Higher Malate Dehydrogenase Activity Research Articles

Aerobic and anaerobic poise of white swimming muscles of the deep-diving scalloped hammerhead shark: comparison to sympatric coastal and deep-water species

Scalloped hammerhead sharks (Sphyrna lewini) routinely perform rapid dives to forage on mesopelagic prey. These deep dives consist of intensive swimming followed by recovery periods in the surface mixed layer. Swimming muscle temperature profiles suggest that S. lewini suppresses gill function as a means to reduce convective heat loss during dives into cool water. Such intensive swimming behavior coupled with reduced respiration prompted us to test whether the aerobic and anaerobic metabolic capacities of the white swimming muscle tissue of this species are greater than those of other shark species from the same region. The activities of key enzymes used in aerobic and anaerobic metabolism provide an indirect indicator of the metabolic potential (“poise”) of a tissue. Here we measured the maximal activities [international units (µmol substrate converted to product per min, U) per gram of wet tissue mass at 10°C] of the citric acid cycle enzymes citrate synthase (CS) and malate dehydrogenase (MDH) and glycolytic enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH) from white swimming muscle of S. lewini. Enzyme activities, and ratios of these enzyme activities that indicate relative indexes of aerobic to anaerobic capacity, were compared to those measured in three sympatric coastal carcharhinid sharks and two deep-dwelling species, Echinorhinus cookei and Hexanchus griseus. This is the first report of swimming-muscle enzyme activity for these deep-dwelling species. In comparison to the other species, S. lewini had significantly higher activities of both LDH and MDH in the white muscle, and a higher MDH/CS ratio. The high LDH activities suggest that the white muscle of S. lewini relies on relatively high rates of anaerobic ATP production, with would result in build up of high lactate levels, during deep foraging dives. High MDH activity in S. lewini white muscle suggests the potential for lactate levels to be rapidly reduced when aerobic conditions are restored while in the surface mixed layer between dives. These biochemical characteristics may enable S. lewini to swim rapidly while suppressing gill function during deep dives and thereby exploit a very different ecological niche from sympatric shark species (e.g., coastal carcharhinids) and hunt more rapidly via faster swimming for deep-water prey compared to species that permanently inhabit deep depths.

Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.

Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90°C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8M; however, in the presence of 4M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90% residual activity even after heating for 6h in boiling water.

The localization of nitrite reductase, glutamate synthase and malate metabolism enzymes in Pisum arvense L. roots

Centrifugation of a homogenate made from <em>Pisum arvense</em> L. roots in a sucrose density gradient enabled the separation of the plastid fraction from mitochondria and microsomes. The presence of nitrite reductase and glutamate synthase was demonstrated in the plastids. Malic enzyme activity was not linked with any organelle fraction and was found only in the cytosol. High malate dehydrogenase activity was found in the mitochondria fraction, although its activity was also determined in plastids. The results suggest that malic acid metabolism in plastids may be the source of reduced pyridine nucleotides for reactions catalysed by nitrite reductase and glutamate synthase.

Central Carbon Metabolism in Marine Bacteria Examined with a Simplified Assay for Dehydrogenases

A simplified assay platform was developed to measure the activities of the key oxidoreductases in central carbon metabolism of various marine bacteria. Based on microplate assay, the platform was low-cost and simplified by unifying the reaction conditions of enzymes including temperature, buffers, and ionic strength. The central carbon metabolism of 16 marine bacteria, involving Pseudomonas, Exiguobacterium, Marinobacter, Citreicella, and Novosphingobium were studied. Six key oxidoreductases of central carbon metabolism, glucose-6-phosphate dehydrogenase, pyruvate dehydrogenase, 2-ketoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and isocitrate dehydrogenase were investigated by testing their activities in the pathway. High activity of malate dehydrogenase was found in Citreicella marina, and the specific activity achieved 22 U/mg in cell crude extract. The results also suggested that there was a considerable variability on key enzymes' activities of central carbon metabolism in some strains which have close evolutionary relationship while they adapted to the requirements of the niche they (try to) occupy.

Malate dehydrogenase in plants: Its genetics, structure, localization and use as a marker

This article is an overview of literature data on the structure, properties, functions and genetic control of the enzyme malate dehydrogenase (MDH) in plants. In most of the plant entities studied, this enzyme is highly polymorphic, which means that malate dehydrogenase has multiple molecular forms. It has been found that MDH polymorphism in each species is genetically determined by several loci with multiple alleles. A readily identifiable phenotypic manifestation and a high activity of malate dehydrogenase in diverse organs and tissues make this enzyme a reliable and convenient genetic marker, which can effectively be used in special, ecological and population genetics.

