Pyrimidine synthesis in Neurospora crassa: regulation of enzyme activities

The regulation of several enzymes involved in pyrimidine biosynthesis in Neurospora crassa has been studied. Elevation of ATCase (l-aspartate carbamoyltransferase) activity is found in all pyrimidine-requiring mutants when they are starved for uridine. DHOase (dihydroorotase) is an unstable enzyme, and it is impossible to conclude what type of regulation, if any, controls this enzyme. DHOdehase (dihydroorotate dehydrogenase) activity shows a marked elevation in uridine-starved pyr-2 cultures, a mutant blocked late in the pathway. Several mutants blocked early in the pathway show much smaller increases in DHOdehase activity and possible explanations for this are discussed. Differences in the modes of regulation of the pyrimidine biosynthetic pathways in various organisms are compared.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Redoxal as a new lead structure for dihydroorotate dehydrogenase inhibitors: a kinetic study of the inhibition mechanism

Mitochondrial dihydroorotate dehydrogenase (DHOdehase; EC 1.3.99.11) is a target of anti-proliferative, immunosuppressive and anti-parasitic agents. Here, redoxal, (2,2'-[3,3'-dimethoxy[1, 1'-biphenyl]-4,4'-diyl)diimino]bis-benzoic acid, was studied with isolated mitochondria and the purified recombinant human and rat enzyme to find out the mode of kinetic interaction with this target. Its pattern of enzyme inhibition was different from that of cinchoninic, isoxazol and naphthoquinone derivatives and was of a non-competitive type for the human (K(ic)=402 nM; K(iu)=506 nM) and the rat enzyme (K(ic)=116 nM; K(iu)=208 nM). The characteristic species-related inhibition of DHOdehase found with other compounds was less expressed with redoxal. In human and rat mitochondria, redoxal did not inhibit NADH-induced respiration, its effect on succinate-induced respiration was marginal. This was in contrast to the sound effect of atovaquone and dichloroallyl-lawsone, studied here for comparison. In human mitochondria, the IC(50) value for the inhibition of succinate-induced respiration by atovaquone was 6.1 microM and 27.4 microM for the DHO-induced respiration; for dichlorallyl-lawsone, the IC(50) values were 14.1 microM and 0.23 microM.     

Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Sequence of the URA1 gene encoding dihydroorotate dehydrogenase from the basidiomycete fungus Agrocybe aegerita.

The URA1 gene encoding dihydroorotate dehydrogenase (DHOdehase) from the edible basidiomycete, Agrocybe aegerita, has been cloned by complementation of the Escherichia coli pyrD mutation. The nucleotide sequence of a 1531-bp genomic fragment carrying URA1 revealed two uninterrupted open reading frames (ORFs) separated by 61 bp. The larger ORF can encode a 328-amino acid (aa) DHOdehase that has 53% homology with the corresponding protein from E. coli. Comparison with other DHOdehase aa sequences showed essentially conservation of the cofactor-binding site of flavoproteins.     

Biosynthetic Dihydroorotate Dehydrogenase from Lactobacillus bulgaricus

This paper describes the first detailed study on a dihydroorotate dehydrogenase involved in pyrimidine biosynthesis. In most organisms the enzyme is membrane-bound; however, a soluble dihydroorotate dehydrogenase was produced in relatively high levels when the anaerobe, Lactobacillus bulgaricus, was released from repression. The enzyme was purified 213-fold over derepressed levels with a 39% recovery of enzyme units. The enzyme showed only one minor protein contaminant when analyzed by polyacrylamide electrophoresis. It was characterized as a flavoprotein containing only flavine mononucleotide as the prosthetic group. Molecular weight estimations by gel filtration gave a value of approximately 55,000, which is one-half that of the degradative enzyme described by others. During aerobic oxidation of dihydroorotate, the rates of oxygen consumption, orotate formation, and hydrogen peroxide formation were equal, as would be expected in a flavoprotein-catalyzed reaction. The enzymatic activity with ferricyanide as acceptor was optimum around pH 7.7. The stimulation of enzymatic activity over a wide pH range by ammonium sulfate was attributed to an effect on the maximum velocity of the reaction. As analyzed by polyacrylamide electrophoresis, inactivation of the enzyme by visible light resulted in the appearance of a second protein band with lowered specific activity. The purified enzyme used redox dyes, oxygen, or cytochrome c as electron acceptors but was not active with pyridine nucleotides. Flavine adenine dinucleotide has been implicated at the active site for pyridine nucleotide reduction in the degradative enzyme. The biosynthetic enzyme lacks this flavine and the associated activity.     

