cells of CAM plants is discussed. New address: Institut für Pflanzenphysiologie und Zellbiologie, Freie Universität Berlin, Königin-Luise-Straße 12-16a. D-1000 Berlin 33     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Properties of leaf NAD malic enzyme from plants with C4 pathway photosynthesis

C₄ acid decarboxylation in one group of C₄-pathway species is mediated by an NAD malic enzyme. This paper reports on the partial purification and properties of this enzyme from three species of this group, Atriplex spongiosa, Amaranthus edulis, and Panicum miliaceum. Depending upon the conditions, the Atriplex spongiosa enzyme was 5–30% as active with NADP compared with NAD but the enzyme from the other species was specific for NAD. The enzyme from each species had an absolute requirement for Mn²⁺ that could not be replaced by Mg²⁺, and activity was increased several fold by low concentrations of either CoA or acetyl CoA. For the enzyme from Atriplex spongiosa and Amaranthus edulis, there was cooperativity for malate binding and the activators CoA and acetyl CoA functioned to increase the affinity of malate for the enzyme. The Hill coefficients for malate binding were approximately 2 and 4, respectively. However, with the enzyme from Panicum miliaceum, cooperative binding of malate was not apparent and activators operated by increasing V rather than the affinity for malate. Bicarbonate inhibited the enzyme from Atriplex spongiosa and Amaranthus edulis and its effect was inversely related to the concentrations of malate, NAD, and activators. The possible significance of these various allosteric effects on the regulation of the enzyme in vivo is discussed. Reactant concentrations and other conditions required for maximum activity are reported.     

Regulatory properties of malic enzyme in the oleaginous yeast, Yarrowia lipolytica, and its non-involvement in lipid accumulation

Malic enzyme (EC 1.1.1.40) converts l-malate to pyruvate and CO₂ providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD⁺ as the primary cofactor. K_m values for NAD⁺ and NADP⁺ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP⁺- but not NAD⁺-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP⁺-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.     

Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation

A sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD⁺ or NADP⁺. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP⁺. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD⁺. Relative to nonproliferating cells, NAD⁺-linked malic enzyme activity was slightly reduced and the NADP⁺-linked activity was unchanged in the particulate fraction of serum-stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two-fold increase in the ratio of NAD(P)⁺-linked malic enzyme to NAD⁺-linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP⁺-dependent malic enzyme activity and a decrease in NAD⁺-linked malate dehydrogenase indictated an increase in the ratio of pyruvate-producing to oxalacetate-producing malate oxidase activity in the cytosol of proliterating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.     

Characterization of a malate dehydrogenase in the cyanobacterium Coccochloris peniocystis

A malate dehydrogenase (MDH) was characterized from the cyanobacterium Coccochloris peniocystis. The enzyme was purified approximately 180-fold and had a molecular weight of about 90000. The enzyme had a pH optimum of pH 6.7 to 7.5; a K_m (malate) of 5.6 mM and K_ms for NAD and NADP of 24 M and 178 M, respectively, although similar V_max were obtained with either pyridine nucleotide. Enzyme activity was inhibited by ATP, citrate, oxalacetate, acetyl CoA and CoA. Enzyme assays with uniformly ¹⁴C-labelled malate caused no ¹⁴CO₂ release, indicating this MDH is not a malic enzyme. Electrophoresis and S-200 gel filtration of the partially purified enzyme indicated a single MDH was present in this preparation. A second, less abundant, MDH was present in crude extracts. The presence of MDH in this organism is consistent with the operation of a glyoxylate cycle which, in the absence of a TCA cycle, would provide organic acids required in secondary carbon metabolism. ATP inhibition of MDH may allow for light regulation of MDH activity since, in the light, oxaloacetic acid is generated by phosphoenolpyruvate carboxylase activity.Abbreviations MDH malate dehydrogenase - PEPcase phosphoenolpyruvate carboxylase - MOPS 3-[N-Morpholino] propane sulfonic acid - TRIS Tris(hydroxymethyl)-aminomethane - EDTA Disodium Ethylenadiamine Tetraacetate - MES 2[N-Morpholino]-ethane Sulfonic Acid - EPPS N-2-Hydroxyethylpiperazine Propane - MW Molecular weight - OAA Oxaloacetic acid     

Mercury ions inhibit photosynthetic electron transport at multiple sites in the cyanobacteriumSynechococcus 6301

Inhibition of electron transport activities in the spheroplasts ofSynechococcus 6301 by HgCl² is dependent on the concentration of mercury ions. The inhibition of whole chain electron transport activity occurs at low concentration of Hg²⁺ (6 ΜM@#@). This inhibition occurs mostly due to interaction of Hg²⁺ on plastocyanin. At an elevated concentration (24 ΜM@#@), mercury induces inhibition chiefly in photosystem II catalyzed electron transport. At this concentration it also alters both the absorption and emission characteristics of the phycocyanin. The photosystem I catalyzed electron transport was inhibited by 50% only at high concentrations (36 ΜM@#@) of HgCl₂. However, electron transport catalyzed by photosystems I and II from reduced duroquinone to methylviologen which involves intersystem electron transport is extremely sensitive to mercury (low concentration 6–9 ΜM) like that of whole chain assay indicating that the observed inhibition in whole chain electron transport at low concentrations is mostly contributed by the damage involving other intersystem electron transport carrier(s) like plastocyanin. Thus mercury ions depending on the concentration affects the electron transport at multiple sites in the spheroplasts ofSynechococcus.     

Malate decarboxylation in isolated mitochondria from the crassulacean acid metabolism plant Sedum praealtum

Mitochondria isolated from the Crassulacean acid metabolism plant Sedum praealtum were demonstrated to decarboxylate added malate at basal rates of 30–50 μmol mg^?1 original chlorophyll h^?1. The basal rate could be stimulated markedly by the addition of ADP, oxaloacetic acid, an uncoupler of oxidative phosphorylation, or NAD, with maximum rates of 70–100 μmol mg^?1 original chlorophyll h^?1 observed. These observed rates were high enough to account for a large proportion of the estimated rate of malate decarboxylation in vivo. The major products of malate oxidation by the mitochondria in most cases were found to be pyruvate and CO₂, indicating that malate oxidation in these mitochondria proceeds mainly through NAD malic enzyme rather than NAD malate dehydrogenase. Under conditions employed little of the pyruvate formed was further oxidized, suggesting a fate other than oxidation (conversion to starch) for this pyruvate. Malate decarboxylation by mitochondria and by partially purified NAD malic enzyme was markedly inhibited by NaHCO₃. A possible physiological role is suggested for this inhibition as a feedback control on the enzyme.     

Detection of an NAD+-dependent malic enzyme locus in the atlantic salmonSalmo salar and other salmonid fish

Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP⁺) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD⁺-preferring, MgCl₂-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the generaSalmo, Salvelinus, andOnchoryhncus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance inS. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms for_s MDH-3,4* and_m MEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.This work was supported by a contract to E. V. from Fisheries and Oceans Canada, St. John's, Newfoundland, and a postgraduate award to W. C. J. from the Department of Education for Northern Ireland.