Effects of ATP production on silicon uptake by roots of rice seedlings

The activities of several respiratory metabolism enzymes and ATP concentrations in the roots of four genotypes of rice raised in hydroponic culture and treated with three levels of silicon (Si) (0, 1.25, and 2.00 mM) were studied. ATP concentrations in roots of the four rice cultivars were significantly higher with Si application than in the control. Phosphoglucose isomerase (PGI) activity and the combined activity of glucose-6-phosphate dehydrogenase (G-6-PDH) and 6-phosphogluconate dehydrogenase (6-PGDH) in roots were also significantly enhanced by Si addition, as was malate dehydrogenase (MDH) activity. PGI and MDH activity increased more than 50% and 25%, respectively, in TN1 roots supplied with Si compared to the control. Among the four genotypes, higher MDH activity and combined activity of G-6-PDH and 6-PGDH were detected in roots of TN1 and Bai Xiang Jing, which had higher Si uptake. ATPase activity and ATP concentrations were inhibited by four metabolic inhibitors: HgCl2, 2,4-dinitrophenol (2,4-DNP), malonic acid (C3H4O4), and sodium phosphate (Na3PO4). Succinate dehydrogenase (SDH) activity in roots also decreased significantly upon treatment with C3H4O4. In summary, Si application can stimulate activity of key respiratory metabolism enzymes to provide more ATP for energy-dependent processes, including Si uptake, during plant growth.

Characteristic properties of metabolism of the yeast Yarrowia lipolytica during the synthesis of α-ketoglutaric acid from ethanol

The study of free amino acid content in Yarrowia lipolytica cells grown on ethanol under thiamine deficiency showed that glutamate, alanine, and γ-aminobutyric acid (γ-ABA) occurred in the highest concentrations among the present 17 free amino acids. The culture liquid contained no amino acids. Analysis of the enzymes of oxidative metabolism in the yeast grown under these conditions showed that the cell-free homogenate contained substantial activity of glutamate decarboxylase, γ-ABA transaminase, and succinyl semialdehyde dehydrogenase. This result indicated the formation of succinate from glutamate in a reaction catalyzed by 4-aminobutyrate aminotransferase (γ-aminobutyrate bypass) under severe thiamine deficiency. These studies lead to the conclusion that cultivation of the yeast Y. lipolytica on ethanol under thiamine deficiency causes adaptive stress-induced metabolic changes. Increase of ammonium nitrogen consumption and excretion of α-ketoglutaric acid are indicative of physiological changes, the functioning of the γ-aminobutyrate bypass and high activity of malate dehydrogenase are manifestations of metabolic changes, and increased activities of the transamination reactions reflect the changes in nitrogen metabolism.

Effect of short-term starvation of adult Antarctic krill, Euphausia superba, at the onset of summer

We investigated the effect of short-term starvation (18 days) on the physiology of adult Euphausia superba from the Lazarev Sea at the onset of summer. Metabolic data, elemental and biochemical composition as well as morphological parameters revealed that, at the beginning of the experiment, krill was in transition from winter to summer physiology, with some features typical for late winter/spring (low lipid reserves, low C:N ratio, elevated O:N ratio) and others for summer (high respiration rates, high MDH activity, large green digestive gland, short intermoult period (IMP) and fast growth). Starvation reduced body reserves drastically by more than 1% C per day. In relative terms, lipids (40%) and glycogen (30%) were reduced most and proteins by 10% of the initial value. Absolute consumption, however, was approximately 4% DM for lipids and proteins each, whereas contribution of glycogen was negligible. Within lipids, triacylglycerols (TAG) and phospholipids (PL) fell most dramatically from already low levels by 84% and 39%, respectively. Phosphatylcholine (PC) constituted 57% of PL and declined by 46%. As a result, proportions of the lipid classes changed with sterols increasing relatively. Metabolite changes were similar in cephalothorax and abdomen, although TAG in the cephalothorax fell more drastically. High metabolic activity at the beginning of starvation was quickly reduced to reach 53% after 18 days, accompanied by a reduction in the abdominal activity of malate dehydrogenase (MDH) by 25%. Our results may provide some explanation why recruitment of some year-classes of krill fails. Despite execution of the experiment in spring (i.e. transitional physiology state) and its short duration, some changes in the activity of metabolic enzymes in the abdomen, representing lipolytic, glycolytic and proteolytic pathways, respectively, were measured. Rising activities of 3-hydroxyacyl-CoA dehydrogenase (HOAD) and glyceralaldehyde-3-phosphate dehydrogenase (GAPDH) indicated increased lipolytic and glycolytic fluxes, respectively, whereas declining glutamate dehydrogenase (GluDH) activity suggests reduced proteolytic flux. Activities of other enzymes from protein catabolism, alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT), however, remained unchanged. Ratios calculated from these trends indicated a declining importance of protein use during the course of starvation compared with consumption of lipids and glycogen. These results suggest that constant-proportion enzymes from different catabolic pathways, and calculated ratios thereof, may be useful in detecting shifts between the consumption of different body reserves.

