Malic dehydrogenase enzyme

Further evidence of like character in support of the participation of coenzyme I in reactions associated with the reduction of dehydroascorbic acid has been advanced by Waygood (1950). Cell-free extracts of wheat seedlings were found to contain a malic dehydrogenase enzyme, reducing coenzyme I, as well as ascorbic oxidase and peroxidase enzymes. When to such extracts malic acid, coenzyme I, and ascorbic acid were added, together with a fixative for the oxalacetate formed in the reaction, the system absorbed oxygen in excess of that required for the complete oxidation of ascorbic acid. In this system methylene blue could replace ascorbic acid. 

When linked enzyme assays are used, the exogenous added enzymes may also be contaminated with small traces of the primary enzyme whose activity is measured, thereby leading to falsely high activities In this instance it is also desirable to make certain that the added enzymes are free of any undesirable activity, i.e, pig heart malic dehydrogenase should be free of GOT activity when used for GOT assays (17),... 

The enzyme reagent is added, malic dehydrogenase glucose-6-dehydrogenase... 

NADH as an end product. This implicates oxidized malic acid, either pyruvic or oxaloacetic acid, as another end product. By adding commercial preparations of L-lactic dehydrogenase or malic dehydrogenase to the reaction mixture, Morenzoni (90) concluded that the end product was pyruvic acid. Attempts were then made to show whether two enzymes—malate carboxy lyase and the classic malic enzyme, malate oxidoreductase (decarboxylating), were involved or if the two activities were on the same enzyme. The preponderance of evidence indicated that only one enzyme is involved. This evidence came from temperature inactivation studies, heavy-metal inhibition studies, and ratio measurements of the two activities of partially purified preparations of Schiitz and Radlers malo-lactic enzyme (76, 90). This is not the first case of a single enzyme having two different activities (91). 

Linked oxidation and decarboxylation. Metabolic pathways often make use of oxidation of a (3-hydroxy acid to a (3-oxoacid followed by decarboxylation in the active site of the same enzyme. An example is conversion of L-malate to pyruvate (Eq. 13-45). The Mg2+ or Mn2+-dependent decarboxylating malic dehydrogenase that catalyzes the reaction is usually called the malic enzyme. It is found in most organisms.237-240 While a concerted decarboxylation and dehydrogenation may sometimes occur,241-242 the enzymes of this group appear usually to operate with bound oxoacid intermediates as in Eq. 13-45. 

Malic dehydrogenase—Prepare this reagent just before use and store at 4°C. Dilute commercially available enzyme in 0.10 M potassium phosphate buffer, pH 7.5, in which 1 mg/ml bovine serum albumin has been dissolved. The final concentration of malic dehydrogenase should be 200 units/ml. Prepare a total of 20 ml of this reagent. 

R. K. Gerding and R. G. Wolfe, J. Biol. Chem., 244 1164 (1%9). Malic Dehydrogenase. VIII. Large Scale Purification and Properties of Supernatant Pig Heart Enzyme. 

Following are a set of assay conditions for marker enzymes of mitochondria (citrate synthetase, malic dehydrogenase, fumarase, and succinate dehydrogenase) and glyoxysomes (citrate synthetase, malate synthetase, and malic dehydrogenase). Some or all of these activities may be assayed across the density gradient. Their quantitative distribution is shown in Table 9-2. 

Both are abundant in skeletal muscle, myocardium, liver, and erythrocytes, so that hemolysis must be avoided, and in serum they may be assayed spectrophotometrically by their conversion of phosphate-buffered pyruvate to lactate (R6, W16) or oxalacetate to malate (S25) at the expense of added NADH2, when the rate of decrease of optical density at 340 m x thus measmes the serum activities of the respective enzymes. Recently, however, the reverse reaction has been found best for serum lactic dehydrogenase assay (A2a). In conventional spectrophotometric units the normal ranges are 100-600 units per ml for lactic dehydrogenase (W16) and 42-195 xmits per ml for malic dehydrogenase (S25) as before, one conventional spectrophotometric unit per ml = 0.48 pmoles/ minute/liter (W13). 

