Malic enzyme development

Multiple genes encode the enzymes involved in metronidazole reduction, and the expression of their corresponding proteins during resistance development is only known for the dominant isoforms. The potential role of other isoforms of malic enzyme, PFOR, and ferredoxin in metronidazole activation is unknown. It was found that the dominant ferredoxin (ferredoxin 1), one of seven ferredoxin genes identified in the I vaginalis genome (Carlton et al. 

Goodridge and co-workers (see Ref. 76) have developed a system of chick embryo hepatocytes which, when cultured in defined medium, respond to T3, insulin and glucagon. Low concentrations of T3 (K50 4xl0 n M) increase by 15-fold the malic enzyme level and 7-fold the concentration of its mRNA [77]. Insulin alone had no effect both on the enzyme and the mRNA levels, whereas in combination with T3 it caused an 11-fold increase in malic enzyme mRNA levels. Glucagon almost completely abolished the stimulatory effect caused by insulin + T3. Experiments performed with puromycin showed that this inhibitor of protein translation blocks the accumulation of malic enzyme mRNA stimulated by T3 suggesting that most of the T3 effect on malic enzyme takes place at a post-transcriptional step. Glucagon had no effect on transcription but caused malic enzyme mRNA to decay... 

The C4 cycle can be viewed as an ATP-dependent C02 pump that delivers C02 from the mesophyll cells to the bundle-sheath cells, thereby suppressing photorespiration (Hatch and Osmond, 1976). The development of the C4 syndrome has resulted in considerable modifications of inter- and intracellular transport processes. Perhaps the most striking development with regard to the formation of assimilates is that sucrose and starch formation are not only compartmented within cells, but in C4 plants also may be largely compartmented between mesophyll and bundle-sheath cells. This has been achieved together with a profound alteration of the Benson-Calvin cycle function, in that 3PGA reduction is shared between the bundle-sheath and mesophyll chloroplasts in all the C4 subtypes. Moreover, since C4 plants are polyphyletic in origin, several different metabolic and structural answers have arisen in response to the same problem of how to concentrate C02. C4 plants have three distinct mechanisms based on decarboxylation by NADP+-malic enzyme, by NAD+-malic enzyme, or by phosphoenolpy-ruvate (PEP) carboxykinase in the bundle-sheath (Hatch and Osmond, 1976). 

Knowles and co-workers observed (V/K) at C-1 of glyoxalate by analyzing the isotopic composition of the malate produced in the reaction at early and late stages (23). In order to measure this ratio accurately they developed a procedure to convert C-2 of malate to carbon dioxide. Through a series of enzymic reactions, C-2 was converted to the carbonyl carbon of the acetyl group of acetyl-carnitine (through the reaction sequence malate — pyruvate — acetyl-CoA - acetylcarnitine). The labeled carbon then was carried flirough another cycle back to malate, but in this case the label was in the carboxylate carbon at C-4 (acetyl-carnitine acetyl-CoA 4-[ 3C]malate). Malic enzyme converts this to pyruvate and labeled carbon dioxide, whose isotopic ratio is determined by mass spectrometry. 

C4 plants of NADP malic enzyme type are characterized by dimorphic leaf chloto-plasts, i.e. their bundle sheath chloroplasts are almost agranal, and are lacking or at least largely depleted in photosystem II activity (15). On the other side, grana structures and the capability of linear electron transport have been found in bundle sheath chloroplasts of developing leaves (16) implying that photosystem II and, concomitantly, its constituent poly-... 

These two forms possessed anatomical structures of culms clearly differing from each other. The terrestrial forms had an unusual Kranz type of anatomy which is characterized by the presence of colourless mestome sheath cells intervening between the mesophyll cells and the Kranz cells (1,2,3). The chloroplasts with well-developed grana and many large mitochondria were scattered in the Kranz cells, although the terrestrial forms had biochemical features of the NAD-malic enzyme C4 subtype (2,3). The submersed forms possessed large spherical mesophyll cells and reduced vascular bundles, which are characteristic of submersed aquatic plants (2,4). Kranz cells contained relatively smaller chloroplasts than the Kranz cells of the terrestrial forms. 

TPNH and in the transport cycle that brings acetyl CoA out of the mitochondria. The contribution of the malic enzyme or other similar malo-lactic decomposing activities in the secondary fermentation of wine is also appreciated, but less well understood. But it can be said that this fermentation is crucial in the development of fine Burgundy and Bordeaux wines, and of the premium red wines of California, such as Cabernet Sauvignon. 

Bacteria do not transform all of the malic acid contained in the grape. From the start, during alcoholic fermentation, yeasts metabolize a maximum of 30% of the malic acid. The product, pyruvate, then enters one of many yeast metabolic pathways—notably leading to the formation of ethanol. This malo-alcoholic fermentation is catalyzed at the first stage by the malic enzyme. The bacteria must develop a sufficient population before malolactic fermentation can truly start. The production of L-lactic acid is coupled with the decrease in malic acid (Figure 6.3). 

