ISSN:
0739-4462
Keywords:
insect hemolymph proteins
;
fluorescence spectroscopy
;
native electrophoresis
;
root weevils
;
Coleoptera
;
Curculionidae
;
citrus
;
Chemistry
;
Food Science, Agricultural, Medicinal and Pharmaceutical Chemistry
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
Notes:
A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1-5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 Mr. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 Mr, while the lower band (band II) yielded only one size subunit of 75,000 Mr on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a KD of 1.5 μM.
Additional Material:
10 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/arch.940110202
