Malate isolation

OC-Hydroxycarboxylic Acid Complexes. Water-soluble titanium lactate complexes can be prepared by reactions of an aqueous solution of a titanium salt, such as TiCl, titanyl sulfate, or titanyl nitrate, with calcium, strontium, or barium lactate. The insoluble metal sulfate is filtered off and the filtrate neutralized using an alkaline metal hydroxide or carbonate, ammonium hydroxide, amine, or alkanolamine (78,79). Similar solutions of titanium lactate, malate, tartrate, and citrate can be produced by hydrolyzation of titanium salts, such as TiCl, in strongly (>pH 10) alkaline water isolation of the... 

Substantial amounts of polymalatase have been isolated from plasmodial extracts. This may refer to stored enzyme before secretion, because /3-poly(L-malate) is not degraded in plasmodia [24]. Several other fungi were found to secrete /3-poly(L-malate) degrading activities to L-malic acid (Ratberger, Molitoris and Holler, unpublished results). These enzymes have not yet been purified and characterized. 

Acid-catalyzed hydration of isolated double bonds is also uncommon in biological pathways. More frequently, biological hydrations require that the double bond be adjacent to a carbonyl group for reaction to proceed. Fumarate, for instance, is hydrated to give malate as one step in the citric acid cycle of food metabolism. Note that the requirement for an adjacent carbonyl group in the addition of water is the same as that we saw in Section 7.1 for the elimination of water. We ll see the reason for the requirement in Section 19.13, but might note for now that the reaction is not an electrophilic addition but instead occurs... 

D. L. Jones, A. M. Prabowo, and L, V, Kochian, Kinetics of malate tran.sport and decomposition in acid soils and isolated bacterial populations the effect of microorganisms on root exudation of malate under A1 stress. Plant Soil /((2 239 (1996). 

Poly-j3-malate is readily degraded completely to L-malic acid under both acid and base conditions [108], and it can also be hydrolyzed by enzymes within the cell [105,106]. Recently, several bacteria were isolated which were able to utilize poly-/i-malate as sole carbon source for growth [109]. Because the polymer is biodegradable and bioadsorbable, it is of considerable interest for pharmaceutical applications, especially in controlled-release drug delivery systems [97,98]. Chemical routes to poly-/ -malate are expected to provide materials with various properties [110]. 

Poly(L-malate) [poly(malic acid) (PMA)], is a water-soluble polyanion produced by slime molds and some yeasts such as Physarum polycephalum or Aureobasidium pullulans, respectively. Its function and metabolism has been studied during the last few years [122-125]. Recently, several PMA-degrad-ing bacteria have been isolated, and a cytoplasmic membrane-bound PMA hydrolase was purified from Comamonas acidovans strain 7789 [126] that... 

Abnormalities of the respiratoiy chain. These are increasingly identified as the hallmark of mitochondrial diseases or mitochondrial encephalomyopathies [13]. They can be identified on the basis of polarographic studies showing differential impairment in the ability of isolated intact mitochondria to use different substrates. For example, defective respiration with NAD-dependent substrates, such as pyruvate and malate, but normal respiration with FAD-dependent substrates, such as succinate, suggests an isolated defect of complex I (Fig. 42-3). However, defective respiration with both types of substrates in the presence of normal cytochrome c oxidase activity, also termed complex IV, localizes the lesions to complex III (Fig. 42-3). Because frozen muscle is much more commonly available than fresh tissue, electron transport is usually measured through discrete portions of the respiratory chain. Thus, isolated defects of NADH-cytochrome c reductase, or NADH-coenzyme Q (CoQ) reductase suggest a problem within complex I, while a simultaneous defect of NADH and succinate-cytochrome c reductase activities points to a biochemical error in complex III (Fig. 42-3). Isolated defects of complex III can be confirmed by measuring reduced CoQ-cytochrome c reductase activity. 

Pritchard and co-workers (147) reported the preparation of an acyclic chiral sulfite, 98, which is formed as a mixture of diastereo-mers in the reaction between racemic methyl chlorosulfite and (+)-diphenacyl malate. The pure diastereomers of 98 were isolated by fractional crystallization. 

There have been a number of isolated studies of metal-ion catalyzed nucleophilic reactions of other groupings. Particularly interesting is the induced nucleophilic attack on olefins. Hydration is normally very sluggish. Enzymes can speed up such reactions. Aconitase, an iron-containing enzyme, catalyzes the isomerization of citric acid to isocitric acid, through the intermediacy of cis-aconitic acid. A possible mechanism has been suggested based on the following Co(III) model chemistry. Rapid cyclization of the maleate ester produces Ai and AS chelated malate half ester ... 