Ultrastructural demonstration of succinic dehydrogenase and cytochrome oxidase activity in sporozoites of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in salivary glands of tick vectors (Rhipicephalus bursa, Hyalomma anatolicum excavatum)

Salivary gland stages ("sporozoites") of Babesia ovis and Theileria annulata (Apicomplexa: Piroplasmea) in female ixodid ticks were studied for ultracytochemical activity of the respiratory enzymes, succinic dehydrogenase (SDH), and cytochrome oxidase. Both SDH and cytochrome oxidase were demonstrated in the sporozoites and the mitochondria in these stages. Identified in this way the final reaction product of SDH was located mainly at the inner side of the mitochondrial boundary, though it was also visible in the internal space of the organelle. Cytochrome oxidase activity always was confined to the wall of mitochondria. This enzyme was demonstrated also in the erythrocyte stage of B. ovis. The cytochemical results indicate respiratory potential of the piroplasmean stages studied. Cristate or typical protozoan mitochondria have not been observed in sporozoites of Babesia or Theileria. This report is the first demonstration of mitochondria, or mitochondrialike activity in Babesia.     

Radiation-induced inactivation of dihydroorotate dehydrogenase in dilute aqueous solution.

The inactivation of dihydroorotate dehydrogenase by gamma irradiation in dilute aqueous solution has been investigated. The activity of the enzyme decreased exponentially as a function of the absorbed dose under aerated and nitrous oxide-saturated conditions. The contributions of the individual radical species derived from water radiolysis were estimated from the inactivation results observed under aerated, argon-saturated, and nitrous oxide-saturated conditions. The hydrogen atom and hydroxyl radical were found to be important in enzyme inactivation. The effect of selected inorganic radical anions such as Br.2-, I.2-, and (SCN).2- on the enzyme activity was also studied, and the results implicate the possible involvement of cysteine and tyrosine residues in the catalytic activity of dihydroorotate dehydrogenase. Changes in the kinetic parameters (Michaelis-Menten constant, Km, and maximal velocity, Vmax) due to irradiation under the conditions investigated suggest that radiation-induced inactivation is due to modification of the substrate binding sites and that of the active site residues in the enzyme. Evidence for the reduction of iron-sulfur centers in the enzyme during the inactivation process has been put forward from the difference spectrum of the irradiated dihydroorotate dehydrogenase. It has also been shown by electrophoretic studies that radiation-induced inactivation was not due to any fragmentation of the protein structure or the formation of any intermolecular crosslinking.     

INTRACELLULAR LOCALIZATION OF PHENOL SULPHOTRANSFERASE IN RAT BRAIN

—The intracellular localization of phenol sulphotransferase in rat brain was studied The distribution pattern found after differential centrifugation closely resembles that of lactate dehydrogenase and does not change during postnatal development. The distribution of the enzyme in discontinuous and continuous sucrose gradients, however, shows a deviation from the lactate dehydrogenase pattern and a shift towards a higher sucrose concentration during development. In the adult the phenol sulphotransferase coincides with monoamine oxidase, succinate dehydrogenase and β-glucuronidase. Disruption experiments, purification of mitochondria and electron microscopy exclude localization of phenol sulphotransferase in mitochondria. These studies support the idea of phenol sulphotransferase as a cytoplasmic enzyme with a preferential binding to or localization in oligodendroglial cells or, more probably, a specific type of synaptosomes.     

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and α-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.     

Submitochondrial localization and function of enzymes of glutamine metabolism in avian liver

Glutamine synthetase (EC 6.3.1.2) was localized within the matrix compartment of avian liver mitochondria. The submitochondrial localization of this enzyme was determined by the digitonin-Lubrol method of Schnaitman and Greenawalt (35). The matrix fraction contained over 74% of the glutamine synthetase activity and the major proportion of the matirx marker enzymes, malate dehydrogenase (71%), NADP-dependent isocitrate dehydrogenase (83%), and glutamate dehydrogenase (57%). The highest specific activities of these enzymes were also found in the matrix compartment. Oxidation of glutamine by avian liver mitochondria was substantially less than that of glutamate. Bromofuroate, an inhibitor of glutamate dehydrogenase, blocked oxidation of glutamate and of glutamine whereas aminoxyacetate, a transaminase inhibitor, had little or no effect with either substrate. These results indicate that glutamine metabolism is probably initiated by the conversion of glutamine to glutamate rather than to an alpha-keto acid. The localization of a glutaminase activity within avian liver mitochondria plus the absence of an active mitochondrial glutamine transaminase is consistent with the differential effects of the transaminase and glutamate dehydrogenase inhibitors. The high glutamine synthetase activity (40:1) suggests that mitochondrial catabolism of glutamine is minimal, freeing most of the glutamine synthesized for purine (uric acid) biosynthesis.     

