Xanthine oxidoreductase and xanthine oxidase in human cornea

Xanthine oxidoreductase (xanthine dehydrogenase + xanthine oxidase) is a complex enzyme that catalyzes the oxidation of hypoxanthine to xanthine, subsequently producing uric acid. The enzyme complex exists in separate but interconvertible forms, xanthine dehydrogenase and xanthine oxidase, which generate reactive oxygen species (ROS), a well known causative factor in ischemia/reperfusion injury and also in some other pathological states and diseases. Because the enzymes had not been localized in human corneas until now, the aim of this study was to detect xanthine oxidoreductase and xanthine oxidase in the corneas of normal post-mortem human eyes using histochemical and immunohistochemical methods. Xanthine oxidoreductase activity was demonstrated by the tetrazolium salt reduction method and xanthine oxidase activity was detected by methods based on cerium ion capture of hydrogen peroxide. For immunohistochemical studies. we used rabbit antibovine xanthine oxidase antibody, rabbit antihuman xanthine oxidase antibody and monoclonal mouse antihuman xanthine oxidase/xanthine dehydrogenase/aldehyde oxidase antibody. The results show that the enzymes are present in the corneal epithelium and endothelium. The activity of xanthine oxidoreductase is higher than that of xanthine oxidase, as clearly seen in the epithelium. Further studies are necessary to elucidate the role of these enzymes in the diseased human cornea. Based on the findings obtained in this study (xanthine oxidoreductase/xanthine oxidase activities are present in normal human corneas), we hypothesize that during various pathological states, xanthine oxidase-generated ROS might be involved in oxidative eye injury.     

Distribution of xanthine oxidoreductase activity in human tissues--a histochemical and biochemical study.

Localization of the activity of both the dehydrogenase and oxidase forms of xanthine oxidoreductase were studied in biopsy and postmortem specimens of various human tissues with a recently developed histochemical method using unfixed cryostat sections, poly-(vinyl alcohol) as tissue stabilizator, 1-methoxyphenazine methosulphate as intermediate electron acceptor and Tetranitro BT as final electron acceptor. High enzyme activity was found only in the liver and jejunum, whereas all the other organs studied showed no activity. In the liver, enzyme activity was found in sinusoidal cells and both in periportal and pericentral hepatocytes. In the jejunum, enterocytes and goblet cells, as well as the lamina propria beneath the basement membrane showed activity. The oxidase activity and total dehydrogenase and oxidase activity of xanthine oxidoreductase, as determined biochemically, were found in the liver and jejunum, but not in the kidney and spleen. This confirmed the histochemical results for these organs. Autolytic rat livers several hours after death were studied to exclude artefacts due to postmortem changes in the human material. These showed loss of activity both histochemically and biochemically. However, the percentage activity of xanthine oxidase did not change significantly in these livers compared with controls. The findings are discussed with respect to the possible function of the enzyme. Furthermore, the low conversion rate of xanthine dehydrogenase into xanthine oxidase during autolysis is discussed in relation to ischemia-reperfusion injury.     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Effect of triamcinolone administration on content of flavins in rabbit liver

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH : lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductace EC 1.6.99.3) and -amino acid oxidase ( -amino acid : oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Histochemical study on the distribution of some enzyme activities in the vagal and facial lobes of the goldfish, Carassius auratus.

The histochemical localization of six enzymic activities (acetylcholinesterase, pseudocholinesterase, monoamine oxidase, lactate dehydrogenase, succinate dehydrogenase and glucose-6-phosphate dehydrogenase) has been studied in the vagal and facial lobes of the goldfish, Carassius auratus. These encephalic centers are hypertrophic in Cyprinidae, corresponding to the dominance of gustatory function. Acetylcholinesterase shows a complex laminar distribution in the vagal lobes and a peculiar cellular localization in vagal motor neurons. Monoamine oxidase activity is mainly evident in fibrous tracts coming to or leaving from the lobes. Among oxidative enzymes examined, lactate dehydrogenase and succinate dehydrogenase exhibit distribution patterns respectively similar to those observed for acetylcholinesterase and monoamine oxidase. Some features on enzymes distribution in the gustatory centers of Carassius are in agreement with the enzymatic patterns well known in higher vertebrates.     

Effects of riboflavin deficiency on oxidative phosphorylation, flavin enzymes and coenzymes in rat liver.

