Benzphetamine demethylase and aniline hydroxylase activities were determined with various hemoproteins including indoleamine 2,3-dioxygenase in a cytochrome P-450-like reconstituted system containing NADPH, NADPH-cytochrome P-450 reductase, and O2. The highest specific activities, almost comparable to those of liver microsomal cytochrome P-450, were detected with indoleamine 2,3-dioxygenase from the rabbit intestine. The indoleamine 2,3-dioxygenase-catalyzed benzphetamine demethylation reaction was inhibited by catalase but not by superoxide dismutase. Exogenous H2O2 or organic hydroperoxides was able to replace the reducing system and O2. The stoichiometry of H2O2 added to the product formed was essentially unity. These results indicate that the dioxygenase catalyzes the demethylation reaction by the so-called "peroxygenation" mechanism using H2O2 generated in the reconstituted system. On the other hand, the dioxygenase-catalyzed aniline hydroxylation reaction was not only completely inhibited by catalase but also suppressed by superoxide dismutase by about 60%. Although the O2- and H2O2-generating system (e.g. hypoxanthine-xanthine oxidase) was also active as the reducing system, neither exogenous H2O2 nor the generation of O2- in the presence of catalase supported the hydroxylation reaction, indicating that both H2O2 and O2- were essential for the hydroxylation reaction. However, typical scavengers for hydroxyl radical and singlet oxygen were not inhibitory. These results suggest that a unique, as yet unidentified active oxygen species generated by H2O2 and O2- participates in the dioxygenase-mediated aniline hydroxylation reaction.

Aniline Compounds↗

PubMed1983

Induction of indoleamine 2,3-dioxygenase in alveolar interstitial cells of mouse lung by bacterial lipopolysaccharide.

The cellular localization of indoleamine 2,3-dioxygenase was studied in the mouse lung after induction by lipopolysaccharide treatment. No significant indoleamine 2,3-dioxygenase activity was detected in alveolar macrophages and type II epithelial cells, which were recovered by alveolar lavages and trypsin-treatment, respectively. To determine this enzyme activity in other types of lung cells, we prepared monodispersed lung cells (6.5 X 10(7) cells/lung) by incubation with 0.1% collagenase and 0.1% trypsin. In a Percoll isopycnic gradient, the dispersed cells were distributed with two peaks at the densities of 1.040 and 1.080 g/ml. The enzyme activity was recovered exclusively in the lighter fractions. As examined by electron microscopy or more quantitatively by using various marker enzyme activities, endothelial cells (angiotensin-converting enzyme as a marker enzyme of these cells), alveolar interstitial cells (prostaglandin dehydrogenase), type I epithelial cells, type II epithelial cells, alveolar macrophages (beta-glucuronidase), Clara cells (coumarin hydroxylase), and polymorphonuclear leucocytes (arylsulfatase) were distributed with peaks at the densities of 1.033, 1.040, 1.042, 1.045, 1.070, 1.082, and 1.093 g/ml, respectively. The distribution pattern of the indoleamine 2,3-dioxygenase activity exactly coincided with that of alveolar interstitial cells. The localization of this enzyme in alveolar interstitial cells was immunohistochemically confirmed with the anti-indoleamine 2,3-dioxygenase antibody.

Animals↗

PubMed1983

Lectin-binding heterogeneity of alphafetoprotein (AFP). An observation in nude mouse xenografts of endodermal sinus tumors and in pediatric surgical patients.

We determined by affinity chromatography on concanavalin A-Sepharose the carbohydrate variant patterns of alphafetoprotein in the sera of 15 infants and children with endodermal sinus tumors (five cases), a neonatal mature teratoma (one case), hepatoblastomas (two cases), pancreatic carcinoma (one case), biliary atresia (four cases), neonatal hepatitis (one case) and neonatal hyperbilirubinemia (one case), in the sera from four normal neonates, and in the sera from two kinds of nude mice bearing human endodermal sinus tumors. Sera from patients with endodermal sinus tumors and pancreatic carcinoma were found to contain a relatively high proportion (48.4 +/- 4.5 and 52.6%) of alphafetoprotein which did not bind to concanavalin A. Sera from nude mice with human endodermal sinus tumors contained AFP, 96.2% of which did not bind to concanavalin A. Sera from patients with other lesions (nine cases) and from normal neonates, whose AFPs are all presumed to be of hepatic origin, contained much less (5.9 +/- 3.6%) of the concanavalin A non-binding AFP variant. These results indicate that human AFP has three distinct patterns of reactivity with concanavalin A and that studies in xenograft models may give important information relating to the glycosylation and secretion process of AFP.

Adolescent↗

PubMed1983