Complete inhibition of dihydro-orotate oxidation and superoxide production by 1,1,1-trifluoro-3-thenoylacetone in rat liver mitochondria.

1,1,1-Trifluoro-3-thenoylacetone was shown to cause complete inhibition of dihydroorotate oxidation in rat liver mitochondria as measured by orotate formation and the rate of dihydro-orotate-dependent reduction of 2,6-dichlorophenol-indophenol or cytochrome c. The inhibition by trifluorothenoylacetone was dose-dependent, and a concentration of 1 mM completely inhibited dihydro-orotate dehydrogenase activity. 1,10-Phenanthroline, another iron-chelating agent, also caused total inhibition of the liver enzyme. Whereas the iron chelators inhibited 100% of dihydro-orotate dehydrogenase activity in liver mitochondria, they inhibited only a maximum of 72% in the case of the brain enzyme. The inhibition by trifluorothenoylacetone was not prevented by addition of phenazine methosulphate or ubiquinone. Dihydro-orotate dehydrogenase-mediated generation of superoxide was abolished when the enzyme was fully inhibited by trifluorothenoylacetone or when the electron-transport system was blocked by antimycin A. These results suggest that the iron component(s) of dihydro-orotate dehydrogenase is of strategic importance for catalytic activity and transfer of reducing equivalents from the primary enzyme to the electron-transport chain. Furthermore, the study indicates that production of superoxide radicals during dihydro-orotate dehydrogenase-catalysed oxidation of dihydro-orotate may be at the cytochrome b-c1 segment of the electron-transport chain (as a consequence of autooxidation of ubisemiquinone) rather than at a site on the primary enzyme.     

Purification and properties of the bovine liver mitochondrial dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase has been purified 6,000-fold from bovine liver mitochondria to apparent homogeneity in six steps. Electrophoretic migration of the homogeneous enzyme on sodium dodecyl sulfate-polyacrylamide gels reveals a subunit Mr of 42,000. By contrast to the well-characterized, cytosolic dihydroorotate oxidases (EC 1.3.3.1), the purified bovine dehydrogenase is a dihydroorotate:ubiquinone oxidoreductase. Maximal rates of orotate formation are obtained using coenzymes Q6 or Q7 as cosubstrate electron acceptors. Concomitant with substrate oxidation, the enzyme will reduce simple quinones, such as benzoquinone, but at significantly lower rates (10-15%) than that obtained for reduction of coenzyme Q6. Enzyme-catalyzed substrate oxidation is not supported by molecular oxygen. The specificity of the purified enzyme for dihydropyrimidine substrates has also been explored. The methyl-, ethyl-, t-butyl-, and benzyl-S-dihydroorotates are substrates, but 1- and 3-methyl and 1,3-dimethyl methyl-S-dihydroorotates are not. Competitive inhibitors include product orotate, 5-methyl orotate, and racemic cis-5-methyl dihydroorotate.     

Protein kinase activity at the inner membrane of mammalian mitochondria.

This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.     

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.     

Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase.

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro-synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N-terminal segment that resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.     

Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase

Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.     

Carvedilol inhibits the exogenous NADH dehydrogenase in rat heart mitochondria

There are several reports on the oxidation of external NADH by an exogenous NADH dehydrogenase in the outer leaflet of the inner membrane of rat heart mitochondria. Until now, however, little was known about its physiological role in cellular metabolism. The present work shows that carvedilol (?1-[carbazolyl-(4)-oxy]-3-[2-methoxyphenoxyethyl)amino]-pro - panol-(2)?) is a specific inhibitor of an exogenous NADH dehydrogenase in rat heart mitochondria. Carvedilol does not affect oxygen consumption linked to the oxidation of succinate and internal NADH. It is also demonstrated that the inhibition of exogenous NADH dehydrogenase by carvedilol is accompanied by the inhibition of alkalinization of the external medium. In contrast to the addition of glutamate/malate or succinate, exogenous NADH does not generate a membrane potential in rat heart mitochondria, as observed with a TPP(+) electrode. It is also demonstrated that the oxygen consumption linked to NADH oxidation is not due to permeabilized mitochondria, but to actual oxidase activity in the inner membrane. The enzyme has a K(m) for NADH of 13 microM. Carvedilol is a noncompetitive inhibitor of this external NADH dehydrogenase with a K(i) of 15 microM. Carvedilol is the first inhibitor described to this organospecific enzyme. Since this enzyme was demonstrated to play a key role in the cardiotoxicity of anticancer drugs of the anthracycline family (e.g., adriamycin), we may suggest that the administration of carvedilol to tumor patients treated with adriamycin might be of great help in the prevention of the cardioselective toxicity of this antibiotic.     