Riboflavin deficiency in rats caused a decrease in the activities of hepatic succinate dehydrogenase (50 %), L-α-glycerophosphate dehydrogenase (50 %) and xanthine oxidase (70 %). It also reduced to 50 % the rate of mitochondrial oxidation of succinate, β-hydroxybutyrate, α-ketoglutarate, glutamate, pyruvate and malate without changing ADP : O ratios, thus showing that riboflavin deficiency interferes with electron transport along the respiratory chain without noticably affecting phosphorylation.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Oscillations of enzyme activities during the cell-cycle of a glucose-repressed fission-yeast Schizosaccharomyces pombe 972h

1. Increased specific activities of cytochrome c oxidase, catalase, succinate dehydrogenase, succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase and malate dehydrogenase were observed during glucose de-repression of Schizosaccharomyces pombe. 2. The cell-cycle of this organism was analysed by three different methods: (a) harvesting of cells at intervals from a synchronous culture, (b) separation of cells by rate-zonal centrifugation into different size classes and (c) separation of cells by isopycnic-zonal centrifugation into different density classes. 3. Measurement of enzyme activities during the cell-cycle showed that all the enzymes assayed [cytochrome c oxidase, catalase, acid p-nitrophenylphosphatase, NADH-dehydrogenase, NADH-cytochrome c oxidoreductase, NADPH-cytochrome c oxidoreductase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase (NADP) and fumarate hydratase] show periodic expression as ;peaks'. 4. Cytochrome c oxidase shows a single maximum at 0.67 of a cycle, whereas succinate dehydrogenase exhibits two maxima separated by 0.5 of a cell-cycle. 5. All other enzymes assayed showed two distinct maxima per cell-cycle; for catalase, malate dehydrogenase and NADPH-cytochrome c oxidoreductase there is the possibility of multiple fluctuations. 6. The single maximum of cytochrome c oxidase appears at a similar time in the cycle to one maximum of each of the other enzymes studied, except for NADH dehydrogenase. 7. These results are discussed with reference to previous observations on the expression of enzyme activities during the cell-cycle of yeasts.     

A sensitive fluorometric assay for measuring xanthine dehydrogenase and oxidase in tissues

The conversion of xanthine dehydrogenase to a free radical producing oxidase is an important component of oxygen-mediated tissue injury. Current assays for these enzymes are of limited sensitivity, making it difficult to analyze activities in organ biopsies or cultured cells. The xanthine oxidase-catalyzed conversion of pterin (2-amino-4-hydroxypteridine) to isoxanthopterin provides the basis for a fluorometric assay which is 100-500 times more sensitive than the traditional spectrophotometric assay of urate formation from xanthine. Enzyme activity as low as 0.1 pmol min-1 ml-1 can be measured with the fluorometric pterin assay. Xanthine oxidase is assayed in the presence of pterin only, while combined xanthine dehydrogenase plus oxidase activity is determined with methylene blue which replaces NAD+ as an electron acceptor. The relative proportions and specific activities of xanthine oxidase and dehydrogenase determined by the fluorometric pterin assay are comparable with the spectrophotometric measurement of activities present in rat liver, intestine, kidney, and plasma. The assay has been successfully applied to brain, human kidney, and cultured mammalian cells, where xanthine dehydrogenase and oxidase activities are too low to detect spectrophotometrically.     

The effects of storage on the retention of enzyme activity in cryostat sections. A quantitative histochemical study on rat liver

Summary The effect of storage of unfixed cryostat sections from rat liver for 4 h, 24 h, 3 days and 7 days at -25°C was studied on the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, xanthine oxidoreductase, glutamate dehydrogenase, succinate dehydrogenase (all demonstrated with tetrazolium salt procedures), glucose-6-phosphatase (cerium-diaminobenzidine method), 5-nucleotidase (lead salt method), dipeptidyl peptidase II, acid phosphatase (both simultaneous azo coupling methods), d-amino acid oxidase (cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure) and catalase (diaminobenzidine method). The effect of drying of the cryostat sections at room temperature for 5 and 60 min was investigated as well. The enzyme activities were quantified by cytophotometric measurements of test and control reactions. The test minus control reaction was taken as a measure for specific enzyme activity. It was found that the activities of all the enzymes investigated, with one exception, were affected neither by storage of the cryostat sections at -25°C for up to 7 days, nor by drying of the sections at room temperature for up to 60 min. The exception was xanthine oxidoreductase, whose activity was reduced by 20% after 5 min drying of sections or after 4 h storage. Therefore, only incubations for xanthine oxidoreductase activity have to be performed immediately after cutting cryostat sections, whereas for the other enzymes a considerable margin appears to exist.     

Comparative study of chicken liver xanthine dehydrogenase and bovine liver xanthine oxidase. dehydrogenase activity of xanthine oxidase (author's transl)]

A method to purify bovine liver xanthine oxidase in described, with which samples of 256-fold specific activity with respect to the initial homogenate are obtained. Bovine liver xanthine oxidase and chicken liver xanthine dehydrogenase with oxygen as electron acceptor exhibit similar profile in pKM and log V versus pH plots. With NAD+ as electron acceptor a different profile in the pKM xanthine plot is obtained for chicken liver xanthine dehydrogenase. However three inflection points at the same pH values appear in all plots. Both enzymes are irreversibly inhibited by pCMB and reversibly by N-ethylmaleimide and by iodoacetamide, with competitive and uncompetitive type inhibitions respectively. These results suggest that NAD+ alters the enzymatic action since its binding to the enzyme antecedes the binding of xanthine to the xanthine oxidase molecule, without undergoing itself any modification. 0.15 M DDT of DTE treatment of bovine liver xanthine oxidase gives to the enzyme a permanent activity with NAD+ without modifying its activity with oxygen. The enzyme thus treated produces parallel straight lines in Lineweaver-Burk plots.     

