Developmental changes in the concentration of ?-bungarotoxin binding sites in the brain of the tobacco hornworm, Manduca sexta.

A moiety in the brain of Manduca sexta larvae that binds ?-bungarotoxin (?-BGT) specifically has been characterized and the concentration of binding sites determined during larval-larval and larval-pupal development. The protein nature of this moiety was determined by its susceptibility to proteolytic digestion and its location in the membrane ascertained by differential centrifugation and subsequent detergent extraction. The association of specific binding approaches equilibrium after 15 min of incubation (K(1) = 4.7 x 10(4) M(?1).s(?1)), while the half life of the receptor-ligand complex is 55 min, and the rate of dissociation is 3.5 x 10(?4).s(?1). Saturation of binding occurs at an ?-BGT concentration of 90 nM. The saturation curve yielded a B(max) of 2.5 pmol ?-BGT binding sites/mg brain protein, a concentration some 25-250 times greater than values reported for mammalian nervous tissue. Scatchard plot analysis of saturation binding revealed a K(D) of 16 nM, the highest recorded for insect nervous tissue, but within the range of published data for other invertebrate nervous tissues. A lower K(D) of 7.4 nM was obtained from on-off kinetics. Pharmacological analysis revealed that nicotinic ligands competed weakly with ?-BGT for binding sites and the K(i) obtained for the various agonists and antagonists used was ? 10-fold greater than those reported previously for the insect nicotinic acetylcholine receptor. The developmental study of ?-BGT binding sites revealed specific changes in the concentration of this binding protein that appeared to correlate temporally with the release of the brain neurohormone, prothoracicotropic hormone, a neuropeptide that elicits insect molting. Since a detergent-solubilized brain extract was used in these studies, the noted fluctuations in the concentration of binding sites probably reflect synthesis and degradation of the binding protein. These data indirectly implicate the cholinergic system with the release of the brain neurohormone and, therefore, suggest a critical role for the cholinergic system in insect molting.