Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.     

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.Supported by U.S. Public Health Service grants 5F2 HD-35,531 and GM-09966.     

Effect of bicarbonate and oxaloacetate on malate oxidation by spinach leaf mitochondria

Mitochondria isolated from spinach leaves oxidized malate by both a NAD⁺-linked malic enzyme and malate dehydrogenase. In the presence of sodium arsenite the accumulation of oxaloacetate and pyruvate during malate oxidation was strongly dependent on the malate concentration, the pH in the reaction medium and the metabolic state condition.Bicarbonate, especially at alkaline pH, inhibited the decarboxylation of malate by the NAD⁺-linked malic enzyme in vitro and in vivo. Analysis of the reaction products showed that with 15 mM bicarbonate, spinach leaf mitochondria excreted almost exclusively oxaloacetate.The inhibition by oxaloacetate of malate oxidation by spinach leaf mitochondria was strongly dependent on malate concentration, the pH in the reaction medium and on the metabolic state condition.The data were interpreted as indicating that: (a) the concentration of oxaloacetate on both sides of the inner mitochondrial membrane governed the efflux and influx of oxaloacetate; (b) the NAD⁺/NADH ratio played an important role in regulating malate oxidation in plant mitochondria; (c) both enzymes (malate dehydrogenase and NAD⁺-linked malic enzyme) were competing at the level of the pyridine nucleotide pool, and (d) the NAD⁺-linked malic enzyme provided NADH for the reversal of the reaction catalyzed by the malate dehydrogenase.     

Enzymes of the tricarboxylic acid cycle in Obeliscoides cuniculi (Nematoda; Trichostrongylidae) during parasitic development

The specific activities of the enzymes of the tricarboxylic acid cycle; citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were determined in early fifth-stage, young and mature adult Obeliscoides cuniculi, the rabbit stomach worm. ∝-Ketoglutarate dehydrogenase activity could not be determined in any fraction. Fumarate reductase activity was found only in the mitochondrial fraction while all other enzymes, including an NADP-dependent malic enzyme were localized in the cytoplasm. Glutamate dehydrogenase, acid and alkaline phosphatase activities were also recorded. High levels of those enzymes acting in the “reversed” direction, i.e. MDH and fumarase relative to the enzymes of the “forward” direction, i.e. citrate synthase, aconitase and isocitrate dehydrogenase suggests that under anaerobic conditions a modified tricarboxylic acid cycle can operate. Some variations in specific activities were apparent as the worms matured but no qualitative differences were observed.     

Malate Oxidation in Plant Mitochondria via Malic Enzyme and the Cyanide-insensitive Electron Transport Pathway

Malate oxidation in plant mitochondria proceeds through the activities of two enzymes: a malate dehydrogenase and a NAD⁺-dependent malic enzyme. In cauliflower, mitochondria malate oxidation via malate dehydrogenase is rotenone- and cyanide-sensitive. Addition of exogenous NAD⁺ stimulates the oxidation of malate via malic enzyme and generates an electron flux that is both rotenone- and cyanide-insensitive. The same effects of exogenous NAD⁺ are also observed with highly cyanide-sensitive mitochondria from white potato tubers or with mitochondria from spinach leaves. Both enzymes are located in the matrix, but some experimental data also suggest that part of malate dehydrogenase activity is also present outside the matrix compartment (adsorbed cytosolic malate dehydrogenase?). It is concluded that malic enzyme and a specific pool of NAD⁺/NADH are connected to the cyanide-insensitive alternative pathway by a specific rotenone-insensitive NADH dehydrogenase located on the inner face of the inner membrane. Similarly, malate dehydrogenase and another specific pool of NAD⁺/NADH are connected to the cyanide- (and antimycin-) sensitive pathway by a rotenone-sensitive NADH dehydrogenase located on the inner face of the inner membrane. A general scheme of electron transport in plant mitochondria for the oxidation of malate and NADH can be given, assuming that different pools of ubiquinone act as a branch point between various dehydrogenases, the cyanide-sensitive cytochrome pathway and the cyanide-insensitive alternative pathway.     

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.     

