Mitochondrial malate dehydrogenase

Lactate dehydrogenase Cytoplasmic malate dehydrogenase Mitochondrial malate dehydrogenase Ehrlich ascites tumour cells... 

The second electron shuttle system, called the malate-aspartate shuttle, is shown in Figure 21.34. Oxaloacetate is reduced in the cytosol, acquiring the electrons of NADH (which is oxidized to NAD ). Malate is transported across the inner membrane, where it is reoxidized by malate dehydrogenase, converting NAD to NADH in the matrix. This mitochondrial NADH readily enters the electron transport chain. The oxaloacetate produced in this reaction cannot cross the inner membrane and must be transaminated to form aspartate, which can be transported across the membrane to the cytosolic side. Transamination in the cytosol recycles aspartate back to oxaloacetate. In contrast to the glycerol phosphate shuttle, the malate-aspartate cycle is reversible, and it operates as shown in Figure 21.34 only if the NADH/NAD ratio in the cytosol is higher than the ratio in the matrix. Because this shuttle produces NADH in the matrix, the full 2.5 ATPs per NADH are recovered. 

In pigeon, chicken, and rabbit liver, phospho-enolpymvate carboxykinase is a mitochondrial enzyme, and phosphoenolpyruvate is transported into the cytosol for gluconeogenesis. In the rat and the mouse, the enzyme is cytosolic. Oxaloacetate does not cross the mitochondrial inner membrane it is converted to malate, which is transported into the cytosol, and convetted back to oxaloacetate by cytosolic malate dehydrogenase. In humans, the guinea pig, and the cow, the enzyme is equally disttibuted between mitochondria and cytosol. 

Mitochondrial L-malate dehydrogenase (bovine heart muscle)1501... 

The malate-aspartate shuttle is the most important pathway for transferring reducing equivalents from the cytosol to the mitochondria in brain. This shuttle involves both the cytosolic and mitochondrial forms of aspartate aminotransferase and malate dehydrogenase, the mitochondrial aspartate-glutamate carrier and the dicarboxylic acid carrier in brain (Fig. 31-5) [69]. The electrogenic exchange of aspartate for glutamate and a... 

In an earlier spectrophotometric study of this enzyme, a red shift of the reduced nicotinamide absorbance had been noted in the difference spectrum of the binding of reduced coenzyme to the purified protein. Fisher et al.92> had pointed out that this is characteristic of most B-stereo-specific dehydrogenases, so Biellman et al. have made a successful prediction for Fisher. Fisher s suggestion that the supernatant and mitochondrial forms of malate dehydrogenase have different stereospecificities for NAD+ has not been substantiated, however 89>. 

In common with cholesterol synthesis described in the next section, fatty acids are derived from glucose-derived acetyl-CoA. In the fed state when glucose is plentiful and more than sufficient acetyl-CoA is available to supply the TCA cycle, carbon atoms are transported out of the mitochondrion as citrate (Figure 6.8). Once in the cytosol, citrate lyase forms acetyl-CoA and oxaloacetate (OAA) from the citrate. The OAA cannot re-enter the mitochondrion but is converted into malate by cytosolic malate dehydrogenase (cMDH) and then back into OAA by mitochondrial MDH (mMDH) Acetyl-CoA remains in the cytosol and is available for fatty acid synthesis. 

C. J. R. Thorne and N. O. Kaplan, Physicochemical properties of pig and horse heart mitochondrial malate dehydrogenase, J. Biol. Chem. 238, 1861-1868 (1963). 

J. Muller, M.-F. Manent, and G. Pfleiderer, Importance of tyrosine for structure and function of mitochondrial malate dehydrogenases, Biochim. Biophys. Acta 742, 189-196 (1983). 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

In the malate shuttle (left)—which operates in the heart, liver, and kidneys, for example-oxaloacetic acid is reduced to malate by malate dehydrogenase (MDH, [2a]) with the help of NADH+HT In antiport for 2-oxogluta-rate, malate is transferred to the matrix, where the mitochondrial isoenzyme for MDH [2b] regenerates oxaloacetic acid and NADH+HT The latter is reoxidized by complex I of the respiratory chain, while oxaloacetic acid, for which a transporter is not available in the inner membrane, is first transaminated to aspartate by aspartate aminotransferase (AST, [3a]). Aspartate leaves the matrix again, and in the cytoplasm once again supplies oxalo-acetate for step [2a] and glutamate for return transport into the matrix [3b]. On balance, only NADH+H"" is moved from the cytoplasm into the matrix ATP is not needed for this. 

In this pathway the electrons for the drug reduction are generated by the oxidative decarboxylation of malate catalyzed by the NAD-dependent malic enzyme (malate dehydrogenase (decarboxylating)). The NADH produced by this reaction is reoxidized by an enzyme with NADH ferredoxin oxidoreductase activity that has been recently identified as a homologue of the NADH dehydrogenase (NDH) module of the mitochondrial respiratory complex I (Hrdy et al. 2004 and see Hrdy et al., this volume). The... 

Because the mitochondrial membrane has no transporter for oxaloacetate, before export to the cytosol the oxaloacetate formed from pyruvate must be reduced to malate by mitochondrial malate dehydrogenase, at the expense of NADH ... 

The standard free-energy change for this reaction is quite high, but under physiological conditions (including a very low concentration of oxaloacetate) AG 0 and the reaction is readily reversible. Mitochondrial malate dehydrogenase functions in both gluconeogenesis and the citric acid cycle, but the overall flow of metabolites in the two processes is in opposite directions. 

However, the urea cycle also causes a net conversion of oxaloacetate to fumarate (via aspartate), and the regeneration of oxaloacetate (Fig. 18-12) produces NADH in the malate dehydrogenase reaction. Each NADH molecule can generate up to 2.5 ATP during mitochondrial... 

OAA is unable to directly cross the inner mitochondrial membrane it must first be reduced to malate by mitochondrial malate dehydrogenase. Malate can be transported from the mitochondria to the cytosol, where it is reoxidized to oxaloacetate by cytosolic malate dehydrogenase (see Figure 10.3). 

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. 

Mitochondrial fractions may be characterized by testing for the presence of known enzyme activities as previously discussed. The relative purity of each fraction can be estimated by measuring the specific activity of marker enzymes. Table El0.1 identifies marker enzymes for the matrix and membranes. Malate dehydrogenase, the tricarboxylic acid cycle enzyme that catalyzes the interconversion of malate and oxaloacetate (Equation E10.1), serves as a marker for the matrix enzymes. 

Malate dehydrogenase activity would be expected in intact mitochondria, but not in SMPs. The activity of this enzyme in mitochondrial fractions may be estimated by a spectrophotometric assay. Oxaloacetate and NADH are incubated, and the disappearance of NADH is monitored at 340 nm. NAD+ does not have strong absorption at this wavelength. Note that the reverse reaction is studied because the reaction as shown above is very unfavorable in thermodynamic terms ACT = +30 kj/mol). 

Now calculate the proportion of total mitochondrial protein (malate dehydrogenase and monoamine oxidase) present in the submitochondrial fraction. 

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