Mitochondrial NADP-dependent malic enzyme of cod heart. Rate of forward and reverse reaction

The mitochondrial NADP-dependent malic enzyme (EC 1.1.1.40) was purified about 300-fold from cod Gadus morhua heart to a specific activity of 48 units (mumol/min)/mg at 30 degrees C. The possibility of the reductive carboxylation of pyruvate to malate was studied by determination of the respective enzyme properties. The reverse reaction was found to proceed at about five times the velocity of the forward rate at a pH 6.5. The Km values determined at pH 7.0 for pyruvate, NADPH and bicarbonate in the carboxylation reaction were 4.1 mM, 15 microM and 13.5 mM, respectively. The Km values for malate, NADP and Mn2+ in the decarboxylation reaction were 0.1 mM, 25 microM and 5 microM, respectively. The enzyme showed substrate inhibition at high malate concentrations for the oxidative decarboxylation reaction at pH 7.0. Malate inhibition suggests a possible modulation of cod heart mitochondrial NADP-malic enzyme by its own substrate. High NADP-dependent malic enzyme activity found in mitochondria from cod heart supports the possibility of malate formation under conditions facilitating carboxylation of pyruvate.     

Pyruvate kinase from Calicophoron ijimai trematodes and its potential inhibition by anthelmintic preparations

It was shown that pyruvate kinase (PK) in the supernatant fraction from Calicophoron ijimai is able to regulate the direction of metabolic flow at glucose break down from phosphoenolpyruvate (PEP) level. The enzyme for activity required substrate, dinucleotides, cations K+ and Mn++. The activity with Mg++ as divalent cation is low. The addition of fructose-1.6-diphosphate (FDP) did not affect the enzyme activity with Mn++, however, increased the affinity for PEP. The velocity of Mg++ activated reaction increased by 8.2 times in the presence of FDP. PK in C. ijimai is sensitive to ATP inhibition, being weakly inhibited by malate. L-alanine did not influence on the enzyme activity. The effect of some anthelminthic preparations on the PK activity was shown.     

The determination of binding parameters when the total and free substrate concentrations are not approximately equal.

The maximal extractable activity of "malic" enzyme (EC 1.1.1.40) in rat islets of Langerhans was similar to that reported for liver. Thus "malic" enzyme may catalyse a near-equilibrium reaction in the cytosol of islets of Langerhans. Measurements of islet content of malate and pyruvate, the metabolite substrate and product of "malic" enzyme, were therefore used to calculate the cytosolic ration of [NADPH]/[NADP+]. This ratio was higher in islets incubated with 20 mM-glucose than in islets incubated with 2 mM-glucose.     

Fermentation and malate metabolism in response to elevated CO2 concentrations in two strawberry cultivars

Concentrations of acetaldehyde, ethanol, ethyl acetate (EA), organic acids and activities and gene expression of alcohol dehydrogenase (ADH; EC 1.1.1.1), pyruvate decarboxylase (PDC; EC 4.1.1.1), alcohol acyltransferase (AAT; EC 1.4.1.14), malate dehydrogenase (MDH; EC 1.1.1.37), malic enzyme (ME; EC 1.1.1.40) and glutamate dehydrogenase (EC 1.4.1.14) were investigated in two strawberry ( Fragaria × ananassa Duch) cultivars with different responses to CO₂ during storage. 'Jewel' fruit treated with CO₂ accumulated acetaldehyde and ethanol but little EA, while 'Cavendish' accumulated little acetaldehyde or ethanol but accumulated EA. In CO₂-treated fruit, PDC activity was positively correlated with EA accumulation in 'Jewel' but not in 'Cavendish', while no differential effect of atmosphere was observed on its gene expression. ADH activity and gene expression show a correlation with ethanol accumulation in 'Cavendish'. In 'Jewel', there was a positive correlation between ADH gene expression and enzyme activity; however, this correlation does not explain ethanol accumulation in this cultivar. EA accumulation did not show any correlation with AAT activity and gene expression in any of the cultivars. Succinate concentrations were highest and those of malate lowest in CO₂-treated fruit of both cultivars, but MDH and ME activities were not affected by CO₂. Gene expression of MDH and ME were not affected by atmosphere in 'Cavendish', although in 'Jewel' the MDH expression was slightly lower in CO₂- than air-treated fruit. The results of this study show that differences in fermentation products and malate accumulation in CO₂-treated strawberry fruit are not consistently correlated with enzyme activities and gene expression.     