Substrate channeling of NADH and binding of dehydrogenases to complex I

The binding of porcine heart mitochondrial malate dehydrogenase and beta-hydroxyacyl-CoA dehydrogenase to bovine heart NADH:ubiquinone oxidoreductase (complex I), but not that of bovine heart alpha-ketoglutarate dehydrogenase complex, is virtually abolished by 0.1 mM NADH. The malate dehydrogenase and beta-hydroxyacyl-CoA enzymes compete in part for the same binding site(s) on complex I as do the malate dehydrogenase and alpha-ketoglutarate dehydrogenase complex enzymes. Associations between mitochondrial malate dehydrogenase and bovine serum albumin were observed. Subtle convection artifacts in short-time centrifugation tests of enzyme association with the Beckman Airfuge are described. Substrate channeling of NADH from both the mitochondrial and cytoplasmic malate dehydrogenase isozymes to complex I and reduction of ubiquinone-1 were shown to occur in vitro by transient enzyme-enzyme complex formation. Excess apoenzyme causes little inhibition of the substrate channeling reaction with both malate dehydrogenase isozymes in spite of tighter equilibrium binding than the holoenzyme to complex I. This substrate channeling could, in principle, provide a dynamic microcompartmentation of mitochondrial NADH.     

Enzymes of carbohydrate metabolism ofCotugnia digonopora and their activity in the presence of anthelmintics,in vitro

Cotugnia digonopora, a fowl cycllophyllidean cestode, was found to possess most of the enzymes, associated with the glycolytic sequence and phosphoenolpyruvate branch point, in the cytosol fraction. Enzymes of malate metabolism were predominantly mitrochondrial. Anthelmintic agents inhibited hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, fumarate reductase, and malic enzyme. In intact worms this effect was significantly reduced. However, the activities of glycogen Phosphorylase and pyruvate kinase were significantly enhanced. Communication No. 4113 from CDRI, Lucknow.     

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.

     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Changes in enzyme pattern of ehrlich ascites tumor cells following serial cultivation in media with increased (hypertonic) NaCl content

Adaptation of Ehrlich ascites tumor cells to serial cultivation in media with progressively elevated (hypertonic) NaCl content (“high NaCl”-tolerant cells) has resulted in progressive increases of the cellular activities of NAD-dependent glycerol-3-phosohate dehydrogenase (EC 1.1.1.8), NAD-dependent malate dehydrogenase (EC 1.1.1.37), glutamate—oxalacetate transaminase (EC 2.6.1.1.), NAD(P)-dependent glutamate dehydrogenase (EC 1.4.1.3), NADP-dependent malate dehydrogenase (EC 1.1.1.40, “malic enzyme”) and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42). The activities of glutamate—pyruvate transaminase (EC 2.6.1.2.) and of glycolytic enzymes as phosphofructokinase (EC 2.7.1.11), glyceradehydephosphate dehydrogenase (EC 1.2.1.12) and lactate dehydrogenase (EC 1.1.1.27) were only slightly and not in progressive manner (in response to the progressive increase of the environmental NaCl concentration) affected. These changes are discussed with respect to a metabolic pattern of these “high NaCl”-tolerant cells which is compatible with increased energy requirements, especially for active cation transport. It is suggested that these increased cellular enzyme activitees reflect an increased transfer of reducing equivalents across mitochondrial membranes (via the “glycerophosphate cycle and the malate—aspartate shuttle”) and possibly a stimulated lipid metabolism. These alterations in the level of enzyme activities must be regarded as an adaptive cellular response to the “high NaCl” enviromment, since readaptation to growth in regular isotonic media resulted in a reversion to the enzyme pattern characteristic of the parent cells.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase     

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     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

The Oxidation of Malate by Isolated Turnip (Brassica napus L.) Mitochondria: II. THE MALATE-OXIDIZING ENZYMES, NUMBER AND LOCATION

The enzymes of malate oxidation in turnip mitochondria havebeen partially purified and some of their properties studied.Turnips contain a cytoplasmic malate dehydrogenase and two mito-chondrialmalate dehydrogenases. These are all distinct isoenzymes withdifferent immunblogical properties but similar molecular weights.The K_m for malate is relatively high (8.3 mM) in the mito-chondrialenzymes. One of the mitochondrial enzymes is located in thematrix while the other is membrane-bound but within the matrixcompartment. This latter enzyme, which retains its NAD and activitywhen submitochondrial particles are prepared, is responsiblefor the first phase of malate oxidation in submitochondrialparticles. Two malic enzymes were isolated: one, an NADP enzyme, is a minorcomponent and was not studied further; the other, immunologicallydistinct from the malate dehydrogenases, is probably locatedin the matrix compartment. The K_m for malate oxidation (1.4mM) is relatively low. This malic enzyme which apparently lacksOAA decarboxylase activity is NAD-specific but is unstable.The possibility of multiple malic enzymes is discussed. Themalic enzymes are responsible for the second NAD-requiring phaseof malate oxidation in submitochondrial particles.     