ENZYMATIC PROPERTIES OF THE INNER AND OUTER MEMBRANES OF RAT LIVER MITOCHONDRIA

Treatment of rat liver mitochondria with digitonin followed by differential centrifugation was used to resolve the intramitochondrial localization of both soluble and particulate enzymes. Rat liver mitochondria were separated into three fractions: inner membrane plus matrix, outer membrane, and a soluble fraction containing enzymes localized between the membranes plus some solublized outer membrane. Monoamine oxidase, kynurenine hydroxylase, and rotenone-insensitive NADH-cytochrome c reductase were found primarily in the outer membrane fraction. Succinate-cytochrome c reductase, succinate dehydrogenase, cytochrome oxidase, β-hydroxybutyrate dehydrogenase, α-ketoglutarate dehydrogenase, lipoamide dehydrogenase, NAD- and NADH-isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and ornithine transcarbamoylase were found in the inner membrane-matrix fraction. Nucleoside diphosphokinase was found in both the outer membrane and soluble fractions; this suggests a dual localization. Adenylate kinase was found entirely in the soluble fraction and was released at a lower digitonin concentration than was the outer membrane; this suggests that this enzyme is localized between the two membranes. The inner membrane-matrix fraction was separated into inner membrane and matrix by treatment with the nonionic detergent Lubrol, and this separation was used as a basis for calculating the relative protein content of the mitochondrial components. The inner membrane-matrix fraction retained a high degree of morphological and biochemical integrity and exhibited a high respiratory rate and respiratory control when assayed in a sucrose-mannitol medium containing EDTA.     

Kinetics of inhibition of human and rat dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar sodium and polyporic acid

Mitochondrially-bound dihydroorotate dehydrogenase (EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. The enzyme has been identified as or surmised to be the pharmacological target for isoxazol, triazine, cinchoninic acid and (naphtho)quinone derivatives, which exerted antiproliferative, immunosuppressive, and antiparasitic effects. Despite this broad spectrum of biological and clinical relevance, there have been no comparative studies on drug-dihydroorotate dehydrogenase interactions. Here, we describe a study of the inhibition of the purified recombinant human and rat dihydroorotate dehydrogenase by ten compounds. 1,4-Naphthoquinone, 5,8-hydroxy-naphthoquinone and the natural compounds juglon, plumbagin and polyporic acid (quinone derivative) were found to function as alternative electron acceptors with 10-30% of control enzyme activity. The human and rat enzyme activity was decreased by 50% by the natural compound lawsone ( > 500 and 49 microM, respectively) and by the derivatives dichloroally-lawsone (67 and 10 nM), lapachol (618 and 61 nM) and atovaquone (15 microM and 698 nM). With respect to the quinone co-substrate of the dihydroorotate dehydrogenase, atovaquone (Kic = 2.7 microM) and dichloroally-lawsone (Kic = 9.8 nM) were shown to be competitive inhibitors of human dihydroorotate dehydrogenase. Atovaquone (Kic = 60 nM) was also acompetitive inhibitor of the rat enzyme. Dichloroally]-lawsone was found to be a time-dependent inhibitor of the rat enzyme, with the lowest inhibition constant (Ki* = 0.77 nM) determined so far for mammalian dihydroorotate dehydrogenases. Another inhibitor, brequinar was previously reported to be a slow-binding inhibitor of the human dihydroorotate dehydrogenase [W. Knecht, M. Loffler, Species-related inhibition of human and rat dihyroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives, Biochem. Pharmacol. 56 (1998) 1259-1264]. The slow binding features of this potent inhibitor (Ki* = 1.8 nM) with the human enzyme, were verified and seen to be one of the reasons for the narrow therapeutic window (efficacy versus toxicity) reported from clinical trials on its antiproliferative and immunosuppressive action. With respect to the substrate dihydroorotate, atovaquone was an uncompetitive inhibitor of human dihydroorotate dehydrogenase (Kiu = 11.6 microM) and a non-competitive inhibitor of the rat enzyme (Kiu = 905/ Kic = 1,012 nM). 1.5 mM polyporic acid, a natural quinone from fungi, influenced the activity of the human enzyme only slightly; the activity of the rat enzyme was decreased by 30%.