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Cholesterol oxidase (cholesterol:oxygen oxidoreductase, EC 1.1.3.6) catalyzes the conversion of sterol delta 5-3 beta-alcohol to the corresponding delta 4-3-ketone with the reduction of oxygen to hydrogen peroxide. Rhodococcus species GK 1, a soil isolated microbe, produces an extracellular and a membrane-bound cholesterol oxidase; the latter is bound to the outer surface of the microbial cell membrane. A simple and sensitive assay is described to measure the two enzyme types; no enzyme extraction is needed for measuring the membrane-bound cholesterol oxidase. In this assay, hydrogen peroxide is reduced by the chromogen 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in the presence of horseradish peroxidase, and the increased absorbance is followed continuously at 600 nm (epsilon m = 1.82 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C). The standardized assay medium contained 46.9 mM sodium-potassium phosphate buffer pH 7.0, 0.16% Triton X-100, 312.5 microM ABTS, 50 micrograms peroxidase (12.5 units at 25 degrees C), 6.25% isopropanol, 306.3 microM cholesterol or other sterols (kept in solution with isopropanol), and cholesterol oxidase. Oxidation of one molecule of cholesterol by cholesterol oxidase gives one molecule of hydrogen peroxide which reacts with two molecules of ABTS. The method is reproducible and the results correlate well with those obtained by measuring the absorbance of delta 4-cholest-3-one at 240 nm (epsilon m = 1.40 x 10(4) M-1.cm-1 at pH 7.0 and 30 degrees C) and by the method of Allain et al. (Clin. Chem. 20, 470-475, 1974).(ABSTRACT TRUNCATED AT 250 WORDS)

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L'application in vitro de l'extrait brut total de vers (Lumbricus terrestris) à différentes dilutions entraîne une stimulation de la rhizogenèse des plantules de haricots (Phaseolus vulgaris). L'effet rhizogène observé est similaire à celui enregistré en présence de l'acide indole acétique, substance de croissance connue chez les végétaux et utilisée ici comme témoin à différentes concentrations. Le fractionnement de l'extrait brut de vers par chromatographie sur colonne a permis d'isoler trois groupes de fractions. Seul le groupe de fractions polaires présente un effet rhizogène notable; cet effet est cependant inférieur à celui observé respectivement en présence de l'extrait brut total de vers et de l'acide indole acétique. Les analyses de l'extrait brut de vers par chromatographie sur couches minces, utilisant des réactifs et des systèmes chromatographiques appropriés, ont révélé l'absence de l'acide indole acétique et la présence probable d'autres dérivés indoliques à comportement chromotographique neutre à basique. Les composés indoliques présumés identifiés sont le méthyl-tryptophane, la sérotonine et l'hydroxyle acide indole acétique. Les analyses par spectrométrie de masse couplées à la chromatographie en phase gazeuse, effectuées après fractionnement et purification du groupe de fractions rhizogènes, ont révélé la présence de l'hydroxyle acide indole carboxylique; celui-ci serait pré-sent sous forme de plusieurs isomères de position. Abstract: In vitro application of total gross extract of earthworms (Lumbricus terrestris) in diverse dilutions stimulates rhizogenesis in young bean plants (Phaseolus vulgaris).The observed effect is similar to that of indol acetic acid, a well-known growth enhancer in plants, used here as a control in various concentrations. Fragmentation of worm extract by column chromatography results in three groups of fractions. Only the polar group of fractions has a significant rhizogenous effect, which is, however, inferior to that observed in the presence of total gross extract of worms or of indol acetic acid. Gross extract analyses using thin layer chromatography, with appropriate chromatography systems and reagents, revealed that indol acetic acid is not present, but is probably replaced by other indol-derived substances that have a neutral to basic chromatographic behaviour. These presumed indol-derived substances are identified as methyltryptophane, serotonin, and hydroxy-indol acetic acid. Analyses using mass spectrometry combined with gas chromatography, following fragmentation and purification of the group of rhyzogenous fractions, have revealed the presence of hydroxy-indol carboxylic acid, which seems to take the form of several isomeres.

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