The influence of freezing and freeze-drying of tissue specimens on enzyme activity

Summary In the presented study the influence of freezing and freeze-drying on enzyme activity is described. Attention is paid to 16 enzymes which can be used for quantitative enzyme histochemical techniques.With the exception of succinate dehydrogenase only, no significant inactivation during freezing and freeze-drying procedures could be demonstrated with lactate dehydrogenase, malate dehydrogenase (NAD⁺), malate dehydrogenase (decarboxylating) (NADP⁺), isocitrate dehydrogenase (NADP⁺), glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADH-oxydoreductase, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome c oxidase, phosphoglucomutase, glucosephosphate isomerase, glucose-6-phosphatase, acid phosphatase, -glucuronidase and non specific aryl esterase. Therefore the results supply a sound foundation for those quantitative enzyme histochemical techniques in which tissue specimens are frozen or frozen-dried before enzyme estimations are performed.     

Xanthine oxidase activities: evidence for two catalytically different types.

Xanthine dehydrogenase (EC 1.2.1.37) from mouse small intestine was accompanied by 20% as much xanthine oxidase (EC 1.2.3.2) activity (dehydrogenase-associated oxidase). NAD⁺ and oxygen did not compete as electron acceptors. Upon incubation at 37 °C, the dehydrogenase activity was gradually transformed to oxidase activity. Unexpectedly, the oxidase thus formed (dehydorgenase-derived oxidase) had catalytic properties different from those of the dehydrogenase-associated oxidase. The activation energy for the dehydrogenase-associated oxidase was 20,600 cal/mol, whereas that for the dehydrogenase-derived oxidase was 13,500 cal/mol. The activation energy for the dehydrogenase was 14,000 cal/mol. Between pH 6.4 and 8.5, the activity of the dehydrogenase-associated oxidase was essentially pH independent, whereas the activities of the dehydrogenase-derived oxidase and the dehydrogenase were enhanced with increasing pH. Use of the transformation inhibitor, dithiothreitol, and the protease inhibitor, diisopropylfluorophosphate, showed that these catalytic differences were not the result of partial proteolysis of the enzyme. The data demonstrate the existence of two catalytically different types of mammalian xanthine oxidase activities: A dehydrogenase-associated oxidase and a dehydrogenase-derived oxidase.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Quantitative succinate-dehydrogenase histochemistry. I. A Methodological study on mammalian and fish muscle.

In enzyme histochemistry formazan production can be used as a measure for oxidative enzyme activity. The formazan deposits can be measured quantitatively per cell with a scanning and integrating microspectrophotometer. Optimal conditions are described for the estimation of histochemical succinate dehydrogenase activity in sections of fish bodymusculature and mouse soleus and plantaris muscle. It is shown that when proper measuring conditions are chosen a ditetrazolium salt (TNBT) can be used in quantitative enzyme histochemistry and that the optimal conditions for the histochemical succinate dehydrogenase reaction in muscle fibres of fish and mouse muscle are somewhat different for these two species. The differences in pH, temperature and succinate sensitivity are the most prominent.     

A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.     

Effects of plant flavonoids on Manduca sexta (tobacco hornworm) fifth larval instar midgut and fat body mitochondrial transhydrogenase

The reversible, membrane-associated transhydrogenase that catalyzes hydride-ion transfer between NADP(H) and NAD(H) was evaluated and compared to the corresponding NADH oxidase and succinate dehydrogenase activities in midgut and fat body mitochondria from fifth larval instar Manduca sexta. The developmentally significant NADPH-forming transhydrogenation occurs as a nonenergy- or energy-linked activity with energy for the latter derived from either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++-dependent ATPase. In general, the plant flavonoids examined (chyrsin, juglone, morine, quercetin, and myricetin) affected all reactions in a dose-dependent fashion. Differences in the responses to the flavonoids were apparent, with the most notable being inhibition of midgut, but stimulation of fat body transhydrogenase by morin, and myricetin as also noted for NADH oxidase and succinate dehydrogenase. Although quercetin inhibited or stimulated transhydrogenase activity depending on the origin of mitochondria, it was without effect on either midgut or fat body NADH oxidase or succinate dehydrogenase. Observed sonication-dependent increases in flavonoid inhibition may well reflect an alteration in membrane configuration, resulting in increased exposure of the enzyme systems to the flavonoids. The effects of flavonoids on the transhydrogenation, NADH oxidase, and succinate dehydrogenase reactions suggest that compounds of this nature may prove valuable in the control of insect populations by affecting these mitochondrial enzyme components.