The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi.

In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane.

A alpha-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes.

alpha-glycerophosphate dehydrogenase (alpha-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance.

A alpha-glycerophosphate dehydrogenase is present in Trypanosoma cruzi glycosomes

alpha-glycerophosphate dehydrogenase (alpha-GPDH-EC.1.1.1.8) has been considered absent in Trypanosoma cruzi in contradiction with all other studied trypanosomatids. After observing that the sole malate dehydrogenase can not maintain the intraglycosomal redox balance, GPDH activity was looked for and found, although in very variable levels, in epimastigotes extracts. GPDH was shown to be exclusively located in the glycosome of T. cruzi by digitonin treatment and isopycnic centrifugation. Antibody against T. brucei GPDH showed that this enzyme seemed to be present in an essentially inactive form at the beginning of the epimastigotes growth. GPDH is apparently linked to a salicylhydroxmic-sensitive glycerophosphate reoxidizing system and plays an essential role in the glycosome redox balance

Purification and properties of phosphoglucose isomerases of Trypanosoma cruzi.

Glucosephosphate isomerase (PGI; EC 5.3.1.9) of Trypanosoma cruzi epimastigotes was found in about the same proportion in the glycosome and the cytosol. This subcellular distribution is similar to that of Leishmania mexicana, but contrasts with that of T. brucei bloodstream form, where the enzyme is essentially restricted to the glycosome. Glucosephosphate isomerase was highly purified from a glycosome-enriched fraction and to about 70% purity from the soluble extract. Both enzymes displayed Michaelis-Menten-Henri kinetics. Km values for fructose 6-phosphate were 0.125 +/- 0.07 and 0.80 +/- 0.10 mM for the glycosomal and the cytosolic PGIs, respectively. Erythrose-4-phosphate, 6-phosphogluconate and mannose-6-phosphate were inhibitors for both PGIs. Phosphogluconate and erythrose phosphate showed higher affinity for cytosolic PGI than for glycosomal PGI, by 2.5- and 4-fold respectively. The PGIs differed slightly in their isoelectric point (7.1 +/- 0.15 and 7.5 +/- 0.12) and optimum pH range. Both PGIs also differed in their chromatographic properties (ion-exchange and phenyl Sepharose), indicating a difference in charge and hydrophobicity, with the glycosomal enzyme being more hydrophobic. The molecular mass of both PGIs was 186,000 +/- 9000 Da, which is higher than that of other known PGIs, including those from T. brucei and other trypanosomatids. The molecular mass of the subunit, 63 kDa, is similar to that of PGIs from other sources. It appears that PGIs from T. cruzi are trimeric, in contrast with all other known PGIs which are dimeric.

Isolation and identification of menaquinone-9 from purified nitrate reductase of Escherichia coli.

On the basis of the observation that nitrate reductase from Escherichia coli is sensitive to UV irradiation with an action spectrum indicative of a naphthoquinone (F. Brito and M. Dubourdieu, Biochem. Int. 15:1079-1088, 1987), we extracted and characterized quinone components from two different preparations of purified nitrate reductase. A soluble form of nitrate reductase, composed of alpha and beta subunits, was purified after release from the membrane fraction by heat treatment, and a detergent-solubilized form, containing alpha, beta, and gamma (cytochrome bNR) subunits, was purified in the presence of Triton X-100. Extraction of soluble alpha beta form with chloroform-methanol yielded several UV-absorbing components, which were characterized as menaquinone-9 with an oxidized side chain and further photodestruction products of the menaquinone. The total amount of menaquinone extracted into the organic phase was estimated to be 0.97 mol/mol of alpha beta dimer. Extraction of the detergent-solubilized alpha beta gamma form by a similar procedure yielded two naphthoquinone-like components which were characterized by mass spectrometry as the oxidized forms of menaquinone-9 and demethylmenaquinone-9. In this case, the molar ratio of total naphthoquinone to the alpha beta dimer was estimated to be greater than 6:1. When cytochrome bNR and detergent were eliminated from the detergent-solubilized enzyme by heat treatment and ion-exchange chromatography, only menaquinone-9 could be identified in the organic extract of the active alpha beta product. These results suggest that menaquinone-9 is specifically bound to the alpha beta dimer and may be the UV-sensitive component in the pathway of electron transfer catalyzed by nitrate reductase.

Molybdenum uptake in Escherichia coli K12.

