NADP-linked malic enzyme and malate metabolism in ageing tomato fruit

Malate dehydrogenase (oxaloacetate-decarboxylating) (NADP⁺) EC 1.1.1.40, malic enzyme, has been purified 40-fold to a homogeneous state using affinity chromatography and gel permeation chromatography. The M_r is 260–265 K with four subunits each of 64–65 K. The enzyme has some competitive or non-competitive inhibitors, particularly some of the Krebs cycle acids and exhibits a rapid rise in activity at the same time as activity of the enzymes of the Krebs cycle are decreasing in the tomato mitochrondrion. The malic enzyme is restricted to the cytosol. The relevance of this information to malate metabolism in plants is discussed.     

Rapid purification and radioimmunoassay of cytosolic malic enzyme

A very rapid and highly effective procedure has been devised for the isolation of homogeneous malic enzyme from rat liver cytosol. A combination of precipitation with 10 to 20% polyethylene glycol, ion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Procion Red HE-3B Agarose was used to prepare 3 to 4 mg of homogeneous malic enzyme from the livers of two rats in 18 h. In addition to introducing the advantages of simplicity, speed, and high yield (31%) the new method eliminates potentially denaturing steps (heat treatment, ethanol fractionation) and prolonged dialysis procedures used in other purification schemes. Malic enzyme purified by this new method was use to immunize rabbits. The resulting antibodies bound purified rat liver and mouse liver malic enzymes with very similar affinities and also avidly complexed cytosolic malic enzyme from two murine cell lines, 3T3-L1 preadipocytes and 3T3-C2 fibroblasts. When purified malic enzyme was incubated with lactoperoxidase, glucose oxidase and Na 125I 1.8 atoms of 125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated 125I-malic enzyme were used to construct a sensitive, competitive binding radioimmunoassay for the measurement of malic enzyme mass in the range of 1 to 100 ng.     

Purification and partial characterization of ubiquitin-activating enzyme from Saccharomyces cerevisiae

Ubiquitin-activating enzyme was purified from the yeast Saccharomyces cerevisiae by covalent affinity chromatography on ubiquitin-Sepharose followed by HPLC anion-exchange chromatography. Enzyme activity was monitored by the ubiquitin-dependent ATP: 32PPi exchange assay. The purified enzyme has a specific activity of 1.5 mumol 32PPi incorporated into ATP.min-1.mg-1 at 37 degrees C and pH 7.0 under standard conditions for substrate concentrations as described by Ciechanover et al. (1982) J. Biol. Chem. 257, 2537-2542. The catalytic activity showed a maximum at pH 7.0. Its molecular weight both in non-denaturing and in SDS-gel electrophoresis was estimated to be 115 kDa, suggesting a monomeric form. The isoelectric point determined by gel electrofocusing was approximately 4.7. Two protein bands differing slightly in electrophoretic mobility could be distinguished when SDS gels were loaded with very small amounts of purified E1 and immunoblotted, the one with higher molecular weight being clearly predominant. The same two bands were also found in anti-E1 immunoblots of crude yeast lysates prepared under broad protease inhibition.     

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.     

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     

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.     

The effect of NADP-dependent malic enzyme expression and anaerobic C4 metabolism in Escherichia coli compared with other anaplerotic enzymes

AIMS: To understand the modification of C4-metabolism under anaerobic glycolysis condition by overexpressing anaplerotic enzymes, which mediating carboxylation of C3 into C4 metabolites, in Escherichia coli. METHODS AND RESULTS: Anaplerotic NADP-dependent malic enzyme (MaeB), as well as the other anaplerotic enzymes, including phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate carboxykinase (Pck) and NAD-dependent malic enzyme (MaeA), were artificially expressed and their C4 metabolism was compared in E. coli. Increasing MaeB expression enhanced the production of C4 metabolites by 2.4 times compared to the wild-type strain in anaerobic glucose medium with bicarbonate supplementation. In MaeB expression, C4 metabolism by supplementing 10 g l(-1) of NaHCO(3) was three times than that by no supplementation, which showed the greatest response to increased CO(2) availability among the tested anaplerotic enzyme expressions. CONCLUSIONS: The higher C4 metabolism was achieved in E. coli expressing increased levels of the NADPH-dependent MaeB. The greatest increase in the C4 metabolite ratio compared to the other tested enzymes were also found in E. coli with enhanced MaeB expression as CO(2) availability increased. SIGNIFICANCE AND IMPACT OF THE STUDY: The higher C4 metabolites and related biomolecule productions can be accomplished by MaeB overexpression in metabolically engineered E. coli.     

