High Malic Enzyme Activity Research Articles

Purification and properties of malic enzyme from herring Clupea harengus spermatozoa

Herring spermatozoa exhibit higher activity of malic enzyme (ME) than Atlantic salmon (Salmo salar), brown trout (Salmo trutta), carp (Cyprinus carpio) and African catfish (Clarias gariepinus) spermatozoa. Two molecular forms of ME are present in herring spermatozoa: an NAD-preferring malic enzyme with very high activity and an NADP-specific malic enzyme with much lower activity (ratio about 33:1). NAD-preferring ME was purified by chromatography on DEAE-Sepharose, Red Agarose and Sephadex G-200 to a specific activity of 36 μmol/min/mg protein and NADP-specific ME on DEAE-Sepharose and 2'5'-ADP Sepharose. The molecular mass for NAD-preferring and NADP-specific ME determined by SDS-PAGE was equal to 61 and 64 kDa, respectively. High activity of ME suggests adaptation of herring spermatozoa to metabolism at high oxygen tension for herring spawn.

Cytosolic and Mitochondrial Malic Enzyme Isoforms Differentially Control Insulin Secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.

Enzyme activities in fish spermatozoa with focus on lactate dehydrogenase isoenzymes from herring Clupea harengus

The activities of NAD- and NADP-dependent dehydrogenases and creatine kinase were compared in extracts of spermatozoa from herring ( Clupea harengus), carp ( Cyprinus carpio) and catfish ( Clarias gariepinus). The activity of malic enzyme in herring spermatozoa was approximately 5 and 36 times higher than in carp and catfish spermatozoa. In contrast, lactate dehydrogenase activity in herring spermatozoa was very low. Herring spermatozoa possess two isoenzymes of lactate dehydrogenase: LDH-A 2B 2 and LDH-B 4. Both herring spermatozoa isozymes were separated, partly purified and characterized by kinetic and physico-chemical properties. The pH optima and K m values for pyruvate reduction were 7.1, 7.25, 7.6 and 0.22, 0.07, 0.09 mM for LDH-A 4, LDH-A 2B 2 and LDH-B 4, respectively. The isoenzymes also have different thermostabilities. High activity of malic enzyme in herring spermatozoa suggests adaptation to metabolism at high oxygen tension.

High malic enzyme activity in tumor cells and its cross-reaction with anti-pigeon liver malic enzyme serum.

Rabbit antibodies against pigeon liver malic enzyme (EC 1.1.1.40) were prepared. The antiserum gave single precipitation line with crude pigeon liver extract. Cross reaction was observed with partially purified malic enzyme or crude extract from chicken liver. Positive cross reaction was also observed with the concentrated cytosolic fraction of two human carcinoma cell lines which were demonstrated to contain high malic enzyme activity. All other proteins examined did not react with the antibodies. When purified pigeon liver malic enzyme was mixed with the antiserum in vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP+ or L-malate. Metal Mn2+ gave little protection.

Mitochondrial malic enzyme from crustacean and fish muscle

1. 1. In contrast to mammalian skeletal muscle mitochondria, the only substrate that crustacean and fish mitochondria oxidize at a high rate is malate. 2. 2. The mitochondria isolated from muscles of fish and crayfish exhibit a high activity of malic enzyme. 3. 3. Assuming that malic enzyme is responsible for the conversion of malate to pyruvate in animal muscle, it could be expected that the mitochondria which possess high activity of this enzyme should oxidize malate very rapidly when oxygen is available. 4. 4. Some properties of different molecular forms of malic enzyme are reviewed.

Malic enzyme in brown adipose tissue—purification, some properties and possible physiological role

1. 1. High activity of malic enzyme ( l-malate: NADP + oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) has been shown in brown adipose tissue isolated from cold-adapted rats. The enzyme was located mainly within extramitochondrial compartment. 2. 2. The extramitochondrial malic enzyme has been partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34. Specific activity of the purified enzyme was 40 μmol · min −1 per mg of protein which corresponds to about 175-fold purification. 3. 3. The enzyme was shown to carboxylate pyruvate in the presence of high concentration of KHCO 3 and pyruvate at about 80% of the rate of forward reaction. The K m values determined at pH 7.2 for malate, NADP and Mn 2+ were 0.18 mM, 8.6 μM and 14.5 μM respectively. The K m values for pyruvate, NADPH and KHCO 3 were 3.1 mM, 6.3 μM and 24.3 mM respectively. 4. 4. In isolated mitochondria, pyruvate, bicarbonate and NADPH (the substrates for malic enzyme) and extramitochondrial malic enzyme were able to replace added malate in supplying oxaloacetate, a condensing partner for acetyl-CoA formed during β-oxidation of fatty acids. 5. 5. Possible metabolic role of extramitochondrial malic enzyme in brown adipose tissue is discussed.