Cytosolic ratio of malate dehyrogenase/lactate dehydrogenase activity in peripheral leukocytes of race horses with training

The activities of the enzymes involved in the malate–aspartate shuttle and m RNA expression of malate dehydrogenase (MDH), a crucial enzyme for the NADH shuttle that produces ATP in glucose metabolism in the peripheral leukocytes of horses, were measured to investigate the change in metabolic states with training. There were no significant differences in plasma glucose and immunoreactive insulin concentrations between race horses and riding horses, used as a comparable reference. The cytosolic and mitochondrial MDH activities in leukocytes of race horses were significantly higher than those of riding horses. High activities of MDH in leukocytes of race horses were confirmed by RT - PCR analysis on the total RNA extracted from the whole blood. The cytosolic ratio ofMDH /lactate dehydrogenase (LDH) activity (M/L ratio) in leukocytes of race horses was significantly higher than in those of riding horses. Increase in the M/L ratio was considered to reflect elevation of energy metabolism in animal tissues. The M/L ratio may be a useful parameter to evaluate the difference in metabolic states between race horses and riding horses.

Activities of enzymes in the malate-aspartate shuttle in the peripheral leukocytes of dogs and cats.

The activities of the enzymes involved in the malate-aspartate shuttle and the expression of malate dehydrogenase (MDH), a rate-limiting enzyme in the NADH shuttle that produces ATP in glucose metabolism in leukocytes, were determined to investigate the differences in this shuttle system in the peripheral leukocytes of dogs and cats. There were no significant differences between dogs and cats in plasma glucose, immunoreactive insulin, free fatty acid or triglyceride concentrations. The activities of cytosolic and mitochondrial MDH and of mitochondrial glutamate dehydrogenase (GLDH) in canine leukocytes were significantly higher than in feline leukocytes. High activities of MDH in canine leukocytes were confirmed by RT-PCR analysis on the total RNA extracted from leukocytes. It was concluded that there were significant differences between dogs and cats in the NADH shuttle system.

Variability of Malate Dehydrogenase among Cotton Cultivars with Differing Fiber Traits

Little is understood about the biochemical basis of differences in quantitative traits of cotton (Gossypium hirsutum L.) fiber. Malate dehydrogenase (MDH) is implicated as an enzyme that may have effect on extension growth during the development of cotton fiber. Our objectives were, first, to determine if variability among cultivars exists for MDH, and, second, to determine if variability in MDH can be correlated with quantitative traits of fiber. Twenty‐four cultivars G. hirsutum were chosen. Polyacrylamide gel electrophoresis was used to search for MDH allozyme variation in etiolated cotyledons. Additionally, MDH activity in developing fiber was measured. All cultivars were grown in 1990 at two sites near Stoneville, MS. At 12 days post anthesis (DPA), immature bolls were harvested and the fiber separated from the ovules. An extract was prepared from the fiber and assayed for MDH activity and protein concentration. Other bolls were allowed to mature and open naturally, and from these bolls fiber was taken and tested for eight traits: elongation, maturity (ratio of wail thickness to fiber diameter), micronaire reading (fiber fineness), perimeter, 2.5 and 50% span length, strength, and wall thickness. Allozyme variation for MDH was not detected. The MDH specific activity ranged from 13.8 to 18.8 µmol NADH min−1 mg−1 protein, with a mean of 16.4 μmol NADH min−1 mg−1 protein in 12 DPA fiber. Variation among cultivars for MDH specific activity and all fiber traits was significant (P < 0.05). Though implicated in extension growth, MDH specific activity was not correlated with the length of mature cotton fiber. Only correlations between MDH specific activity and three traits of fiber walls (maturity, r = −0.476; micronaire, r −0.469; and wall thickness, r = −0.475 were significant. Cotton cultivars vary significantly for MDH activity in developing fibers and high MDH activity is indicative of low micronaire reading, thin fiber walls, and decreased maturity.