Cottonseed malate synthase : purification and immunochemical characterization

Malate synthase (EC 4.1.3.2), an enzyme unique to the glyoxylate cycle, was purified to homogeneity from cotyledons of 72-hours, darkgrown cotton (Gossypium hirsutum L.) seedlings. Homogeneity of the enzyme was assessed by silver staining SDS-PAGE gels. Purification was accomplished by using a single buffer medium through six steps involving one ammonium sulfate fractionation and chromatography on three columns (Sephacryl S-300, DEAE Sephacel, Phenyl Sepharose). Large-scale preparation of glyoxysomes, a main step in all other published procedures, was not involved. The purified enzyme and that extracted from glyoxysomes appears to be a dodecamer with a native molecular weight of 750,000 (sedimentation coefficient of >20 Svedberg units [S] on sucrose gradients) composed of identical subunits (molecular weight approximately 63,000). The monomer (5S) occurs in the cytosol. Polyclonal antibodies raised in rabbits were judged to be monospecific for malate synthase by immunotitration, double immunodiffusion, and western blotting. Double immunodiffusion experiments revealed only partial immunological identity between the 5S (cytosolic) and 20S (glyoxysomal forms, although complete identity was observed between the 5S form in immature and germinated seeds, and the 20S form in immature and germinated seeds. Cross-reactivity of the cotton antimalate synthase serum was observed with extracts from five other oilseeds. Western blot analyses showed that malate synthase protein was not present in immature seeds prior to appearance of enzyme activity, but when present, subunit molecular weight was indistinguishable in immature, desiccated, and germinated seeds.     

Galactosylceramide beta-galactosidase in Krabbe disease: partial purification and characterization of the mutant enzyme

Galactosylceramide β-galactosidase (EC 3.2.1.46) has been partially purified from liver of a patient who died of Krabbe disease. Approximately 700-fold purification was achieved by solubilization, adsorption with immobilized concanavalin A, gel filtration through Bio-Gel A-1.5m and chromatography on immobilized sphingosine. The relative increase in crossreacting material and residual galactosylceramidase and lactosylceramidase I activities of the mutant enzyme was essentially identical to that obtained for the enzyme partially purified by the same procedure from normal liver control. An apparent molecular weight of about 750,000 and similar electrophoretic mobilities were observed for both enzymes. In contrast, catalytic properties and stability of the enzyme protein were severely affected in the mutant as compared to the normal enzyme. The apparent K_m values of the mutant enzyme for β-galactosidase activities toward galactosylceramide and lactosylceramide in the presence of pure sodium taurocholate were 14 and 4 times, respectively, higher than the normal values. Incubation for 4 min at 52 °C or dialysis against 1.3 m urea caused a 50% loss of residual enzymatic activity of the mutant enzyme, whereas a 35-min incubation or dialysis against 5.6 m urea was required for 50% inactivation of the normal enzyme. These findings indicate that the mutation in Krabbe disease leads to synthesis of normal quantities of catalytically and structurally altered protein.     

The purification and characterization of arginase from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, ornithine transcarbamoylase and arginase form a regulatory multienzyme complex (Hensley, P. (1988) Curr. Top. Cell. Regul. 29, 35-75). In this complex, arginase acts as a negative allosteric effector for ornithine transcarbamoylase. Before an analysis of the factors which promote and stabilize complex formation, arginase was purified in milligram quantities from a plasmid-containing, enzyme-overproducing, protease-deficient yeast strain and its physical characterization undertaken. The purified enzyme has a specific activity of 885 mumol urea min-1 mg-1 and a Km for arginine of 15.7 mM. The ultraviolet spectrum has a maximum absorbance at 279 nm, and the steady-state fluorescence emission spectrum has a maximum intensity at 337 nm, suggesting that the 3 tryptophans/polypeptide chain are in a relatively hydrophobic environment. Arginase has a weakly bound manganese responsible for the maintenance of the catalytic activity and is known to be heat activated in the presence of manganese. This effect is half-maximal at 12.1 microM manganese. In addition to a catalytic requirement for manganese, the presence of a more tightly bound metal is suggested from sedimentation studies. The native trimeric enzyme has a sedimentation coefficient of 5.95 S. Removal of the weakly associated metal results in no change in the sedimentation coefficient. However, dialysis with EDTA causes the s-value to decrease to 4.65 S, suggesting that under these conditions, the trimeric enzyme may partially dissociate. An analysis of CD spectra shows that significant spectral changes result from the removal of both the weakly bound metal and dialysis against EDTA.     

Overexpression, purification, and partial characterization of Saccharomyces cerevisiae processing alpha glucosidase I

The gene encoding yeast processing alpha glucosidase I, CWH41, was overexpressed in Saccharomyces cerevisiae AH22, resulting in a 28-fold increase in expression of the soluble form of the enzyme. The soluble enzyme results from proteolytic cleavage between residues Ala 24 and Thr 25 of the transmembrane sequence of the membrane-bound form of the enzyme. This cleavage could be partially inhibited by addition of leupeptin and pepstatin during the enzyme isolation. The enzyme was purified to a final specific activity of 8550 U/mg protein using a combination of ammonium sulfate precipitation, anion exchange, concanavalin A, and gel filtration chromatography. The soluble form of the enzyme is a monomer with a molecular weight of 98 kDa by SDS-PAGE, and 89 kDa by gel filtration. The molecular weight decreased by approximately 5 kDa after treatment with N-glycosidase F, indicating that it is a glycoprotein. Soluble glucosidase I was sensitive to diethyl pyrocarbonate and not affected by N-ethylmaleimide, suggesting that mechanistically it is more similar to the plant than the mammalian form of the enzyme.     