Malate dehydrogenase and lactate dehydrogenase in trematodes and turbellarians

Studies have been made on the activity and properties of malate and lactate dehydrogenases from the cattle rumen trematodes Eurytrema pancreaticum, Calicophoron ijimai and the turbellarian Phagocata sibirica which has a common free-living ancestor with the trematodes. All the species studied have a highly active malate dehydrogenase, its activity in the reaction of reducing oxaloacetate being 6-14 times higher than in the reaction of malate oxidation. The affinity of malate dehydrogenase to oxaloacetate was found to be higher than that to malate. The activity of lactate dehydrogenase (reducing the pyruvate) was lower than the activity of malate dehydrogenase, the difference being 50 times for C. ijimai, 4 times for E. pancreaticum and 10 times for P. sibirica.     

Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+).     

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)     

Purification and characterization of a novel mannitol dehydrogenase from Lactobacillus intermedius

Mannitol 2-dehydrogenase (MDH) catalyzes the pyridine nucleotide dependent reduction of fructose to mannitol. Lactobacillus intermedius (NRRL B-3693), a heterofermentative lactic acid bacterium (LAB), was found to be an excellent producer of mannitol. The MDH from this bacterium was purified from the cell extract to homogeneity by DEAE Bio-Gel column chromatography, gel filtration on Bio-Gel A-0.5m gel, octyl-Sepharose hydrophobic interaction chromatography, and Bio-Gel Hydroxyapatite HTP column chromatography. The purified enzyme (specific activity, 331 U/mg protein) was a heterotetrameric protein with a native molecular weight (MW) of about 170 000 and subunit MWs of 43 000 and 34 500. The isoelectric point of the enzyme was at pH 4.7. Both subunits had the same N-terminal amino acid sequence. The optimum temperature for the reductive action of the purified MDH was at 35 degrees C with 44% activity at 50 degrees C and only 15% activity at 60 degrees C. The enzyme was optimally active at pH 5.5 with 50% activity at pH 6.5 and only 35% activity at pH 5.0 for reduction of fructose. The optimum pH for the oxidation of mannitol to fructose was 7.0. The purified enzyme was quite stable at pH 4.5-8.0 and temperature up to 35 degrees C. The K(m) and V(max) values of the enzyme for the reduction of fructose to mannitol were 20 mM and 396 micromol/min/mg protein, respectively. It did not have any reductive activity on glucose, xylose, and arabinose. The activity of the enzyme on fructose was 4.27 times greater with NADPH than NADH as cofactor. This is the first highly NADPH-dependent MDH (EC 1.1.1.138) from a LAB. Comparative properties of the enzyme with other microbial MDHs are presented.     

Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.     

On the cellular localization of phosphoenolpyruvate carboxylase in Sorghum leaves

The localization of phosphoenol pyruvate carboxylase (EC 4.1.1.3.1.) in the leaf cells of Sorghum vulgare was investigated by using three techniques: the conventional aqueous and non aqueous methods gave conflicting results; the immunocytochemical techniques clearly showed that the enzyme is predominantly located in the cytoplasm of mesophyll cells.Abbreviations PEP phosphoenol pyruvate - PAG polyacrylamide gel - NADP MDH NADP malate dehydrogenase - FITC fluorescein isothiocyanate - SAB serum albumine bovine - DTT dithiothreitol - MDH malate dehydrogenase - ME malic enzyme - PBS phosphate buffer saline - PAP peroxidase anti-peroxidase     

High malic enzyme activity in tumor cells and its cross-reaction with antipigeon liver malic enzyme serum

Summary 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 antiserumin vitro, a time-dependent inactivation of the enzyme activity was observed. Protection of the enzyme activity against antiserum inactivation was afforded by NADP⁺ orL-malate. Metal Mn²⁺ gave little protection.     

