Characterization of azadirachtin binding to Sf9 nuclei in vitro.
[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 x 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 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) >> 11-methoxy 22,23-dihydroazadirachtin (6.67 x 10(-7) M). [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:461-473 (1997).]
Nisbet
A J
AJ
Department of Zoology, University of Aberdeen, Tillydrone Ave., Aberdeen AB24 2TZ, United Kingdom.
Mordue Luntz
A J
AJ
Grossman
R B
RB
Jennens
L
L
Ley
S V
SV
Mordue
W
W
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000502:physiology; D018929:Cell Culture Techniques; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D007306:Insecticides / Q000378:metabolism* / Q000494:pharmacology; D036701:Limonins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018411:Spodoptera / Q000187:drug effects / Q000254:growth & development*; D013379:Substrate Specificity; D014315:Triterpenes / Q000378:metabolism* / Q000494:pharmacology
D007306:Insecticides; D036701:Limonins; D011500:Protein Synthesis Inhibitors; D014315:Triterpenes; C010329:azadirachtin