Muscle enzyme adaptations to added load during training and nontraining hours in rats

The effects of added load (20% of body mass) on the selected enzyme activities of red and white quadriceps femoris (QF), soleus, and gastrocnemius muscles of rats were studied. The rats were divided into sedentary control (SC), sedentary control with added load (SC+AL), endurance training (ET), and endurance training with added load (ET+AL) groups (n = 10 rats/group). After 6 wk, the SC+AL group had 57% higher (P less than 0.001) beta-glucuronidase (beta-GU) activity and 24% lower (P less than 0.05) citrate synthase activity in white QF than SC. Citrate synthase activity was also decreased in red QF (P less than 0.05) after the added load was used during nontraining hours. The training with added load induced similar but more pronounced changes than normal endurance training, especially in white QF. The ET+AL group demonstrated higher citrate synthase activity in white QF (P less than 0.001) and gastrocnemius (P less than 0.01) and higher malate dehydrogenase activity (P less than 0.05) and beta-GU activity (P less than 0.001) in white QF than the ET group. ET+AL rats also had higher phosphofructokinase (P less than 0.01) and lower creatine kinase (P less than 0.001) activity in white QF than ET rats. In conclusion, the added load without training had minor adaptive influences on muscles. The added load during training hours seemed to be an effective means of influencing the activation and adaptation in muscles that contain fast glycolytic fibers.

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

Studies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.

Studies on the anaerobic energy metabolism in the foot muscle of marine gastropod Patella caerulea (L.)

1. 1. The foot muscle of P. caerulea has a complete sequence of glycolytic enzymes. The low activitiy of hexokinase, in comparison with the activities of glycogen phosphorylase and phosphofructokinase, indicate that glycogen is the main fuel oxidized. 2. 2. The reduction of aspartate content in combination with the accumulation of alanine and the presence of considerable activities of glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase indicates a coupled metabolism of glycogen and aspartate during exposure to air. 3. 3. From the changes in the concentration of the metabolites during exposure to air it appears that up to the second hour of anaerobiosis alanine, lactate and glutamate are the end-products which accumulate in the foot muscle of P. caerulea, whereas from the second to the fourth hour only succinte and alanine accumulate. 4. 4. The low activities of the Krebs cycle enzymes as well as the absence of α-ketoglutarate dehydrogenase activity suggest that the Krebs cycle is not in operation. 5. 5. The absence of opine dehydrogenases shows that the end products octopine, alanopine and strombine are not accumulated in the foot muscle under anaerobiosis. 6. 6. The high activity of malate dehydrogenase in the direction of malate formation in combination with the low activity of d-lactate dehydrogenase and the absence of opine dehydrogenases suggests that the former dehydrogenase is coupled 1:1 to glyceraldehyde-3-phosphate dehydrogenase.

Capacity for fermentation in roots and Rhizobium nodules of Pisum sativum L.

The aim of this work was to compare the capacities for fermentation and synthesis of malate from phosphoenolpyruvate in roots and Rhizobium nodules of Pisum sativum. The nodules and the cortices and apices of roots had similar activities of glycolytic enzymes and enzymes of ethanolic and lactic fermentation when expressed on a protein basis. The activity of phosphoenolpyruvate carboxylase was similar in nodules and apices, and three to four fold lower in cortices. All three tissues had very high activities of malate dehydrogenase, significant activity of NADP-malic enzyme, and no detectable activity of phosphoenolpyruvate carboxykinase. These results do not support the belief that nodules have a substantially greater capacity to convert phosphoenolpyruvate to malate than roots, or that there are major qualitative differences in the pathways of fermentation of nodules and roots.

The function of redox shuttles during aerobic glycolysis in two strains of Ehrlich ascites tumor cells

The function of glycerophosphate and malate-aspartate shuttles during glucose metabolism in two strains of Ehrlich ascites tumor cells was evaluated by several experimental approaches. The activities of the enzymes involved in these shuttle systems were assayed in the cytosolic and mitochondrial compartments after cell fractionation by the digitonin method. The glycerophosphate shuttle can be ruled out because of the lack of relevant enzymatic activities, and the failure of glucose to increase rotenone-inhibited respiration. Analysis of glycolytic flux in the presence of aminooxyacetate indicates that the activity of malate-aspartate shuttle may be very low. Balance studies of glucose uptake and lactate production suggest the existence of other pathways for the reoxidation of cytosolic NADH, which are acetyl-CoA dependent. Estimation of citrate synthase and ATP citrate lyase, in addition to the observed high activity of malate dehydrogenase, suggests a malate-citrate shuttle.