Molybdenum uptake was examined in Escherichia coli K12 using the radionuclide 99Mo. The molybdenum uptake system was characterized in an unusual chlD strain, which appeared to be normal in uptake of the MoO4(2-) ion but altered in subsequent molybdenum processing. As a consequence, molybdenum could be chased from cells in the chlD strain, while it was irreversibly assimilated in the wild-type strain. Molybdenum uptake showed a biphasic kinetic curve, with a very rapid binding followed by a slow uptake phase. The uptake appeared to involve an active transport system. Molybdenum, probably in the form of molybdate, accumulated by a factor of about 30 in the cells. An energy source was necessary and uptake was inhibited by arsenate, but not by CCCP (carbonyl cyanide m-chlorophenylhydrazone). The uptake system saturated with a Km of 2.5-2.7 x 10(-8) M. Uptake seemed to depend on a periplasmic binding protein, since cold shock treatment and arsenate abolished uptake. A molybdate binding protein activity was detected in the periplasmic fluid with a KD of 9 nM. Sulphate inhibited uptake and the uptake activity was pH dependent, with an apparent pK of 6.7. These results imply that molybdate transport belongs to the family of energy-dependent periplasmic binding protein systems. An explanation for the peculiar behaviour of the chlD strain used in this work is proposed.

The narJ gene product is required for biogenesis of respiratory nitrate reductase in Escherichia coli.

Respiratory nitrate reductase purified from the cell membrane of Escherichia coli is composed of three subunits, alpha, beta, and gamma, which are encoded, respectively, by the narG, narH, and narI genes of the narGHJI operon. The product of the narJ gene was deduced previously to be a highly charged, acidic protein which was not found to be associated with any of the purified preparations of the enzyme and which, in studies with putative narJ mutants, did not appear to be absolutely required for formation of the membrane-bound enzyme. To test this latter hypothesis, the narJ gene was disrupted in a plasmid which contained the complete narGHJI operon, and the operon was expressed in a narG::Tn10 insertion mutant. The chromosomal copy of the narJ gene of a wild-type strain was also replaced by the disrupted narJ gene. In both cases, when nar operon expression was induced, the alpha and beta subunits accumulated in a form which expressed only very low activity with either reduced methyl viologen (MVH) or formate as electron donors, although an alpha-beta complex separated from the gamma subunit is known to catalyze full MVH-linked activity but not the formate-linked activity associated with the membrane-bound complex. The low-activity forms of the alpha and beta subunits also accumulated in the absence of the NarJ protein when the gamma subunit (NarI) was provided from a multicopy plasmid, indicating that NarJ is essential for the formation of the active, membrane-bound complex. When both NarJ and NarI were provided from a plasmid in the narJ mutant, fully active, membrane-bound activity was formed. When NarJ only was provided from a plasmid in the narJ mutant, a cytosolic form of the alpha and beta subunits, which expressed significantly increased levels of the MVH-dependent activity, accumulated, and the alpha subunit appeared to be protected from the proteolytic clipping which occurred in the absence of NarJ. We conclude that NarJ is indispensible for the biogenesis of membrane-bound nitrate reductase and is involved either in the maturation of a soluble, active alpha-beta complex or in facilitating the interaction of the complex with the membrane-bound gamma subunit.

An ultraviolet light sensitive target in nitrate reductase of Escherichia coli K-12.

Ultraviolet light was shown to inactivate purified nitrate reductase in the presence of reduced benzyl viologen. Loss of activity was not complete, reaching 60 to 70%. Photolysis was maximum at 345 nm. The differential spectrum between native and irradiated enzyme exhibited absorption bands at 216, 275, 314 and 365 nm. The photosensitive electron carrier could be extracted by organic solvents. It had the following absorption bands: 225, 275 and 285 nm. It was reduced by Nile blue A but not by methylene blue. The precise nature of this light sensitive molecule could not be determined although the results support the idea that this chromophore might be a naphthoquinone.

Tetrathionate reductase of Salmonella thyphimurium: a molybdenum containing enzyme.

Use of radioactive molybdenum demonstrates that the tetrathionate reductase of Salmonella typhimurium is a molydenum containing enzyme. It is proposed that this enzyme shares with other molybdo-proteins, such as nitrate reductase, a common molybdenum containing cofactor the defect of which leads to the loss of the tetrathionate reductase and nitrate reductase activities.

Periplasmic cytochrome C552 of Escherichia coli K12. Oligomeric structure and its role in nitrite reduction.

Perisplasmic cytochrome C552 was purified from a cold shock fluid. We demonstrated that this protein could reduce nitrite into ammonium. The reducing equivalents could be donated by formate or NADH through some segment of the membrane respiratory chain. FAD addition was necessary when pure cytochrome was used to reduce nitrite. The predominant form was found to have a molecular weight of about 65 to 70 K daltons, composed of three 21,000 d. subunits. Hemes may or may not be present on the subunit.

[Parasitic appendicitis. Apropos of 4 cases of acute appendicitis]. / Les appendicites parasitaires. A propos de 4 observations d'appendicite aiguë.

The authors present 4 observations of acute appendicitis with the presence of parasites at the micropathological examination. To this end, they study the literature in order to try to show the frequency of parasitic appendicitis on the one hand and what it does on the other, which is still controversial in the physiopathological of appendicitis. According to the authors, an aetiology of parasitic involvement is evident in 1.9% to 25% cases of appendicitis. The most frequent intestinal parasites in this area are the pinworms (54.78%), the whipworms (13%) and the ascaris (9%).

Reconstitution "in vitro" of a formate or NADH dependent nitrite reductase activity starting from cytochrome C552 and membrane vesicles from Escherichia coli K-12.