Figure 2. Isozymer banding pattern in PAG of Protogonyaulax tamarensis morphotypes stained for malic enzyme (ME/NADP-dependent malate dehydrogenase).400-406 English Bay isolates ( indicates clonal). 255 Lummi Island (Washington State).

In studies with Isolated plant mitochondria, flavones, flava-nones, cinnamic acids, and benzoic acids were shown to Inhibit the oxidation of succinate, malate, and NADH Inhibition was... 

The hydrogenosomal membrane displays selective permeability, thereby presenting an effective barrier to pyridine nucleotides and coenzyme A (Cerkasovov et al. 1978 Lindmark and Muller 1973 Muller 1973 Steinbiichel and Muller 1986). Transport of metabolic substrates and products across the hydrogenosomal membrane remains to be studied, but isolated T. foetus hydrogenosomes were shown to readily accumulate both radiolabeled pyruvate and malate (our unpublished data). 

In Leuconostoc oenos ML 34, we have shown oxaloacetic acid decarboxylation manometrically (6, 7, 8). We were also able to demonstate fluorometrically the enzymatic production of reduced NAD with malic acid as a substrate, but, of course, were unable to do so with oxaloacetic acid since no NADH could be formed from this substrate. It is likely that this oxaloacetic acid decarboxylation activity, as in Lactobacillus plantarum, is distinct from the activity causing the malic-lactic transition. It is also possible that oxaloacetic acid decarboxylation is caused by a malic enzyme. However, there is no verified NAD dependent malic oxidoreductase (decarboxylating) enzyme which does so (12). For example, Macrae (31) isolated a malic enzyme from cauliflower bud mitochondria which showed no activity with oxaloacetic acid. Similarly, Saz (32) isolated a malic enzyme from Ascaris lumbricoides which is also inactive toward oxaloacetic acid. True, the Enzyme Commission (12) lists an enzyme described as L-malate NAD oxidoreductase (decarboxylating) (E.C. 1.1.1.38) which is said to be capable of decarboxylating oxaloacetic acid, but its description dates back to the studies of Ochoa and his group, and we now feel this listing may be improper. 

In discussion of nomenclature of malic acid decomposing enzymes, mention should be made of malate-lactate transhydrogenase. This enzyme, isolated from Micrococcus lactiyticus (VielloneUa alcalescens), a bacterium found in vertibrates, can catalyze reversibly the conversion of L-malic and pyruvic acids to L-lactic and oxaloacetic acids with NAD as coenzyme (36). 

Another example is provided by malic acid, a chiral molecule which also contains a prochiral center (see Eq. 9-74). In this case replacement of the pro-R or pro-S hydrogen atom by another atom or group would yield a pair of diastereoisomers rather than enantiomers. Therefore, these hydrogen atoms are diastereotopic. When L-malic acid is dehydrated by fumarate hydratase (Chapter 13) the hydrogen in the pro-R position is removed but that in the pro-S position is not touched. This can be demonstrated by allowing the dehydration product, fumarate, to be hydrated to malate in 2HzO (Eq. 9-74). The malate formed contains deuterium in the pro-R position. If this malate is now isolated and placed with another portion of enzyme in H20, the deuterium is removed cleanly. The fumarate produced contains no deuterium. 

Transaminases participate in metabolism of most of the amino acids, over 60 different enzymes have been identified.142163 Best studied are the aspartate aminotransferases, a pair of cytosolic and mitochondrial isoenzymes which can be isolated readily from animal hearts. Their presence in heart muscle and brain in high concentration is thought to be a result of their functioning in the malate-aspartate shuttle... 

Many of the biochemical processes that generate chemical energy for the cell take place in the mitochondria. These organelles contain the biochemical equipment necessary for fatty acid oxidation, di- and tricarboxylic acid oxidation, amino acid oxidation, electron transport, and ATP generation. In this experiment, a mitochondrial fraction will be isolated from beef heart muscle. The mitochondria will be analyzed for protein content and fractionated into submitochondrial particles. Each fraction will be analyzed for malate dehydrogenase and monoamine oxidase activities. 

Students will isolate intact mitochondria from beef heart and fractionate them to prepare submitochondrial particles. Each fraction will be characterized by protein estimation by the biuret method and measurement of malate dehydrogenase and monoamine oxidase activity. 

Immobilied Cibacron Blue 3G-A has been found very efficient for the isolation of human serum albumin interferons produced in tissue cultures phosphogly-cerate kinase from Saccharomyces cerevisiae and alcohol dehydrogenase from equine liver. Immobilized Procion Red HE-3B and Prcxaon Blue MX-4GD have been successfully applied for the isolation of 3-hydroxybutyrate and malate dehydrogenases... 

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