Reductase activity measurement

Reductase Activity Measurement. Reductase activity was measured by the reduction of 200 /iM veratraldehyde to veratryl alcohol in the presence of 250 /tM NADPH. The reaction was carried out in 20 raM Tris buffer pH 7.4 (optimal pH = 6) with 20% glycerol and 1 mM EDTA. The decrease in absorbance of NADPH at 365 nm was measured. NADPH was slowly oxidized in those mixtures which were not purified by ion exchange even in the absence of veratraldehyde. This unspecific reaction was considered in the calculation of the reductase activity. One unit of reductase reduced 1 /imol min-1 of veratraldehyde to veratryl alcohol at room temperature (25°C). 

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. 

The marker enzymes used in this experiment are as follows vanadate-sensitive H+-ATPase (plasma membrane), nitrate-sensitive H+-ATPase or pyrophosphatase (tonoplast), TritonX-100 stimulated-UDPase or IDPase (Golgi complex), antimycin A-insensitive NADPH cytochrome c reductase (ER), and cytochrome c oxidase (mitochondria inner membrane). NADH cytochrome c reductase activity is found to be 10 times higher than NADPH cytochrome c reductase activity. Chlorophyll content can be measured as the chloroplast marker. The chlorophyll content is calculated by the following equation. Before measurement, auto zero is performed at 750 ran. 

Honda M, Tint GS, et al (1996) Measurement of 3/ -hydroxysteroid A7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate a new method for the diagnosis of the Smith-Lemli-Opitz syndrome. J. Lipid Res 37 2433-2438... 

Chlorobium liimcola . thiosulfatophilum (22) and ThiQgaBfia roseopersicina (461 do not contain siroheme sulfite reductases, although sulfite reductase activity was measured in the last organism. 

Rutin, quercetin 3-rutinoside, i.e., 3-rhamnoglucoside, has been reported to suppress glycation,609 but it is only sparingly soluble in water. Nagasawa el al.6W have therefore examined the water-soluble 4G-a-D-glucopyranosyl derivative (G-rutin), obtained from a sugar factory. Streptozotocin-treated rats were fed for 4 weeks on a 20% casein diet, when they showed an increase in fructosyllysine obtained on hydrolysis of their protein however, when their diet had been supplemented with 0.2% G-rutin, the increase was reduced by 20% for kidney protein. Supplementation reduced AGE accumulation, as measured with an anti-AGE monoclonal antibody, in the serum and kidney proteins to the level of the control rats. Supplementation inhibited aldose reductase activity in the kidney, but not in the liver. 

Stubbs, A. P., Murphy, G. M., and Wilkinson, M. L., 1991. Isocratic high-performance liquid chromatographic measurement of optimal 5a-steroid reductase activity in Hep-G2 cells. J. Chromatogr. 570, 293-299 (1991). 

Fia. 5. Kinetics of the resolution of complex I as a function of temperature of the incubation medium. The release of menadione reductase activity was measured as an index of resolution 2 min after incubation of complex I with 0.47 Af NaC10< at the temperatures indicated. From Davis and Hatefi (69). 

The activities involved in yeast fatty acid biosynthesis are covalently linked as separate domains of two multifunctional polypeptides, a and p, encoded by the fas2 and fasl genes, respectively (Fig. 2) [57,58]. The functionalities associated with the 220 kDa a subunit include -ketoacyl synthase activity, -ketoacyl reductase activity, and an AGP domain which bears a phosphopantetheinylated serine. The 208 kDa -subunit has acetyl and malonyl CoA transacylase, palmi-toyl transferase, -hydroxyacyl-enzyme dehydratase, and enoyl acyl-enzyme reductase activities. The two subunits can be readily dissociated, and the individual activities maybe measured [57]. 

Determination of reductase activity in milk indicates whether there is bacterial contamination. The reductase activity is measured by the decolorization rate of methylene blue. Acceptable milk decolorizes... 

Figure 1 Induction of hepatic aminoazo dye N-demethylase and reductase activities. Rats (50 g) were injected once with 1 mg of 3-methylcholanthrene (3-MC). N-Demethylase activity was determined in fortified liver homogenates by measuring the metabolism of 3-methyl-4-monomethylaminoazobenzene to 3-methyl-4-aminoazobenzene (3-methyl-AB). Reductase activity was determined by measuring the reduction of the azo linkage of 4-dimethylaminoazobenzene (DAB). Demethylase activity is expressed as fig of 3-methyl-AB formed per 50 mg of liver per 30 min. Reductase activity is expressed as fig of DAB reduced per 30 mg of liver per 30 min. Each point is the average of the activities for two or three rats. Taken from Ref. (4).

Fig. 11 Effect of specific inhibitors of the respiratory chain on the nitrite reductase activity of rat liver mitochondria. Mitochondria were incubated with nitrite for 2 h under argon in the presence of succinate or glutamate/malate (Glu/Mal). Nitric monoxide derived from nitrite was trapped with hemoglobin. The concentration of NO-hemoglobin complexes was measured using low temperature ESR spectroscopy. Other details are described in Kozlov et al. [46]. CON control, ROT rotenone, TTFA thenoyltrifluoroacetone, MYX myxothiazol, AA antimycin A...

Table 10. Effect of erythrocuprein on cytochrome-c reductase activity of flavoproteins. The cytochrome-c reductase activity was measured in air-equilibrated solutions containing 0.1 M pyrophosphate, pH 8.5, in the presence of 3.33 X 70-5 M cytochrome c and 10 fig bovine catalase. The concentration of erythrocuprein in this assay mixture was 0.62 fiM. The temperature was 25° (150)...

Similar problems have compUcated the quantitation of HMG-CoA reductase activity as a measure of the rate of cholesterol synthesis. For example, it has not been established as firmly for the intestine as for the hver that this enzyme is rate limiting to the overall synthesis of the cholesterol molecule [23]. Furthermore, HMG-CoA reductase in both the intestine and liver is subject to a phosphorylation-dephosphorylation reaction that modifies enzyme activity and is involved in the activation of an inactive (phosphorylated) to an active (dephos-phorylated) form during homogenization of the mucosa [24,25]. Finally, HMG-CoA reductase is incompletely recovered from the mucosa during preparation of micro-somes [26,27] and is very sensitive to inactivation by proteases present in the intestine [28]. These various technical problems have led to considerable confusion with respect to various aspects of intestinal cholesterol synthesis and have made it difficult to interpret quantitatively some of the results presented below. 

Takeuchi et al. measured cholesterol 7a-hydroxylase and HMG-CoA reductase activity in fasted rats refed glucose [237]. The administration of glucose to these rats resulted in increased cholesterol synthesis after 1 h and increased cholesterol 7a-hydroxylase activity after 2 h. These effects were not noted in rats pretreated with an inhibitor of cholesterol biosynthesis, suggesting that the effects on the cholesterol 7a-hydroxylase were secondary to those on cholesterologenesis. It was suggested that the stimulatory effect of glucose was due to increased availability of cholesterol for the cholesterol 7a-hydroxylase. 

Earlier studies with rat-liver microsomes indicated that NADB-dehydrogenase systems readily reduced ammineruthenium(III) complexes. Crane has since discovered plasma membrane NADH-dehydrogenase enzymes on a number of different t3rpes of cells.Experiments with pig erythrocytes showed that ammineruthenium(IIl) ions are fairly readily reduced by the plasma membrane NADS-dehydrogenase. As expected, the reduction rates are a function of the metal ion s reduction potential and its intrinsic electron-transfer rate. Comparison of the hexammineruthenium(III) reductase activities of subcellular fractions of mouse liver revealed the plasma membrane and Golgi apparatus to be about equivalent (when measured per mg of protein) and the endoplasmic reticulum to be 3-4 times more effective It... 

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