Glutamic-oxalacetate transaminase (GOT) is released into the blood stream as a result of myocardial infarction. The enzyme is assayed in serum by following the decrease in the absorbance of NADH in the malic dehydrogenase (MDH)-coupled reaction sequence shown below. 

A reaction mixture contained excess aspartate (i.e., lOO times its value), 0.1 ml of serum, 0.3 /rmole of NADH, and an excess of malic dehydrogenase in a total volume of 0.9 ml. The reaction was started by adding an excess of a-ketoglutarate in 0.1 ml. After a short lag, the absorbance decreased at a rate of 0.04 A unit/min. The cuvette had a light path of 1 cm. Calculate the concentration of GOT in the patient s serum (i.e., the specific activity of the serum in terms of enzyme units/ml). 

Many metabolic processes such as glycolysis, Krebs cycle reactions, photosynthesis, protein synthesis, and lipid metabolism are affected by exposure to F. Much of the action of F on these processes can be attributed to F-dependent inhibition of enzymes. Examples of enzymes shown to be inhibited by F include enolase, phosphoglucomutase, phosphatase, hexokinase, PEP carboxylase, pyruvate kinase, succinic dehydrogenase, malic dehydrogenase, pyrophosphatase, phytase, nitrate reductase, mitochondrial ATPase, urease (Miller et al. 1983), lipase (Yu et al. 1987), amylase (Yu et al. 1988), invertase (Yu 1996 Ouchi et al. 1999), and superoxide dismutase (SOD) (Wilde and Yu 1998). 

Fig. 1-13. Krebs cycle. A condensing enzyme, B Aconitase, C Isocitric dehydrogenase, a-ketogluta-rate decarboxylase, E Succinic dehydrogenase, F F"u-marase, G Malic dehydrogenase...

Various relationships between enzymes of the Krebs cycle and mitochondria are possible. For instance, all enzymes could be enclosed within mitochondrial structures or the enzymes could take part in the structural build-up of the cell. There is no evidence demonstrating that all enzymes of the Krebs cycle are part of the mitochondria. The existence of enzymes with multiple catalytic properties (isocitric dehydrogenase, aconitase, and malic dehydrogenase) and the failure to separate the multiple steps of an overall reaction (pyruvic and a-ketoglutarate oxidation) are sometimes taken as evidence for the participation of the enzyme in the building-up of the mitochondrial structure, but these arguments do not take into account the limitations of the actual biochemical methods, and, therefore, conclusions based upon them are premature. 

In spite of the absence of integral mitochondria, the red cell contains some of the enzymes (fumerase, isocitric dehydrogenase, malic dehydrogenase, and cytochrome oxidase) functioning in the Krebs cycle and electron transport. These enzymes probably represent mitochondrial remnants, and their presence in the mature erythrocyte may be a consequence of their greater stability. Similarly, enzymes concerned with... 

Malic Dehydrogenase. The oxidation of the hydroxy dicarboxylic acid, malic acid, is catalyzed by two different enzymes a simple dehydrogenase, employing pjrridine nucleotides to oxidize malate to oxalacetate (VII), and an enzyme that catalyzes an oxidative decarboxylation (VIII). 

The former is called malic dehydrogenase. Although it was reported early that the enzyme is specific for DPN, in fact malic dehydrogenase from several sources has been found to react with TPN at 5-7 per cent of the rate of the DPN reaction. The affinities for DPN and TPN are quite similar, Kn equals about 10 at neutral pH. The equilibrium of the reaction at neutral pH values lies far to the side of malate and DPN, but, as discussed previously, at higher pH values the equilibrium of reactions in which H+ participates is shifted, and near pH 10 the oxidation of malate proceeds to a considerable extent and at a good rate. Even at low pH values, however, the oxidation of malate is readily carried out when coupled with an effective system for oxidizing DPNH or removing oxalacetate (as citrate formation). 

The equilibrium constant favours the reaction occurring from left to right but the reverse reaction occurs if the TPN formed is continuously removed. The oxaloacetic acid is shown in brackets because it does not appear in the free form during the reaction. Under the action of the enzyme it is decarboxylated straight away the enzyme must not be confused with malic dehydrogenase which requires DPN as its coenzyme. 

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