El aislamiento en Escherichia coli K-12 de un cytocromo C552, libre de contaminantes particulados, permitio la reconstitucion "in vitro" de una actividad nitrito reductasa que requiere vesiculas de membrana, cytocromo C552 y formiato (o NADH) como donadores de electrones. La via de transferencia de electrones desde el formiato o el NADH incluye un segmento de las cadenas respiratorias membranales del nitrato y el oxigeno que carece de cytocromos de tipo b. Este segmento contiene NADH y formiato deshidrogenasas, y un transportador redox sensible al 2-heptil-4hidroxiquinolina-N-oxido (HQNO). Evidencias indirectas sugieren que este transportador es probablemente una quinona que reaccionaria directamente, o indirectamente por medio de un transportador desconocido, con el cytocromo C552 periplasmico soluble. En este sistema el citocromo C552 parece actuar directamente sobre el nitrito como una nitrito reductasa terminal

Chlorate metabolism by whole cells and membrane vesicles of Escherichia coli K-2.

Se siguio la reduccion del clorato por Escherichia coli K-12 mediante la utilizacion de clorato marcado 36 C1-, preparado por oxidacion anodica del ion cloruro. Las celulas redujeron el nitrato y el clorato a velocidades similares. Las vesiculas de membrana y las celulas no acumularon clorato ni sus productos de reduccion (C102-, C10-, C1). El bajo nivel de radioactividad enlazado a celulas o vesiculas puede ser atribuido principalmente a absorcion inespecifica. Estos resultados son discutidos en relacion con la localizacion del sitio enlazante del clorato (y el nitrato) sobre la nitrato reductasa membranal de E. coli

Localization and characterization of cytochromes from membrane vesicles of Escherichia coli K-12 grown in anaerobiosis with nitrate.

Cytochromes b of anaerobically nitrate-grown Escherichia coli cells are analysed. Ascorbate phenazine methosulfate distinguishes low and high potential cytochromes b. Reduction kinetics performed at 559 nm presents a very complex pattern which can be analysed assuming that at least four b-type cytochromes are present. The electron transport chain from formate to oxygen would contain a low potential cytochrome b-556, a cytochrome b-558 associated to the oxidase, and a cytochrome d as the principle oxidase. Cytochrome o is also present, but seems to be functional only at low oxygen concentrations. A cytochrome b-556 associated to nitrate reductase is shown to belong to a branch of the formate-oxidase chain. 2-N-Heptyl-4-hydroxyquinoline-N-oxide affects the reduction kinetics in a very complex way. One inhibition site is in evidence between cytochrome b-558 and cytochrome d; another between the cytochrome associated to nitrate reductase and the nitrate reductase. A third inhibition site is located in the common part of the formate-nitrate and the formate-oxidase systems. Ascorbate phenazine methosulfate is shown to donate electrons near cytochrome b-558.

Amino acid sequence of Desulfovibrio vulgaris flavodoxin.

The complete amino acid sequence for the 148-amino acid flavodoxin from Desulfovibrio vulgaris was determined to be: H3N+-Met-Pro-Lys-Ala-Leu-Ile-Val-Tyr-Gly-Ser-Thr-Thr-Gly-Asn-Thr-Glu-Tyr-Thr-Ala-Glu-Thr-Ile-Ala-Arg-Glu-Leu-Ala-Asn-Ala-Gly-Tyr-Glu-Val-Asp-Ser-Arg-Asp-Ala-Ala-Ser-Val-Glu-Ala-Gly-Gly-Leu-Phe-Glu-Gly-Phe-Asp-Leu-Val-Leu-Leu-Gly-Cys-Ser-Thr-Trp-Gly-Asp-Asp-Ser-Ile-Glu-Leu-Gln-Asp-Asp-Phe-Ile-Pro-Leu-Phe-Asp-Ser-Leu-Glu-Glu-Thr-Gly-Ala-Gln-Gly-Arg-Lys-Val-Ala-Cys-Phe-Gly-Cys-Gly-Asp-Ser-Ser-Tyr-Glu-Tyr-Phe-Cys-Gly-Ala-Val-Asp-Ala-IleGlu-Glu-Lys-Leu-Lys-Asn-Leu-Gly-Ala-Glu-Ile-Val-Gln-Asp-Gly-Leu-Arg-Ile-Asp-Gly-Asp-Pro-Arg-Ala-Ala-Arg-Asp-Asp-Ile-Val-Gly-Try-Ala-His-Asp-Val-Arg-Gly-Ala-Ile-COO. This protein is of interest as it was the first flavoenzyme for which high resolution x-ray diffraction studies were published (Watenpaugh, K.D., Sieker, L.C., and Jensen, L.H. (1973) Proc. NAtl. Acad. Sci. U.S.A. 70, 3857-3860). Ser(10), Thr(12), Asn(14), and Thr(15) were shown to bind the phosphate of the FMN while the isoalloxazine ring is positioned between Trp(60) and Tyr(98).