Biochemical studies on an oocyte membrane receptor for the biliprotein, insecticyanin, of the hawkmoth Manduca sexta

Abstract

Sequestration of the blue biliprotein, insecticyanin, into developing oocytes of the hawkmoth, Manduca sexta was investigated using diverse techniques. Oudin's immunodiffusion assays revealed that insecticyanin concentration in mature eggs (29.6 μM) is slightly higher than that in hemolymph (25.8 μM). The endocytotic uptake of insecticyanin was visualized at the light microscopic level using autoradiography. Uptake of ¹²⁵I-insecticyanin by isolated oocytes was ligand specific, saturable with increasing insecticyanin concentration, and sensitive to parameters such as size of oocyte, constituents of media and temperature. Analysis of in vitro uptake data yielded values of K(uptake) (insecticyanin concentration at half-maximal uptake rate) of 4.2 μM and V(max) (maximum rate of uptake) of 1 pmol follicle⁻¹ h⁻¹. Oocyte membrane proteins were efficiently solubilized using 40 mM detergent CHAPS. Labeled insecticyanin was shown to bind to crude follicle membranes and solubilized membrane proteins with high specificity and affinity. The K(d) (equilibrium dissociation constant) was estimated as 40 nM and 17 nM for crude membranes and solubilized membrane proteins, respectively. The B(m) (maximum binding) estimated from crude membrane and solubilized membrane protein was 1.6 and 11.4 pmol/mg proteins respectively. Competition studies showed that binding of labeled insecticyanin to its receptor was blocked by an excess of unlabeled insecticyanin but not by other major hemolymph proteins, lipophorin and vitellogenin of M. sexta. Additional binding experiments demonstrated that receptors for insecticyanin are only present in oocyte membranes, not in fat body, gut tissue or ovariole. The results from co-immunoprecipitation showed that the apparent molecular mass for insecticyanin receptor is approximately 185 kDa on reducing SDS-PAGE gel while chemical crosslinking of the insecticyanin-receptor complex revealed a product with a molecular mass near 1000 kDa. This result suggests that the insecticyanin receptor has a multimeric structure, or that four receptor molecules can bind to one insecticyanin tetramer.