A rapid, efficient procedure for the isolation and purification of the vitellogenin binding protein from locust ovarian membranes is described. After solubilization with the nonionic detergent octyl-P-D-glucoside and removal of the detergent, the binding protein is subjected to affinity chromatography on vitellogenin coupled covalently to Affi-Gel 15. The binding protein is eluted with suramin and EDTA at low pH value. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis reveals a polypeptide with a molecular weight of 156,000 in the eluted fraction. By ligand blotting this polypeptide could be identified as the vitellogenin binding protein. It retains its high-affinity binding properties. The specific binding of vitellogenin increases from 4.8 kg (intact ovarian membranes) to 170.9 pg (affinity purified binding protein) per rng rnembrane protein, which corresponds to a purification factor of 35.
Key words: endocytosis, binding protein, vitellogenin, locust, affinity purification
INTRODUCTIONIn egg-laying animals the predominant yolk protein precursors are called vitellogenins. Insect vitellogenins are synthesized in the fat body released into the hemolymph and transported to the ovaries 11-31. Maturing oocytes sequester vitellogenin by receptor-mediated endocytosis, a process first proposed by Roth and Porter [4] for mosquito oocytes. Receptor-mediated endocytosis is a common cellular process that includes the binding of a macromolecule, e.g., a protein, to a receptor molecule associated with the cell membrane and the subsequent internalization of the receptor-ligand complex [5]. In several insect species the selective uptake of vitellogenin by maturing oocytes could be demonstrated [6-101 as well as the specific binding of vitellogenin to an oocyte membrane bound binding protein [ll-131, presumably the vitellogenin receptor.
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