Malate dehydrogenase assay

A less time consuming alternative to the saponifica-tion/malate dehydrogenase assay is the albeit less specific measurement of the carboxylic ester groups according to Kak c and Vejdelek [10]. The sample (160 /xl) is... 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

The reaction mixture for a coupled assay includes the substrates for the initial or test enzyme and also the additional enzymes and reagents necessary to convert the product of the first reaction into a detectable product of the final reaction. The enzyme aspartate aminotransferase (EC 2.6.1.1), for instance, results in the formation of oxaloacetate, which can be converted to malic acid by the enzyme malate dehydrogenase (EC 1.1.1.37) with the simultaneous conversion of NADH to NAD+, a reaction which can be followed spectropho-tometrically at 340 nm ... 

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

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). 

Use the plot of AiAQ vs. time to calculate AH/min over the linear portion of the curve. Convert the rate in absorbance terms to activity units. One enzyme unit is the amount of malate dehydrogenase that catalyzes the reduction of 1 micromole of oxaloacetate to L-malate in 1 minute under the described assay conditions. The reduction of 1 micromole of oxaloacetate leads to the oxidation of 1 micromole of NADH therefore, Equation E10.4 may be used to calculate the specific activity of malate dehydrogenase. 

B 10. You are required to assay a solution of purified malate dehydrogenase (specific activity = 1.0). What A d34Q/min will you observe if you use 1 mg of enzyme in a 3-mL assay as described in part D of this experiment ... 

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). 

Fig. 2 Heat inactivation of Thermus PEPCs. Heat treatments were carried out with 0.1 mg/ml of wild-type ( , A) and mutant (O, A) Thermus PEPCs at 90 ( , O) and 95T (A, A). The residual activities were assayed at 60°C in the reaction mixture containing 10 mM potassium PEP, 10 mM KHCO3,10 mM MgS04,0.3 mM NADPH, 1.0 mM CoASAc, 0.1 M Ches-KOH, pH 8.6,2.0 U malate dehydrogenase from Thermus sp., and the enzyme. Relative activities were plotted against incubation time.

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

When a linked enzyme assay, known as an indicator reaction, is used to determine the activity of a different enzyme, it is essential that the primary reaction be the rate-limiting step. For example, in the determination of aspartate aminotransferase activity, the indicator reaction is the reduction of the oxaloacetate formed in the aminotransferase reaction to malate by malate dehydrogenase and NADH. The activity of the indicator enzyme must be sufficient to ensure the virtually instantaneous removal of the product of the first reaction, to prevent significant reversal of the first reaction. The measured enzyme is typically acting under conditions of saturation with respect to its substrate however, the concentration of the substrate of the indicator enzyme (i.e., the... 

Product inhibition is a cause of nonlinearity of reaction progress curves during fixed-time methods of enzyme assay. For example, oxaloacetate produced by the action of aspartate aminotransferase inhibits the enzyme, particularly the mitochondrial isoenzyme. The inhibitory product may be removed as it is formed by a coupled enzymatic reaction malate dehydrogenase converts the oxaloacetate to malate and at the same time oxidizes NADH to NADL... 

Catalytic properties and assay of malate dehydrogenase Dehydrogenases probably operate according to an, at least partially, ordered pathway (Banaszak and Bradshaw, 1975) ... 

Using this reaction, Weigelt (1987) and Schubert et al. (1991) employed the LDH-LMO sensor for assaying ALAT and aspartate aminotransferase (ASAT, EC 2.6.1.1). For this purpose, malate dehydrogenase (MDH, EC 1.1.1.37) was additionally immobilized in the enzyme membrane. ALAT was determined as described above, and the ASAT reaction then initiated by substrate injection. MDH reduces the oxaloacetate formed to malate ... 

The main advantage of the proposed wine analysis is its selectivity because only primary amines can be detected using this method. Also, byproducts do not interfere with phenols or thiols. The quality of the wine and its organoleptic characteristics are well defined considering the effects of the malolactic fermentation process. The electrometric methods assure reliable results for the 1-malic and 1-lactic acids assay. The biosensors construction for 1-malic and 1-lactic acids assay in wine are based on malate dehydrogenase and lactate oxidase enzymes.117 The reproducibility of the results as well as the selectivity make it reliable for establishing the quality of the wine. 

Figure 8.18 Colourimetric assay. Malate dehydrogenase (MDH) assay system. Oxalate is onward converted to L-malate by means of enzyme MDH that uses the reverse-colourimetric reductant NADH to effect catalytic reduction.

The specificity of the NAD-dependent malate dehydrogenase (decarboxylase) as used In this radiometric assay was evaluated using... 

The transamination of the a-amino group to a keto acid acceptor (reaction 2) has been demonstrated in a number of higher plant studies (Nahler and Ruis, 1973 Streeter, 1977 Lloyd and Joy, 1978). The product of the transamination is 2-oxosuccinamate. This can be deamidated to oxaloacetate by lettuce and spinach leaf preparations (Meister, 1953). A similar reaction was reported by Streeter (1977) in soybean and pea leaf extracts. On the other hand, Joy (1978) reported that the 2-oxosuccinamate is reduced to 2-hydroxysuccinamate in these leaves in vivo. The apparent discrepancy between the results of Streeter (1977) and those of Joy (1978) may be due to the enzyme assay used by the former. It consisted of the oxidation of NADH in the presence of the enzyme extract and 2-oxosuccinamate. The assumption was that deamidation occurred leading to oxaloacetate which then acted as the substrate of endogenous malate dehydrogenase. The work of Davies (1961) showed that plant malate dehydrogenase is not specific for oxaloacetate, and it is possible that the 2-oxosuccinamate may act as a substrate. Meister (1953). actually measured the production of ammonia from 2-oxosuccinamate by his leaf preparations. 

This enzyme is a pyridoxal protein which is present in excessive amounts in blood serum during diseases of the liver or cardiac muscle. It may be assayed by an ultra-violet method using malate dehydrogenase in an indicator reaction [274]. Alternately, the oxalacetate formed is decomposed to pyruvate which is treated with DNP to form pyruvate-dinitrophenylhydrazone. In the presence of sodium hydroxide, an intense brown colour is produced with an absorption maximum at 505 nm [275]. 

This enzyme also oxidises some other 2-hydroxydicarboxylic acids. It is one of the enzymes of the citric acid cycle in which it catalyses the conversion of malate to oxalacetate. It should not be confused with malate dehydrogenase (decarboxylating) E.C. 1.1.1.40. It may be assayed by u.v. spectroscopy, if the system is driven in reverse... 

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