Cecilia A. McIntosh

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.

The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.

Citrus spp.: In Vitro Culture and the Production of Naringin and Limonin

Citrus is cultivated in many tropical and subtropical regions of the world. Those areas with the highest level of production include parts of the United States (Florida, California, Texas, and Arizona), Asia (Japan, China, India, and other areas), Central America (Mexico), South America (Brazil, Argentina, Chile, Trinidad, and Tobago), the Mediterranean (Spain, Italy, Greece, Cyprus, Israel, and other areas), South Africa, and Australia. These regions help define a “citrus belt” which is contained between 35°N and 35°S (Ziegler and Wolfe 1975). Some of these areas produce citrus on a large scale and export fruit, while others produce fruit mainly for local consumption.

Enzyme Immunoassay of Plant Constituents

The use of immunoassay to quantitate plant derived compounds was first developed in clinical laboratories in the early 1970’s. These first assays were directed toward small organic molecules, e. g., colchicine (Boudene et al., 1975), nicotine (Langone et al., 1973), and morphine (Spector, 1971), which have medicinal or pharmaceutical value. These assays were based on the radioimmunoassay (RIA) and were developed for compounds whose small molecular size renders them non-immunogenic. Assays for low molecular weight compounds of animal origin had been developed earlier (see Erlanger, 1980, and references therein) using synthesized protein-hapten conjugates for immunization. The immunoassays mentioned above demonstrated that the hapten-protein conjugate principle (Landsteiner, 1945) also could be used to induce animals to produce antibodies against compounds of plant origin.