Carbon for FAS could also be produced via ATP-citrate lyase. This activity, which will convert citrate to oxaloacetate plus acetyl-CoA, has been demonstrated in soybean extracts. But there is no current evidence for citrate transport into the plastid nor of localisation of this activity in the plctstid to support citrate cleaving enzyme as a source of carbon for FAS, at least for avocado mesoccirp plastids. Extracts of developing soybean also contain a NADP+-dependent medic enzyme.20 NAD+-dependent malic enzyme, which produces pyruvate ind Cctrbon dioxide from malate, is an enzyme specific to the mitochondrial matrix in higher pleints.21 The localisation of NADP+-malic enzyme in immature soybeans, eind the possibility of pyruvate production other than by pyruvate kinase, and the utilisation of this pyruvate in FAS, remain to be determined. 

Fumarase. The development and use of this immobilized enzyme by Tanabe Seiyaku for production of L-malic acid is very similar to that of aspartase ( 3). Lysed Brevibacterium ammoniagenes or B. flavin cells are treated with bile acid to destroy enzymatic activity which converts fumarate to succinate. As with aspartase, the cells can be immobilized in polyacrylamide or k-carrageenan gels. Using a substrate stream of 1 M sodium fumarate at pH 7.0 and 37°C, L-malic acid of high purity has been produced since 1974 by a continuous, automated process (3,39) for example, using a 1000-L fixed-bed bioreactor, 42.2 kg L-malic acid per hour was produced continuously for 6 months. 

CIC Vitamin C (300 mg/100 g fmit), citric acid and malic acid are responsible for the clean acidic taste of the fresh fruit. A high enzyme activity of a lipoxygenase produces high amounts of (E)-2-hexenal, hexanal and (E)-2-hexenol, all fruity green notes, characteristic of green apples. The cut surfaces develop at prolonged standing 2-heptenal, 2,4-nonadienal and l-octen-3-one, responsible for stale, fatty odour. Methyl butyrate and ethyl butyrate impart the ripe, fruity, estery, fresh, juicy impression. 

Three immobilized enzyme or microbial cell systems currently used industrially in synthesis of chiral amino acids plus one presently under development are described. L-amino acids are produced by enzymatic hydrolysis of DL-acylamino acid with aminoacylase immobilized by ionic binding to DEAE-Sephadex. Escherichia coli cells immobilized by K-carrageenan crosslinked with glutaraldehyde and hexamethylenediamine are used to convert fumaric acid and cimmonia to L-aspartic acid and Brevibacterium flavum cells similarly immobilized are used to hydrate fumaric acid to L-malic acid. The decarboxylation of L-aspcirtic acid by immobilized Pseudomonas dacunhae to L-alanine is currently under investigation. 

If a compound is nonfluorescent, it may be converted to a fluorescent derivative. For example, nonfluorescent steroids may be converted to fluorescent compounds by dehydration with concentrated sulfuric acid. These cyclic alcohols are converted to phenols. Similarly, dibasic acids, such as malic acid, may be reacted with j8-naphthol in concentrated sulfuric acid to form a fluorescing derivative. White and Argauer have developed fluorometric methods for many metals by forming chelates with organic compounds (see Ref. 23). Antibodies may be made to fluoresce by condensing them with fluorescein isocyanate, which reacts with the free amino groups of the proteins. NADH, the reduced form of nicotinamide adenine dmucleotide, fluoresces. It is a product or reactant (cofactor) in many enzyme reactions (see Chapter 24), and its fluorescence serves as the basis of the sensitive assay of enzymes and their substrates. Most amino acids do not fluoresce, but fluorescent derivatives are formed by reaction with dansyl chloride. 

The method of analysis developed is for acetic acid and involves the use of two enzymes. Chiral acetic acid [as 1.26)] is irreversibly condensed as its CoA-derivative with glyoxylic acid (/.27), using the enzyme malate synthase, to give malic acid 1.28). The condensation occurs with loss of hydrogen isotope by a primary kinetic isotope effect (A h kj). This means that loss of H is favoured over loss of D which is in turn favoured over loss of T. The result is a high retention of tritium. 

Ogston has recently put forward an idea which removes the difficulty of ascribing both reactions to one enzyme. It is a development of his theory discussed above of a three-point combination between enzyme and substrate. As already explained, the fumarate molecule in order to yield optically active malic acid must be so placed on the enzyme surface that only one double-bond component can react, but no direction need be exerted on the water in which H and OH are distributed. If it is now assumed that aconitase is constructed analogously to fumarase in that again no direction is exerted on the elements of water when they combine with aconitic acid, it is seen at once that two different compounds arise, namely, citric and isocitric acids. The occurrence of the reverse reaction would follow from the requirement of catalytic reversibility. 

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