Decarboxylation of malate by isolated bundle-sheath cells of certain plants having the C4-dicarboxylic acid cycle of photosynthesis

Bundle-sheath cells isolated by the grinding and filtration procedure of Edwards and Black (1971b) from species of plants having the C4-dicarboxylic acid pathway of photosynthesis were tested for the decarboxylation of malate from the C4-carboxyl position. The bundle-sheath cells, which showed high malic enzyme activity in extracts, decarboxylated 4[(14)C]malate at rates sufficient to be involved in photosynthesis. The malate decarboxylation is dependent on the addition of magnesium or manganese and NADP(+). The activity was increased by raising the temperature from 30 to 50°. The evidence supports the idea that malate may be a carboxyl donor to the reductive pentose-phosphate cycle in bundle-sheath cells in certain C4-dicarboxylic acid pathway plants such as Zea mays L., Sorghum bicolor L., and Digitaria sanguinalis (L.) Scop.

In vitro fatty acid synthesis and enzyme activity in liver and adipose tissue of the mouse ( Mus musculus)

1. 1. The incorporation of glucose- U- 14C into Catty acids by mouse epididymal adipose tissue was much greater than by liver slices from the same animals. 2. 2. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicated that pentose phosphate shunt activity is also greater in adipose tissue than in liver. 3. 3. The high malic enzyme activity in mouse adipose tissue suggests that this enzyme plays an important role in the genera don of NADPH for the support of fatty acid synthesis in this tissue.

Dehydrogenases during postembryonic development of Tribolium confusum Duval (Coleoptera). Malate dehydrogenase and malic enzyme in the particulate and soluble fractions.

Malate dehydrogenase and malic enzyme activities in the particulate (mitochondria) and soluble fraction have been determined in relation to the larval–pupal transformation of Tribolium confusum. The malate dehydrogenase activity in the soluble fraction follows a more or less inverse trend as compared with that in the particulate fraction. The malate dehydrogenase activity in the particulate and soluble fractions of the larva is attributed to different enzymes based on their electrophoretic mobility. A sudden increase in the activity of malate dehydrogenase in the soluble fraction at the end of the larval period is attributed to release of the enzyme from mitochondria by lysis. A further comparison of the larval particulate and pupal soluble malate dehydrogenase is made on the basis of their kinetic behavior. Malic enzyme is NADP-linked, although activity was also noted with NAD. The significance of the high activity of malic enzyme and malate dehydrogenase during pupation is discussed in relation to anaerobic metabolism.

Activity of Dehydrogenase Enzymes during the Metamorphosis of the Mealworm, Tenebrio Molitor Linnaeus1

No evidence was found for formic, beta-hydroxybutyric, choline, or glutamic dehydrogenases in the larva; glutamic1 appeared towards the end of the pupal stage. Succinate and alpha-glycerophosphate did not require DPN for their oxidation, but its addition greatly stimulated utilization of the latter substrate. Alcohol, glucose, glutamic, and malic dehydrogenases could not act without DPN; isocitric dehydrogenase and malic enzyme required TPN. Lactic dehydrogenase did not require DPN in the larva; however, in the pupa and adult its addition increased the oxidation of lactate. Curves for the activities of malic and succinic dehydrogenases and the malic enzyme were U-shaped during metamorphosis. Since the activity of succinic dehydrogenase was lower than those of the other two enyzmes, it could be a determining factor in the U-shaped respiratory curve. The high activity of malic enzyme coupled with a low value for lactic dehydrogenase makes the accumulation of lactate unlikely, since the malic enzyme would convert pyruvate to malate under anaerobic conditions.