Richard L. Mansell

Multiple forms and specificity of coniferyl alcohol dehydrogenase from cambial regions of higher plants

Abstract Alcohol dehydrogenases of 89 species of plants, from the Bryophyta, Pteridophyta, Gymnosperms and Angiosperms were examined by starch gel electrophoresis for their substrate and coenzyme specificities. High activities and multiple bands were observed with EtOH and NAD in most species. The same, but weaker banding patterns were also observed with benzyl alcohol and salicin. When coniferyl alcohol was used as substrate, activity was found only with NADP as coenzyme and the resulting bands were distinct from those obtained with the other substrates. Most plants tested had only one or occasionally a second coniferyl alcohol dehydrogenase band. Salix species were an exception, with multiple bands found in each of the species tested.

Biosynthesis of naringin in Citrus paradisi: UDP-glucosyl-transferase activity in grapefruit seedlings

Abstract A UDP-glucosyltransferase activity capable of position-specific transfer of glucose to the 7-position of naringenin has been isolated from grapefruit seedlings. Leaves are the richest source of this activity on a fresh weight basis. Characteristics include a pH optimum of 6.5–7.5, a temperature optimum of 37°, an apparent E act of 7.76 kcal mol −1 , and an apparent K m for naringenin of 0.08 mM. Hesperitin and 2′,4′,6′,4-tetrahydroxychalcone are also glucosylated with apparent K m s of 0.11 mM and 0.13 mM respectively. Thermal degradation studies indicate that the naringenin and hesperitin glucosylating activities have different stabilities. Chromatofocusing resolves the naringenin glucosylating activity into two peaks with apparent pIs of 4.4 and 3.9. The hesperitin glucosylating activity separates into three peaks with apparent pIs of 4.5, 4.4, and 4.0.

Naringin and Limonin Production in Callus Cultures and Regenerated Shoots from Citrus sp.

Summary Limonin, limonoate A-ring monolactone, and naringin levels in Citrus paradisi Macf. and Citrus sinensis Osbeck were monitored during initiation of callus from hypocotyl sections and during shoot regeneration. The results show that the callus material contained substantially reduced levels of each of these compounds and that shoot development was accompanied by an increase in naringin and limonoate A-ring monolactone. Callus initiated from albedo explants showed a rapid decrease in limonin and naringin during the first three weeks of culture and by week 42 contained an average of 0.1 % of the original naringin and 0.06 % of the original limonin levels. Less than 1.5 % of the original naringin and limonin was lost to the culture medium. Limonin and naringin were still detectable in callus from different explant sources which had been in culture for over 3 years and a wide range of concentrations (over 10 fold) was found.

Radioimmunoassay for the quantitative determination of hesperidin and analysis of its distribution in Citrus sinensis

Abstract A simple and sensitive radioimmunoassay (RIA) for the citrus flavanone hesperidin and other flavonoid 7-rutinosides is described. The assay utilizes antibodies raised against a hesperidin 4- O -carboxymethyl-oxime hapten and a tritiated radiotracer prepared by direct reduction of hesperidin with NaB[3-H]4. The detection limit of the assay is 0.2 ng/0.1 ml (0.3 pmol/0.1 ml and the measuring range extends to 10 ng/0.1 ml (16.4 pmol/0.1 ml). This assay is specific for flavonoid rutinosides, is characterized by a low coefficient of variation, and shows good correlation with HPLC for the quantification of hesperidin in oranges. The application of this method in determining the distribution of hesperidin in leaves, fruit, seeds, and seedlings of Citrus sinensis is also reported.

Competitive solid phase enzyme-linked immunoassay for the quantification of limonin in citrus

A solid-phase enzyme immunoassay for the quantitative determination of the bitter triterpene-lactone, limonin, in citrus juice samples is described. As little as 0.1 ppm of limonin can be detected. Quantitative results are available within 1 h of total assay time. The assay makes use of a limonin-alkaline phosphatase tracer of high immunoreactivity and has been semiautomated using antibody-coated polystyrene microcuvettes, a vertical light path photometer, and a forced-air microplate incubator.

Isolation and partial characterization of three glucosyl transferases involved in the biosynthesis of flavonol triglucosides in Pisum sativum L

Abstract The glucosyl transferases involved in the biosynthesis of flavonol-3-triglucosides in Pisum sativum L. have been further purified and characterized. The reaction sequence is as follows: Flavonol → flavonol-3-monoglucoside → flavonol-3-diglucoside → flavonol-3-triglucoside The reaction kinetics suggested the involvement of three distinct glucosyl transferases which could be demonstrated after elution of the enzyme preparation from DE-52 with a linear KCl gradient. Each enzyme has been characterized as to its pH optimum, stability, substrate specificity, and Km. The results obtained show that the synthesis of flavonol triglucosides in Pisum involves three distinct and highly specific enzymes, and the kinetic data present an explanation for the lack of accumulated mono- and diglucoside derivatives in vivo.

Polyamide column chromatography for resolution of complex mixtures of anthocyanins

Abstract A method for the separation of anthocyanins on polyamide columns is described. This procedure makes possible the detection and enrichment of minor components and the separation of derivatives which are difficult to separate by paper or thin-layer chromatography. Separation of acylated anthocyanins was effected with minimal degradation. Microcolumns gave excellent resolution of extracts from as little as 15 mg tissue.

Partial purification and characterization of a flavonoid-3-β-d-glucosidase from petals of impatiens balsamina

Abstract A flavonoid-3-β- d -glucosidase preparation from petals of Impatiens balsamina was partially purified and characterized. The glucosidase showed a strict requirement for an aromatic aglycone and a single β-linked glycoside. Two pH optima were found at 3·5 and 5·6. When the preparation was incubated with pelargonidin-3-monoglucoside, hydrolysis of the glucoside was followed by a spontaneous decomposition of the aglucone nucleus. Both glucosidase and galactosidase activities were detected.

O-methyltransferase activity from young flower petals of Impatiens balsamina

Abstract Petals from stage 3 flower buds of the purple (LLhhP r P r ) genotype of Impatiens balsamina were examined for O -methyltransferase activity. Acetone powders of the tissue gave active preparations which would methylate caffeic acid to ferulic acid in the presence of S -adenosyl- l -methionine. The biosynthetic role of this enzyme is discussed.

Peroxidase-linked, solid-phase enzyme immunoassay for the determination of picomole levels of limonin☆

Abstract A solid-phase enzyme immunoassay (EIA) for the determination of 0.1–10.0 ng/0.1 ml of the bitter triterpenelactone, limonin, in plant extracts and juice samples is described. As little as 0.15 pmol of limonin can be detected. Quantitative results are available within 30 min of total assay time. The assay makes use of a limonin-horseradish-peroxidase tracer of high immunoreactivity and has been semi-automated using antibody-coated polystyrene optical cuvettes.