Isozymes in the Culture Forms of Leishmania tarentolae*

SYNOPSIS. Leishmania tarentolae grown in Trager's defined medium C, blood brain heart infusion broth and blood agar contained 2 forms of malic dehydrogenase (MDH) after zone electrophoresis in potato starch: one at the point of origin and the other migrating towards the anode. The pH optimum with oxaloacetate as substrate was ? 8.35 for the anodal form and 7.50 for the point of origin enzyme. The Michaelis constant (Km) with oxaloacetate was 1.8–2.8 × 10^?5 M for the anodal form and 4.0 × 10^?5 M for the nonmigratory form. At pH 7.4, both MDHs were inhibited by oxaloacetate concentrations greater than 3.75 × 10^?4 M. Ratios of activity with different NAD analogs were dissimilar. A few of the non-migratory enzyme ratios corresponded with those reported for mitochondrial MDH. There was no correspondence between the ratios shown by anodal MDH and ratios reported either for mitochondrial MDH or for cytoplasmic MDH. The thionicotinamide analog was not utilized by point of origin MDH; however, the anodal form did show greater activity with this analog which is a characteristic of cytoplasmic MDH. Anodal MDH was more stable than non-migratory enzyme. Heat inactivation studies indicated 80% inactivation at 68°C for the anodal form and 100% inactivation at 37°C for the other form. The point of origin enzyme had a half life of about 48 hours at 4°C whereas anodal MDH was stable for at least one week at 4°C. Addition of enzyme stabilizing agents (Cleland's reagent, mercaptoethanol and gelatin) did not prevent breakdown of the non-migrating enzyme. Phosphate buffer increased the activity of the point of origin enzyme but had no effect on anodal MDH. On the basis of the above results, non-migratory enzyme is thought to be a variant of mitochondrial MDH. The characteristics of the anodal MDH do not readily indentify it as a typical mitochondrial or cytoplasmic type and it may be a modified type similar to those found in parasitic protozoa by other workers.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Studies on regulatory functions of malic enzymes. V. Comparative studies of malic enzymes in bacteria.

Screening of four malic enzymes--NAD-linked enzyme [EC 1.1.1.38], NAD, NADP-linked enzyme [EC 1.1.1.39], NADP-linked enzyme [EC 1.1.1.40], and D-malic enzyme--was carried out with cell-free extracts of the following 16 strains of bacteria by the aid of Sepharose 6B column chromatography: 9 strains of enteric bacteria, 3 strains of Pseudomonas, Alcaligenes faecalis, Agrobacterium tumefaciens, Rhodospirillum rubrum, and Clostridium tetanomorphum. All the strains tested contained at least one malic enzyme. The NADP-linked enzyme activity was found in all the strains except C. tetanomorphum, the NAD-linked enzyme activity in 12 strains--8 strains of enteric bacteria, 2 strains of Pseudomonas, Ag. tumefaciens, and C. tetanomorphum--and D-malic enzyme activity in 4 strains--A, aerogenes (IFO 3319 and 12059), Ps. fluorescens, and R. rubrum. The NADP-linked and NAD-linked enzyme activities of two strains of Pseudomonas were not separated by the chromatography. The available evidence suggested that the NAD, NADP-linked enzyme was not present in these 16 strains. The comparative studies of molecular, enzymatic, and serological properties of the malic enzymes in these 16 strains revealed a close similarity of the same types of malic enzymes among enteric bacteria.     

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     

Malate utilization by a group D Streptococcus: physiological properties and purification of an inducible malic enzyme

Growth of Streptococcus faecalis in the presence of l-malate resulted in the induction of a "malic enzyme" [l-malate:nicotinamide adenine dinucleotide (NAD) oxidoreductase (decarboxylating), E.C. 1.1.1.39]. Synthesis of the malic enzyme did not appear to be subject to catabolite repression by intermediate products of glucose or fructose dissimilation. However, malate utilization was inhibited during growth in the presence of glucose or fructose. The purified enzyme was specific for malate as substrate and NAD as cofactor. Mn(+2) or Mg(+2) was required for optimal activity and NH(4)Cl stimulated the reaction rate. Several lines of indirect evidence suggested that the streptococcal malic enzyme was involved primarily with energy production and not biosynthesis.     

