Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

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.     

Evidence for the role of malic enzyme in the rapid oxidation of malate by cod heart mitochondria

Mitochondria isolated from the heart of cod (Gadus morrhua callarias) oxidized malate as the only exogenous substrate very rapidly. Pyruvate only slightly increased malate oxidation by these mitochondria. This is in contrast with the mitochondria isolated from rat and rabbit heart which oxidized malate very slowly unless pyruvate was added. Arsenite and hydroxymalonate (an inhibitor of malic enzyme) inhibited the respiration rate of mitochondria isolated from cod heart, when malate was the only exogenous substrate. Inhibition caused by hydroxymalonate was reversed by the addition of pyruvate. In the presence of arsenite, malate was converted to pyruvate by cod heart mitochondria. Cod heart mitochondria incubated in the medium containing Triton X-100 catalyzed the reduction of NADP+ in the presence of L-malate and Mn2+ at relatively high rate (about 160 nmoles NADPH formed/min/mg mitochondrial protein). The oxidative decarboxylation of malate was also taking place when NADP+ was replaced by NAD+ (about 25 nmol NADH formed per min per mg mitochondrial protein). These results suggest that the mitochondria contain both NAD+- and NADP+-linked malic enzymes. These two activities were eluted from DEAE-Sephacel as two independent peaks. It is concluded that malic enzyme activity (presumably both NAD+- and NADP+-linked) is responsible for the rapid oxidation of malate (as the only external substrate) by cod heart mitochondria.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Purification and properties of cytosolic malic enzyme from human skeletal muscle

1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Two malic enzymes in Pseudomonas aeruginosa

Cell-free extract supernatant fluids of Pseudomonas aeruginosa were shown to lack malic dehydrogenase but possess a nicotinamide adenine dinucleotide (NAD)- or NAD phosphate (NADP)-dependent enzymatic activity, with properties suggesting a malic enzyme (malate + NAD (NADP) --> pyruvate + reduced NAD (NADH) (reduced NADP [NADPH] + CO(2)), in agreement with earlier findings. This was confirmed by determining the nature and stoichiometry of the reaction products. Differences in heat stability and partial purification of these activities demonstrated the existence of two malic enzymes, one specific for NAD and the other for NADP. Both enzymes require bivalent metal cations for activity, Mn(2+) being more effective than Mg(2+). The NADP-dependent enzyme is activated by K(+) and low concentrations of NH(4) (+). Both reactions are reversible, as shown by incubation with pyruvate, CO(2), NADH, or NADPH and Mn(2+). The molecular weights of the enzymes were estimated by gel filtration (270,000 for the NAD enzyme and 68,000 for the NADP enzyme) and by sucrose density gradient centrifugation (about 200,000 and 90,000, respectively).     

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.     

Regulation of coenzyme utilization by mitochondrial NAD(P)-dependent malic enzyme

1. Skeletal muscle mitochondrial NAD(P)-dependent malic enzyme [EC 1.1.1. 39, L-malate:NAD+ oxidoreductase (decarboxylating)] from herring could use both coenzymes, NAD and NADP, in a similar manner. 2. The coenzyme preference of mitochondrial NAD(P)-dependent malic enzyme was probed using dual wavelength spectroscopy and pairing the natural coenzymes, NAD or NADP with their respective thionicotinamide analogues, s-NADP or s-NAD, that have absorbance maxima in reduced forms at 400 nm. 3. s-NAD and s-NADP were found to be good alternate substrates for NAD(P)-dependent malic enzyme, the apparent Km values for the thioderivatives were similar to those of the corresponding natural coenzymes. 4. ATP produced greater inhibition of the NAD or s-NAD linked reactions than of the NADP or s-NADP-linked reactions of skeletal muscle mitochondrial NAD(P)-dependent malic enzyme. 5. At 5 mM malate concentration and in the presence of 2 mM ATP the NADP-linked reaction is favoured and the activity ratios, V(s-NADP)/V(NAD) or V(NADP)/V(s-NAD), are 6 and 26, respectively.     

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.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L.

A divalent cation electrode was used to measure the stability constants (association constants) for the magnesium and manganese complexes of the substrates for the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) from pea stems. At an ionic strength of 26.5 mM and at pH 7.4 the stability constants for the Mg2+-isocitrate and Mg2+-NADP+ complexes were 0.85 +/- 0.2 and 0.43 +/- 0.04 mM-1 respectively and for the Mn2+-isocitrate and Mn2+-NADP+ complexes they were 1.25 +/- 0.07 and 0.75 +/- 0.09 mM-1 respectively. At the same ionic strength but at pH 6.0 the Mg2+-NADPH and Mn2+-NADPH complexes had stability constants of 0.95 +/- 0.23 and 1.79 +/- 0.34 mM-1 respectively. Oxalosuccinate and alpha-ketoglutarate do not form measureable complexes under these conditions. Saturation kinetics of the enzyme with respect to isocitrate and metal ions are consistent with the metal-isocitrate complex being the substrate for the enzyme. NADP+ binds to the enzyme in the free form. Saturation kinetics of NADPH and Mn2+ indicate that the metal-NADPH complex is the substrate in the reverse reaction. In contrast the pig heart enzyme appears to bind free NADPH and Mn2+. A scheme for the reaction mechanism is presented and the difference between the reversibility of the NAD+ and NADP+ enzyme is discussed in relation to the stability of the NADH and NADPH metal complexes.     

Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.     

Structural studies of the pigeon cytosolic NADP(+)-dependent malic enzyme.

Malic enzymes are widely distributed in nature, and have important biological functions. They catalyze the oxidative decarboxylation of malate to produce pyruvate and CO(2) in the presence of divalent cations (Mg(2+), Mn(2+)). Most malic enzymes have a clear selectivity for the dinucleotide cofactor, being able to use either NAD(+) or NADP(+), but not both. Structural studies of the human mitochondrial NAD(+)-dependent malic enzyme established that malic enzymes belong to a new class of oxidative decarboxylases. Here we report the crystal structure of the pigeon cytosolic NADP(+)-dependent malic enzyme, in a closed form, in a quaternary complex with NADP(+), Mn(2+), and oxalate. This represents the first structural information on an NADP(+)-dependent malic enzyme. Despite the sequence conservation, there are large differences in several regions of the pigeon enzyme structure compared to the human enzyme. One region of such differences is at the binding site for the 2'-phosphate group of the NADP(+) cofactor, which helps define the cofactor selectivity of the enzymes. Specifically, the structural information suggests Lys362 may have an important role in the NADP(+) selectivity of the pigeon enzyme, confirming our earlier kinetic observations on the K362A mutant. Our structural studies also revealed differences in the organization of the tetramer between the pigeon and the human enzymes, although the pigeon enzyme still obeys 222 symmetry.     

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.     

NADP-Utilizing Enzymes in the Matrix of Plant Mitochondria

Purified potato tuber (Solanum tuberosum L. cv Bintie) mitochondria contain soluble, highly latent NAD⁺- and NADP⁺-isocitrate dehydrogenases, NAD⁺- and NADP⁺-malate dehydrogenases, as well as an NADPH-specific glutathione reductase (160, 25, 7200, 160, and 16 nanomoles NAD(P)H per minute and milligram protein, respectively). The two isocitrate dehydrogenase activities, but not the two malate dehydrogenase activities, could be separated by ammonium sulfate precipitation. Thus, the NADP⁺-isocitrate dehydrogenase activity is due to a separate matrix enzyme, whereas the NADP⁺-malate dehydrogenase activity is probably due to unspecificity of the NAD⁺-malate dehydrogenase. NADP⁺-specific isocitrate dehydrogenase had much lower K_ms for NADP⁺ and isocitrate (5.1 and 10.7 micromolar, respectively) than the NAD⁺-specific enzyme (101 micromolar for NAD⁺ and 184 micromolar for isocitrate). A broad activity optimum at pH 7.4 to 9.0 was found for the NADP⁺-specific isocitrate dehydrogenase whereas the NAD⁺-specific enzyme had a sharp optimum at pH 7.8. Externally added NADP⁺ stimulated both isocitrate and malate oxidation by intact mitochondria under conditions where external NADPH oxidation was inhibited. This shows that (a) NADP⁺ is taken up by the mitochondria across the inner membrane and into the matrix, and (b) NADP⁺-reducing activities of malate dehydrogenase and the NADP⁺-specific isocitrate dehydrogenase in the matrix can contribute to electron transport in intact plant mitochondria. The physiological relevance of mitochondrial NADP(H) and soluble NADP(H)-consuming enzymes is discussed in relation to other known mitochondrial NADP(H)-utilizing enzymes.     

Characterization of two members of a novel malic enzyme class.

The Gram-negative bacterium Rhizobium meliloti contains two distinct malic enzymes. We report the purification of the two isozymes to homogeneity, and their in vitro characterization. Both enzymes exhibit unusually high subunit molecular weights of about 82 kDa. The NAD(P)(+) specific malic enzyme [EC 1.1.1.39] exhibits positive co-operativity with respect to malate, but Michaelis-Menten type behavior with respect to the co-factors NAD(+) or NADP(+). The enzyme is subject to substrate inhibition, and shows allosteric regulation by acetyl-CoA, an effect that has so far only been described for some NADP(+) dependent malic enzymes. Its activity is positively regulated by succinate and fumarate. In contrast to the NAD(P)(+) specific malic enzyme, the NADP(+) dependent malic enzyme [EC 1.1.1.40] shows Michaelis-Menten type behavior with respect to malate and NADP(+). Apart from product inhibition, the enzyme is not subjected to any regulatory mechanism. Neither reductive carboxylation of pyruvate, nor decarboxylation of oxaloacetate, could be detected for either malic enzyme. Our characterization of the two R. meliloti malic enzymes therefore suggests a number of features uncharacteristic for malic enzymes described so far.     

Nicotinamide-adenine dinucleotide-linked "malic" enzyme in flight muscle of the tse-tse fly (Glossina) and other insects.

1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate