Succinate dehydrogenase assay

A very useful complement to enzyme assays as described above is histochemical study, which can provide additional information [76]. In particular, because it is possible to measure the activity cell per cell, histochemistry permits, in the case of a heteroplasmic population of mitochondria, the detection of even a small number of affected cells, which may have remained undetected by biochemical assays. Spectacular images showing, side-by-side, cells endowed with either high or absent enzyme activity can be obtained. The limitation of the method is in part due to the few activities possibly measured (essentially complex IV, succinic dehydrogenase, and less specifically, ATPase and NADH reductase) and to the fact that it is poorly quantitative. Histochemical investigations are performed under selected conditions (e.g., substrate concentrations, pH), which often differ from those used for enzyme assays, thus possibly introducing discrepancy between the two approaches. 

Fio. 22. Resolution of complex II with respect to succinate dehydrogenase by various chaotropes. Complex II was suspended in 50 mM Tris-HCl, pH 8.0. After addition of 0.6 M chaotrope the concentration of complex II was 8 mg/ml. After addition of the salts, samples were taken at the intervals indicated and assayed for succinate-Qi and succinate-PMS reductase activities. Solid lines, sucdnate-ubiquinone-2(Q) reductase activity dotted line, succinate-PMS reductase activity. Resolution temperature, 0° assay temperature, 38°. The complex II preparations used in the experiments of this and subsequent Figs. 24 and 25 had specific activities between 40 and 45 moles Qj reduced by succinate per min per mg protein. From Davis and Hatefi (169). 

Conditions Complex II was inactivated by 20 min incubation at pH 9.3 and readjusted to pH 7.6 (protein 11.25 mg/ml). Succinate dehydrogenase was prepared and stored as an ammonium sulfate-precipitated pellet for 1 day at —70°. It was dissolved in 50 mM Tris-HCl (pH 8.0) containing 20 mAf succinate and 5 mAf di-thiothreitol before using (protein 21.1 mg/ml). Alkali-treated complex II (0.5 ml) and succinate dehydrogenase (0.6 ml) were mixed together, assayed, and centrifuged for 60 min at 49,000 rpm. The supernatant and the pellet were then separated, the latter was suspended in Tris-succinate-dithiothreitol buffer and both fractions were assayed as indicated. Activities shown are expressed as micromoles of succinate oxidized X min X mg of total protein at. 38°. 

Baginsky and Hatefi (155, 156) showed that loss of reconstitution activity appears to be related to a damage in the iron-sulfur system of the enzyme which is not detectable by assay for iron and labile sulfide content. They obtained a preparation of succinate dehydrogenase from complex II which exhibited no reconstitution activity but had an iron labile sulfide flavin ratio close to 8 8 1. They were then able to reactivate this enzyme for reconstitution by treating it with NajS, ferrous ions, and mercaptoethanol, essentially in the same manner as apoferredoxin had been previously converted to ferredoxin (181, 18Z). The reactivated preparation was able to reconstitute with alkali-treated submitochondrial particles or complex II. Analyses showed that the preparation had acquired additional iron and labile sulfide, but control experiments indicated that reconstitution activity was not a spurious effect. The reactiva-... 

Fia. 32. Activation of succinate dehydrogenase by reduced ubiquinone-10. Phos-pliorylating siibmitochondrial particles (ETPh) were resuspended in a sucrose-Tris-Mg buffer, pH 7.4, at 4 mg protein/ml. Antimycin A (1 nmole/mg) and cyanide (1 mM) were added and the sample placed under an atmosphere of N7 to prevent autoxidation of reduced ubiquinone-10. Ubiquinone-10 was reduced with borohydride, neutralized with dilute acetic acid, and shaken till the first appearance of the yellow color of oxidized ubiquinone-10 to ensure removal of unreacted borohydride, all at 0°. Activation of succinate dehydrogenase was started by adding 50 mI of either reduced ubiquinone-10 (curve A) or of ubiquinone-10 (curve B) in absolute ethanol to 3 ml of enzyme giving 175 mAf final concentration of the quinone. Curve C, no addition. Samples were withdrawn at intervals and assayed immediately at 17 . The horizontal arrow indicates the maximal activation reached with succinate as activator. From Gutman et al. (197). 

Fra. 33. Activation of succinate dehydrogenase by NADH. A preparation of phos-phorylating submitochondrial particles (ETPh) (succinoxidase activity = 1.18 /unoles succinate per min per mg at 30°) was washed by centrifugation in a sucrose-Tris-Mg buffer (pH 7.4) and resuspended in the same buffer at 1 mg of protein/ml. Antimycin A (1 nmole/mg protein) was added to slow the rate of aerobic oxidation of NADH, followed by 0.25 mAf NADH. Oxidation of the latter at 23° was monitored spectro-photometrically at 340 nm (dashed line). Samples were removed periodically and assayed immediately for succinate dehydrogenase activity in the presence of 033 mg of PMS/ml (solid line). At 16 min a second aliquot of 035 mM NADH was added. From Gutman et al. (197). 

Fia. 35. Reconstitution of succinoxidase activity of alkali-inactivated bovine heart ETP with bovine and R. rubrum succinate dehydrogenases (SD). Left-hand trace 114 ng alkali-treated ETP (alk-ETP) and 37 bovine SD per ml. Right-hand trace 172 g alk-ETP and 46 /ig R. rubrum SD per ml. In both cases alk-ETP and SD at 10 times the concentrations indicated above were premixed with 10 mM succinate and preincubated for 3 min at 30° before addition to the assay mixture. Alk-ETP, bovine SD, or R. rubrum SD alone resulted in no oxygen uptake. Where indicated, 5.9 miH TTFA and 0.15 mM PMS were used. Assay temperature 30°. S.A., specific activity. From Hatefi et al. 

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. 

State two reasons why the FAD stimulation test, using assays of succinate dehydrogenase and red blood cells as a possible source of enzyme, would probably not work. 

Fig. 26. Effect of chaotropic agents and freeze-thawing on the resolution of succinate dehydrogenase. The enzyme, at a protein concentration of 1.48 mg/ml in 50 mM sodium phosphate (pH 7.0) and 20 mM succinate, was treated with various chaotropic agents as shown in the figure, and frozen in liquid nitrogen and thawed at room temperature three times. Since in the presence of higher concentrations of chaotropes the larger subunit precipitated after freeze-thawing, all samples were centrifuged for 1 min at 35,000 rpm, and only the clear supernatant was assayed for PMS reductase activity. F-T, freeze-thawing. From Hanstein et al. (166).

Aqueous extracts of cat s claw were tested for cytotoxicity in four in vitro bioassays using Chinese hamster ovary cells and bacterial cells (Santa Maria et al., 1997). Concentrations of 10, 20, 30, 40, 50, 75, and 100 mg/mL were used. The neutral protein assay (measures inhibition of cell growth), the total protein content assay, the tetrazolium assay (measures mitochondrial succinic dehydrogenase activity), and the microtox assay (measures inhibition of light output from a luminescent bacterium) showed no evidence of cytotoxicity. 

Lysine 2,3-aminomutase is not coenzyme 832 dependent, but a further lysine mutase, -lysine mutase (EC 5.4.3.3), catalyzes the coenzyme 812-dependent rearrangement shown in Scheme 68, the product 3,5-diaminohe-xanoic acid 261 having been shown (265) to have the (35,55) configuration. When the reaction was conducted in the presence of [5 - H] coenzyme 8,2 and the )5-lysine 258a was degraded to succinic acid, assay with succinate dehydrogenase showed the latter to be (25)-[2- HJ succinate (266). Thus... 

NADH cytochrome c reductase was isolated from pigeon breast and pig heart muscle. The enzyme was shown to contain four atoms of iron per flavin molecule. NADH cytochrome c reductase, like succinic dehydrogenase, is a ferroflavoprotein. The ratio of iron to flavin is four. The enzyme contains sulfhydryl groups that can be titrated by classical methods, but their oxidation has no effect on the enzymatic activity. In contrast, the removal of the metal leads to a decrease in the ability of the enzyme to reduce cytochrome c. As for succinic dehydrogenase, the structure of the flavin in NADH cytochrome c reductase is not clear. It was demonstrated that it is not flavin mononucleotide, but the identity of the flavin component with flavin adenine dinucleotide is not established in fact, the flavin component differs from the classical FAD by its chromatographic properties and its behavior in enzymic assays. It is not known if it is a structural variation of the flavin nucleotide or if the nucleotide is conjugated to a peptide. 

Assay of Transamination. Since the sum of keto acids and amino acids does not change in a transamination, specific reactions are required to assay the reaction products. Some of the methods used are oxidation of a-ketoglutarate to succinate and determination of succinate with succinic dehydrogenase decarboxylation of oxalacetate with aniline citrate decarboxylation with specific amino acid decarboxylases separation of products on paper chromatograms and spectrophotometric determination of those keto acids that exhibit specific absorption. 

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