Characterization of azadirachtin binding to Sf9 nuclei in vitro

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Abstract
[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 Bmax = 23.87 ± 1.15 pmol/mg protein, Kd = 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). Arch. Insect Biochem. Physiol. 34:461–473, 1997.