Enzymes of pyrimidine biosynthesis in Crithidia luciliae.

Evidence has been obtained for the presence of enzymes of both the de novo and salvage pyrimidine pathways in the protozoan parasite, Crithidia luciliae. Carbamyl phosphate synthetase-II activity could not be unequivocally demonstrated in crude extracts. However, a distinct peak of activity with a molecular weight of approximately 500 000 was observed following chromatography on Sepharose CL-6B. The enzyme preferentially utilised glutamine with respect to ammonia. It was inhibited by UTP and 5-phosphoribosyl-1-diphosphate had a small activating effect. Carbamyl phosphate synthesis by a 'phosphorolytic' citrullinase could not be demonstrated. The ensuing three de novo enzymes could also be separated on Sepharose CL-6B. Approximate molecular weights were estimated: aspartate transcarbamylase (150,000); dihydroorotase (90,000) and dihydroorotate dehydrogenase (70,000). As reported previously, orotate phosphoribosyltransferase and orotidylate decarboxylase were particulate, being associated with the glucosome. Activities of the salvage enzymes, uracil phosphoribosyltransferase, uridine phosphorylase and uridine nucleosidase were observed. All enzymes were cytoplasmic. No uridine kinase activity was detected.

Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline.

The activity of Plasmodium falciparum dihydroorotate dehydrogenase, a particulate, electron transport-linked enzyme involved in de novo pyrimidine synthesis, was depressed when the parasite was cultured in the presence of a therapeutic concentration of tetracycline over a 96 h period. There was no direct inhibitory effect of the antibiotic on the enzyme activity. The activity of glutamate dehydrogenase, which is cytoplasmic in the parasite, was unaffected by tetracycline over the same period. Dihydroorotate dehydrogenase activity was substantially recovered when electron acceptors were added. It is suggested that the effect of tetracycline is manifested at the level of the dehydrogenase and/or the electron transport chain linked to this enzyme.

Localization of dihydroorotate oxidase in myocardium and kidney cortex of the rat. An electron microscopic study using the cerium technique.

Biochemical studies have demonstrated that dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1 or 1.3.99.11) is the sole enzyme of de novo pyrimidine synthesis in mitochondria, whereas the rest of the pathway takes place in the cytosol. The dehydrogenation of dihydroorotate to orotate is linked to the respiratory chain via ubiquinone. In this study, we show for the first time the ultrastructural localization of DHOdehase. Since the purified enzyme was found to act both as dehydrogenase and as oxidase, the cerium capture technique for detecting enzymatically generated hydrogen peroxide could be applied to pin-point the in situ activity of DHOdehase oxidase in mitochondria of rat heart and kidney cortex. Cerium perhydroxide as the final reaction product was detected predominantly in the matrix with some focal condensation along the inner membrane, but not in the intermembrane space. From this pattern of localization, it is concluded that the active site of the membrane-bound enzyme could face the mitochondrial matrix similar to succinate dehydrogenase. The reliability of the applied method for the demonstration of DHOdehase oxidase was demonstrated by the addition of Brequinar sodium to the incubation medium. This quinoline-carboxylic acid derivative is a potent inhibitor of DHOdehase and has proven anti-proliferative activity. The present observations do not ascertain whether the oxidase is permanently active as a constant portion of the enzyme in vivo, similar to xanthine oxidase/dehydrogenase. However, DHOdehase should be considered as a source of radical oxygen species under pathophysiological conditions.

Pyrimidine biosynthesis in Neisseria meningitidis. 1. Demonstration of enzyme activities.

Activities of the five enzymes specific for the pyrimidine biosynthetic pathway, aspartate carbamoyl-transferase (ACTase), dihydroorotase (DHOase), dihydroorotate dehydrogenase (DHOdehase), orotate phosphoribosyltransferase (OMPppase), and orotidine 5'-phosphate decarboxylase (OMPdecase) were found in cell-free extracts of Neisseria meningitidis. Extracts also contained enzyme activities corresponding to the arginine pathway enzyme ornithine carbamoyltransferase (OCTase), and to carbamoylphosphate synthetase (CPSase), the enzyme common to both pathways. N. meningitidis possessed a single glutamine-dependent CPSase.

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.

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 gastrointestinal 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 1:4. This was in accordance with that of 1:5 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 1:15 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.

Immunochemical analysis of membrane vesicles from Escherichia coli.

Membrane vesicles isolated from Escherichia coli ML 308--225 have been analyzed by crossed immunoelectrophoresis, and immunoprecipitates corresponding to the following cellular components have been identified: ATPase (EC 3.6.1,3), two or three NADH dehydrogenases (EC 1.6.99.3), D-lactate dehydrogenase (EC 1.1.1.27), glutamate dehydrogenase (EC 1.4.1.4), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), beta-galactosidase (EC 3.2.1.23), lipopolysaccharide, and Braun's lipoprotein. The cellular origin of many of the vesicle immunogens is determined, and Braun's lipoprotein is used as a marker to quantitate the extent of outer membrane contamination (less than 3%). Membrane antigens are also characterized with regard to their amphiphilic or hydrophilic properties by charge-shift crossed immunoelectrophoresis. Furthermore, the following immunogens cross-react with components in membrane vesicles prepared from Salmonella typhimurium: one of the three NADH dehydrogenases, ATPase, polynucleotide phosphorylase, 6-phosphogluconate dehydrogenase, Braun's lipoprotein, and three unidentified antigens. In the accompanying paper [Owen, P., & Kaback, H. R. (1979) Biochemistry 18 (following paper in this issue)] quantitative immunoadsorption is utilized to establish the topology of the vesicles with respect to the distribution of antigens on the inner and outer faces of the membrane.