Induction of hepatic mitochondrial glycerophosphate dehydrogenase in rats by dehydroepiandrosterone.

Feeding the thermogenic steroid, 5-androsten-3 beta-ol-17-one (dehydroepiandrosterone, DHEA) in the diet of rats induced the synthesis of liver mitochondrial sn-glycerol 3-phosphate dehydrogenase to levels three to five times that of control rats within 7 days. The previously reported enhancement of liver cytosolic malic enzyme was confirmed. The induction of both enzymes was detectable at 0.01% DHEA in the diet, reached plateau stimulation at 0.1 to 0.2%, and was completely blocked by simultaneous treatment with actinomycin D. Feeding DHEA caused smaller, but statistically significant increases of liver cytosolic lactate, sn-glycerol 3-phosphate, and isocitrate (NADP(+)-linked) dehydrogenases but not of malate or glucose 6-phosphate dehydrogenases. The capability of DHEA to enhance mitochondrial glycerophosphate dehydrogenase and malic enzyme was influenced by the thyroid status of the rats; was smallest in thyroidectomized rats and highest in rats treated with triiodothyronine. 5-Androsten-3 beta,17 beta-diol and 5-androsten-3 beta-ol-7,17-dione were as effective as DHEA in enhancing the liver mitochondrial glycerophosphate dehydrogenase and malic enzyme. Administering compounds that induce the formation of cytochrome P450 enzymes enhanced liver malic enzyme activity but not that of mitochondrial glycerophosphate dehydrogenase. Arochlor 1254 and 3-methylcholanthrene also increased the response of malic enzyme to DHEA feeding.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

Evidence for Metabolic Domains within the Matrix Compartment of Pea Leaf Mitochondria : Implications for Photorespiratory Metabolism

The simultaneous oxidation of malate and of glycine was investigated in pea (Pisum sativum) leaf mitochondria. Adding malate to state 4 glycine oxidation did not inhibit, and under some conditions stimulated, glycine oxidation. State 4 oxygen uptake with glycine is restricted because of the control exerted by the membrane potential but reoxidation of NADH by oxaloacetate reduction can still occur. Thus, malate addition stimulates glycine metabolism by producing oxaloacetate. The malate dehydrogenase (EC 1.1.1.37) enzyme fraction remote from glycine decarboxylase (EC 2.1.2.10) oxidizes malate whereas that closely associated with it produces malate, i.e. they function in opposite directions. It is shown that these opposing directions of malate dehydrogenase activity occur within the same mitochondrial matrix compartment and not in different mitochondrial populations. It is concluded that metabolic domains containing different complements of mitochondrial enzymes exist within the one mitochondrial matrix without physical barriers separating them. The differential spatial organization within the matrix may account for the previously reported limited access of some enzymes to the respiratory electron transport chain. The implications for leaf mitochondrial metabolism are discussed.     

Pathways of glucose catabolism in procyclic Trypanosoma congolense

Studies of respiration on glucose in procyclic Trypanosoma congolense in the presence of rotenone, antimycin, cyanide, salicylhydroxamic acid and malonate have indicated the presence of NADH dehydrogenase, cytochrome b-c1, cytochrome aa3, trypanosome alternate oxidase and NADH fumarate reductase/succinate dehydrogenase pathway that contributes electrons to coenzyme Q of the respiratory chain. The rotenone sensitive NADH dehydrogenase, the trypanosome alternate oxidase, and cytochrome aa3 accounted for 24.5 +/- 6.5, 36.2 +/- 4.2 and 54.1 +/- 5.5% respectively of the total respiration. Activities of lactate dehydrogenase, NAD(+)-linked malic enzyme and pyruvate kinase were less than 6 nanomoles/min/mg protein suggesting that they play a minor role in energy metabolism of the parasite. Phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, succinate dehydrogenase, NADP(+)-linked malic enzyme, NADH fumarate reductase, malate dehydrogenase, and alpha-ketoglutarate dehydrogenase and glycerol kinase on the other hand had specific activities greater than 60 nanomoles/min/mg protein. These enzyme activities could account for the production of pyruvate, acetate, succinate and glycerol. The results further show that the amount of glycerol produced was 35-48% of the combined total of pyruvate, acetate and succinate produced. It is apparent that some of the glycerol 3-phosphate produced in glycolysis in the presence of salicylhydroxamic acid is dephosphorylated to form glycerol while the rest is oxidised via cytochrome aa3 to form acetate, succinate and pyruvate.