Sequestration of insecticyanin, a blue hemolymph protein, into the egg of the hawkmoth Manduca sexta. Evidence for receptor-mediated endocytosis.

Sequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated. Immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 microM) is slightly higher than that in hemolymph (25.8 microM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of 125I-insecticyanin by isolated oocytes was saturable. Analysis of in vitro uptake data estimated that the value of K(uptake) (insecticyanin concentration at half-maximal uptake rate) is 4.2 microM and that the Vmax (maximum rate of uptake) is 1 pmol follicle-1 h-1. Labeled insecticyanin was shown to bind to sonicated follicle membranes with high specificity and affinity. The KD (equilibrium dissociation constant) and the Bm (total number of binding sites per follicle), were estimated as 4 x 10(-8) M and 8 x 10(7) respectively. Competition studies showed that binding of labeled insecticyanin to oocyte membranes was blocked by excess amounts of unlabeled insecticyanin but not by lipophorin and vitellogenin of M. sexta. Additional membrane binding experiments demonstrated that receptors for insecticyanin are only present in the oocytes membranes, not in fat body or gut tissue.