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 X 10(6) 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 X 10(-8) M) dihydroazadirachtin (3.16 X 10(-8) M) dansyl dihydroazadirachtin (7.40 X 10(-8) M) DNP-azadirachtin (7.50 X 10(-8) M) biotin dihydroazadirachtin (1.27 X 10(-7) M) much greater than 11-methoxy 22,23-dihydroazadirachtin (6.67 X 10(-7) M)