Characterization of azadirachtin binding to Sf9 nuclei in vitro

✍ Scribed by Alasdair J. Nisbet; A. Jennifer Mordue (Luntz); Robert B. Grossman; Lyn Jennens; Steven V. Ley; William Mordue

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 91 KB
• Volume: 34
• Category: Article
• ISSN: 0739-4462
• DOI: 10.1002/(sici)1520-6327(1997)34:4<461::aid-arch5>3.0.co;2-v

✦ Synopsis

22,23-3 H 2 ]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 ± 0.008 min -1 using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 ± 0.00013 min -1 . An association rate constant of 7.3 × 10 6 M -1 min -1 was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B max = 23.87 ± 1.15 pmol/mg protein, K d = 18.1 ± 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC 50 in parentheses): azadirachtin (1.55 × 10 -8 M) > dihydroazadirachtin (3.16 × 10 -8 M) > dansyl dihydroazadirachtin (7.40 × 10 -8 M) > DNP-azadirachtin (7.50 × 10 -8 M) > biotin dihydroazadirachtin (1.27 × 10 -7 M) >> 11-methoxy 22,23-dihydroazadirachtin (6.67 × 10 -7 M). Arch.