Extracellular cell wall lytic enzyme from Staphylococcus aureus: purification and partial characterization

An autolysin obtained from culture fluid of Staphylococcus aureus strain 8507 was purified 3,000-fold. One milligram of this preparation (S-5DL) will solubilize 12 mg of cell wall in 1 hr. The major activity is N-acetylmuramyl-l-alanine amidase. Recovery of lytic activity in the purified preparation was repeatably only 20% of the starting level. This suggests that other cell wall lytic enzymes may be present in the starting material. The S-5DL enzyme has been compared to freeze-thaw extracted enzyme (AFZ). Both enzymes precipitate in 0.01 m KPO(4) (pH 6.0) and dissolve in 0.1 to 0.7 m NaCl. Fifty per cent of the AFZ activity and 66% of the S-5DL activity bind rapidly to cell walls of S. aureus at 0 C in the presence of magnesium ion. None of the AFZ activity and 66% of the S-5DL activity bind to cell walls at 0 C in the absence of magnesium ion. The cell walls of nine different strains of S. aureus were compared for level of native autolysin activity. These same walls after inactivation of the native autolysin were tested for susceptibility to the S-5DL enzyme.     

Mannitol oxidase: partial purification and characterization of the membrane-bound enzyme from the snail Helix aspersa

Mannitol oxidase, a membrane-bound oxidase has been purified 250-fold from snail digestive gland tissue. The activity is solubilized by a number of ionic, non-ionic, and zwitterionic detergents. Purification of the solubilized enzyme was by polyethylene glycol fractionation and column chromatography using anionic exchange resins, hydroxylapatite, and gel filtration. The enzyme is stabilized by glycerol and remains active for at least one week at -20 degrees. Hydrogen peroxide is the oxygen reduction product and a mannose/hydrogen peroxide stoichiometry of 0.86 was found. D-Arabinitol and D-mannitol were the most active substrates of those tested. Results with these and other substrates suggest that the configuration around carbons-2 and -4 is critical for binding and reactivity. The apparent Km for D-mannitol is 6 mM and for oxygen, 40 microM. The pH optimum for the enzyme is between 8 and 8.5 and the isoelectric point is 5.4-5.6.     

How hexoses and inhibitors influence the malate transport system in Zygosaccharomyces bailii

When grown in fructose or glucose the cells of Zygosaccharomyces bailii were physiologically different. Only the glucose grown cells (glucose cells) possessed an additional transport system for glucose and malate. Experiments with transport mutants had lead to the assumption that malate and glucose were transported by one carrier, but further experiments proved the existence of two separate carrier systems. Glucose was taken up by carriers with high and low affinity. Malate was only transported by an uptake system and it was not liberated by starved malate-loaded cells, probably due to the low affinity of the intracellular anion to the carrier. The uptake of malate was inhibited by fructose, glucose, mannose, and 2-DOG but not by non metabolisable analogues of glucose. The interference of malate transport by glucose, mannose or 2-DOG was prevented by 2,4-dinitrophenol, probably by inhibiting the sugar phosphorylation by hexokinase. Preincubation of glucose-cells with metabolisable hexoses promoted the subsequent malate transport in a sugar free environment. Preincubation of glucose-cells with 2-DOG, but not with 2-DOG/2,4-DNP, decreased the subsequent malate transport. The existence of two separate transport systems for glucose and malate was demonstrated with specific inhibitors: malate transport was inhibited by sodium fluoride and glucose transport by uranylnitrate. A model has been discussed that might explain the interference of hexoses with malate uptake in Z. bailii.Abbreviations 2,4-DNP 2,4-dinitrophenol - 2-DOG 2-deoxyglucose - 6-DOG 6-deoxyglucose - pCMB para-hydroxymercuribenzoate     

The role of malic enzyme in the malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

Mitochondria isolated from human term placenta were able to form citrate from malate as the only added substrate. While mitochondria were incubated in the presence of Mn2+ the citrate formation was stimulated significantly both by NAD+ and NADP+ and was inhibited by hydroxymalonate, arsenite, butylmalonate and rotenone. It is concluded that NAD(P)-linked malic enzyme is involved in the conversion of malate to citrate in these mitochondria. It has also been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate was stimulated by NAD+ and NADP+ and inhibited by arsenite and fluorocitrate. This suggests that the stimulation by malate of progesterone biosynthesis depends not only on the generation of NADPH by NAD(P)-linked malic enzyme, but also on NADPH formed during further metabolism of pyruvate to isocitrate which is in turn efficiently oxidized by NADP+-linked isocitrate dehydrogenase.