Pyruvate dehydrogenase activity measurement

Dihydroxybenzoic acid (DHB) is also a commonly used tool to measure the pharmacological effects of HIF-la stabilization via PHD inhibition. Recently, it was shown that mice pretreated with DHB (100 mg/kg, i.p.) showed a marked resistance to the neurotoxic effects of l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) via protection of dopaminergic cell loss and striatal denervation. Importantly, this protection was seen to coincide with HIF-la stabilization, and the prevention of the MPTP-induced loss of ferroportin and striatal iron. Additionally, in these studies, DHB was also observed to block MPTP-induced reduction in mitochondrial pyruvate dehydrogenase, at both the mRNA level and through the measurement of enzyme activity in midbrain substantia nigra [26]. 

They stated further that, the new adaptive enzyme catalyzing Reaction 3 appears to be similar to the malic enzyme of pigeon liver, although strictly DPN (instead of TPN)-specific. The coenzyme specificity explains the ready occurrence of Reaction 1. Therefore, the authors showed that exogenous NAD was required for the overall reaction (malic acid -> lactic acid), but because this activity was measured manometrically, they never demonstrated the formation of reduced NAD. Similarly, they did not attempt to show that pyruvic acid was the intermediate between L-malic acid and lactic acid. Instead, the formation of pyruvic acid was inferred from the NAD requirement and because the malic acid dissimilation activity remained constant during purification while the lactate dehydrogenase activity decreased (14). In fact, attempts to show any appreciable amounts of pyruvic acid intermediate failed (22). 

Clinical Biochemical Determinations of the Serum Serum lactic dehydrogenase (LDH) and glutamic-pyruvic transaminase (GPT) activities were measured on fresh, refrigerated serum within U8 h of sacrificing the animal. Lactic dehydrogenase was measured according to the method of Amador, Dorfman, and Wacker (12). Serum GPT activity was measured according to the method of Wroblewski and LaDue (13). 

Figure 1. The rate of incorporation of carbon from Glc6P and pyruvate (ImM) into fatty acids and the rate of pyruvate (ImM) uptake by plastids isolated from embryos at three stages in development (A 1.5 mgFW, B 2.5 mgFW and C 3.5 mgFW). A unit of GAPDH (NAPH-dependent glyceraldehyde 3-phosphate dehydrogenase) activity is one />tmol min Each value is the mean SE of measurements from three separate preparations.

Reaction 4 depends upon the operation of a DPN-dependent, lipoic acid dehydrogenase. A protein fraction has been obtained from E. coli which exhibits this property. Measurement of lipoic acid dehydrogenase activity directly with DPN+ and reduced lipoic acid as reductant or reoxidation of DPNH with a-lipoic acid as oxidant was not particularly successful. However, lipoic acid dehydrogenase activity could readily be determined by linking it with lactic acid dehydrogenase in the presence of pyruvate and catalytic amounts of DPN, by measuring the residual —SH groups. 

The isoenzymes associated with heart muscle can convert a-oxobutyrate (which has a similar structure to pyruvate) to a-hydroxybutyrate more effectively than the other LDH isoenzymes. Measurement of serum hydroxybutyrate dehydrogenase activity therefore gives an estimate of heart LDH (see separate entry on hydroxybutyrate dehydrogenase). 

Pyruvate dehydrogenase complex assays PDHc activity was measured on crude liver and brain homogenates using an assay system adapted from Wieland et al.(1971) and Cremer Teal (1974),and already described elsewhere (Raulin et al 1979). Coenzyme A was omitted from the reaction blanks. ... 

The ADP produced by the hydrolysis of ATP is continuously used up by added purified pyruvate kinase, which in the presence of phosphoenol pyruvate produces pyruvate and ATP (Fig. 3.8.6). Pyruvate is then utilized by added lactate dehydrogenase, which in the presence of NADH produces lactate and NAD+. Complex V activity is estimated from the rate of NADH oxidation at 340 nm (e 4870-M 1-cm 1 isosbestic point 380 nm), after subtracting the oligomycin-resistant activity. It should be kept in mind that oligomicyn sensitivity requires the preserved attachment of the Fr component of the enzyme to the membranous F0 component. The attachment is readily lost upon freeze-thaw cycles. Consequently, it is reasonable to measure the activity on fresh material only. 

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). 

Answer The measurement of the activity of alanine aminotransferase by measurement of the reaction of its product with lactate dehydrogenase is an example of a coupled assay. The product of the transamination (pyruvate) is rapidly consumed in the subsequent indicator reaction, catalyzed by an excess of lactate dehydrogenase. The dehydrogenase uses the cofactor NADH, the disappearance of which is conveniently measured by observing the rate of decrease in NADH absorption at 340 nm. Thus, the rate of disappearance of NADH is a measure of the rate of the aminotransferase reaction, if NADH and lactate dehydrogenase are added in excess. 

The activity of the ATP-forming enzyme complex V is usually assessed by determining the reverse reaction ATP — ADP + Pi. The reaction is coupled to reactions catalyzed by pyruvate kinase (ADP + phosphoenolpyruvate —> pyruvate + ATP) and lactate dehydrogenase (pyruvate + NADH — lactate -F NAD+). This final reaction can be followed spectrophotometrically by measuring NADH at 340 nm. The activity of complex V (ATPase) can be derived from the rate of NADH conversion in the presence and absence of the specific complex V inhibitor oligomycin. 

Table 1 Summary of respiratory chain, complex V and PDHc activity assays. All assays are spectrophotometric assays except for the PDHc assay with CO2 detection, which is a radiochemical assay. The specific inhibitor indicated is used for blank measurements. Non-standard abbreviations UQi nbiquinone-Qi DQ decylubiquinone DCIP 2,6-dichlorophenolindophenol, PK pyruvate kinase LDH lactate dehydrogenase AABS / -[p-(aminophenyl)azo]benzene sulfonic acid ArAt arylamine acetyltransferase...

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