Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-³H₂]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 × 10⁶ 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 (IC₅₀ 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).