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.

The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs) . It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity . In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritiated phorbol 12,13dibutyrate ([ 3 H]PDBu) . The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [ 3H] PDBu binding is time dependent, specific, saturable, and reversible . Binding sites in HCs displayed a dissociation constant of 11 .5 nM and a total number of 2 .8 pmol/mg of protein . Autoradiography revealed that [ 3 H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata . Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation . In HCs, the treatment with PDBu in particular affected the amount of 32p incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation . These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation . Key Words: Phorbol ester binding-Protein kinase C-Protein phosphorylation-Retina-Horizontal cells.