Fermentation and the properties of some enzymes of carbohydrate metabolism in the trematode Calicophoron ijimai

Glycogen content, glucose consumption and the production of metabolic end products by Calicophoron ijimai were determined under aerobic and anaerobic conditions. The major end products of fermentation were identified as lactic, acetic, propionic, isobutyric and α-methylbutyric acids, propionic acid predominating. The activities and properties of some of the enzymes of carbohydrate metabolism were determined. The worms showed high phosphoenolpyruvate carboxykinase, malate dehydrogenase and malate dehydrogenase (decarboxylating) but relatively low pyruvate kinase and very low lactate dehydrogenase activities. The pH optima, coenzyme, cofactor and ionic requirements of the enzymes were similar to those of other helminths. Malate dehydrogenase had an 8-fold greater affinity for oxaloacetate than malate, and was about 14 times more active for oxaloacetate reduction than malate oxidation. Phosphoenolpyruvate carboxykinase was 2.4 times more active and had a 2-fold greater affinity for phosphoenolpyruvate and dinucleotide than pyruvate kinase. The low activities of lactate dehydrogenase and pyruvate kinase but high activities of malate dehydrogenase and phosphoenolpyruvate carboxykinase suggest that anaerobic carbohydrate catabolism follows the fumarate reductase pathway.

Characterization of lactate dehydrogenase, malate dehydrogenase, pyruvate kinase, and phosphoenolpyruvate carboxykinase in Romanomermis culicivorax postparasitic larvae and adults

1. 1. Romanomermis culicivorax postparasites 48 hr after emergence had high malate dehydrogenase (MDH) and phosphoenolpyruvate carboxykinase (PEPCK) but relatively low pyruvate kinase (PK) and very little lactate dehydrogenase (LDH) activities. 2. 2. The pH optima, coenzyme, cofactor, and ionic requirements of the enzymes were similar to those of other helminths. 3. 3. MDH had a tenfold greater affinity for oxaloacetate (OAA) than malate and was 9 times more active for OAA reduction than malate oxidation. 4. 4. PK and PEPCK did not differ in their apparent K m s for phosphoenolpyruvate ( 0.9 × 10 −3 M and 1.1 × 10- 3 M , respectively), but PEPCK had a greater affinity for IDP ( K m = 1.2 × 10 −4 M ) than PK for ADP ( K m = 5.8 × 10 −4 M . 5. 5. The low activities of LDH and PK but high activities of MDH and PEPCK suggested that anaerobic carbohydrate catabolism followed the fumarate reductase pathway. 6. 6. PEPCK had a lower pH optimum (5.25) than PK (7.25) suggesting that pH may be an important factor in regulating the PK/PEPCK branchpoint.

Activity patterns of phosphofructokinase, glyceraldehydephosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase in microdissected fast and slow fibres from rabbit psoas and soleus muscle.

Methods for standardized determination of phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activities in nanogram samples of microdissected single fibres of rabbit psoas and soleus muscle are described. Fast and slow fibres in soleus muscle show lower absolute activities of these enzymes than the respective fibre types in psoas muscle. Slow fibres represent a more uniform population in the two muscles according to absolute and relative activities of the enzymes investigated. Slow fibres are characterized by high activities of MDH and relatively low activities of glycolytic enzymes. Fast fibres in the soleus muscle represent a population with high activities of MDH and glycolytic enzymes. Fast fibres in psoas muscle represent a heterogeneous population with high activities of glycolytic enzymes and extremely variable activity of MDH. More than 10-fold differences exist in the MDH activities of the extreme types of this fibre population. Differences in the activity levels of MDH in single fast type fibres but also in the activities of glycolytic enzymes between fast and slow fibres are greater than those reported between extreme white and red rabbit muscles.

Analysis of the enzymatic activity of individual subcellular particles of Acetabularia mediterranea

A simple procedure is described for quantitative spectrophotometric analysis of individual subcellular particles; the procedure is based upon the method proposed earlier to measure the optical density of microdroplets in capillary flow cells. The activity of malate dehydrogenase (MDH) was determined in individual chloroplasts, nuclei, nucleoli, and nuclear membranes of the unicellularalga Acetabularia. Chloroplasts are markedly heterogeneous in their enzymatic activity. A high MDH activity is characteristic of nuclei, insignificant activity is found in nuclear membranes, and no activity is present in nucleoli.