Study of properties of NADP malate dehydrogenase from corn leaves]

Kinetic properties of purified chloroplast isoenzyme of the "malic" enzyme from corn leaves were studied. The enzyme had optimum activity at pH 8.0 and 36 degrees C. Under standart conditions the Michaelis constants for the "malic" enzyme with Mn2+ as cofactor are 0.091 mM for malate and 0.04 mM for NADP. In case of Mg2+ as cofactor they are 0.66 and 0.02 mM respectively. Respective Km values for the cofactors Mn2+ and Mg2+ are 0.018 and 0.091 mM. The activity of the "malic" enzyme was inhibited by reduced NADP and NAD, ATP, ADP, fructose-1,6-diphosphate, oxaloacetic, oxalic, glyoxylic, glycolic and alpha-ketoglutaric acids, as well as by phosphate anions and pyrophosphate. The inhibitory effect of all metabolites and ions is more pronounced in case of Mn, rather than Mg, used as cofactors for the reaction. A possibility of metabolic regulation of NADP-"malic" enzyme activity in the leaves of C4-plants, is discussed.     

Long-term consumption of a diet with moderate medium chain triglyceride content does not inhibit the activity of enzymes involved in hepatic lipogenesis in the rat

The activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), malic enzyme (EC 1.1.1.40), ATP-citrate lyase (EC 4.1.3.8), acetyl-CoA carboxylase (EC 6.4.1.2) and fatty acid synthetase were lower (-25 to -60%) in liver of rats fed during 45 days with a moderate long-chain triglycerides (LCT) content diet (32% of metabolizable energy, ME), than in control rats fed with a low fat diet (LCT, 10% of ME). However, the fall in malic enzyme activity was not significant. In contrast, these activities were higher (+40 to +160%) in rats fed with a diet with a moderate medium-chain triglycerides (MCT) content (32% of ME), than in control rats. Nevertheless, the increase in activity of malic enzyme and ATP-citrate lyase was more important. Contrary to LCTs, MCTs had no inhibitory effect on the activity of enzymes involved in hepatic lipogenesis.     

Mitochondrial malic enzyme from the crayfish abdomen muscle,purification, regulatory properties and possible physiological role

• 1.1. Mitochondrial malic enzyme (l-Malate: NADP oxidoreductase (oxaloacetate decarboxylating) EC 1.1.1.40) has been isolated from abdomen muscle of crayfish Orconectes limosus by chromatography on Sepharose 6B and DEAE cellulose. Specific activity of the purified enzyme was about 5 μmols per min per mg protein, which corresponds to about 30-fold purification.
• 2.2. This enzyme showed extremely small reversiblity, since the reaction in the direction of decarboxylation is at least 37, 190 and 760 times that for the carboxylation at pH 7.0, 7.5 and 8.0 respectively.
• 3.3. Purified enzyme showed allosteric properties, which was more accentuated at more alkaline pH (Hill coefficients were 1.1, 1.7 and 1.8 at pH 7.0, 7.5 and 8.0 respectively). The activity of malic enzyme was increased considerably in the presence of succinate and fumarate.
• 4.4. Mitochondira isolated from abdomen muscle of Orconectes limosus incubated in the presence of malate, fumate and succinate catalysed pyruvate production which was stimulated by ADP and inhibited by respiratory chain inhibitors.
• 5.5. NADH but not NADPH oxidation was catalysed by broken mitochondria or sonic particles. When NADPH and NAD were added simultaneously the rate of oxidation. This suggests the presence of active NADPH:NAD transhydrogenase in mitochondria isolated from the crayfish abdomen muscle.
• 6.6. A possible metabolic role for NADP-linked malic enzyme/transhydrogenase couple in abdomen muscle of crayfish Orconectes limosus is proposed.