Aj. Nisbet et al., CHARACTERIZATION OF AZADIRACHTIN BINDING TO SF9 NUCLEI IN-VITRO, Archives of insect biochemistry and physiology, 34(4), 1997, pp. 461-473
[22,23-H-3(2)]dihydroazadirachtin was incorporated by Sf9 cells in cul
ture and was bound specifically to the nuclear fraction. The observed
association constant of the binding of the radioligand to a purified n
uclear fraction was determined to be 0.037 +/- 0.008 min(-1) using a o
ne-phase exponential association equation, and binding appeared to be
to a single population of sites. The binding was essentially irreversi
ble, and the dissociation constant was estimated to be 0.00065 +/- 0.0
0013 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 recep
tor 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 semisynthe
tic azadirachtin analogues for competition for the binding site was as
follows (IC50 in parentheses): azadirachtin (1.55 x 10(-8) M) > dihyd
roazadirachtin (3.16 x 10(-8) M) > dansyl dihydroazadirachtin (7.40 x
10(-8) M) > DNP-azadirachtin (7.50 x 10(-8) M) > biotin dihydroazadira
chtin (1.27 x 10(-7) M) much greater than ll-methoxy 22,23-dihydroazad
irachtin (6.67 x 10(-7) M). (C) 1997 Wiley-Liss, Inc.