Visualization of the chicken oocyte lipoprotein receptor by ligand blotting with biotinylated plasma and yolk very low density lipoproteins
1994
Elkin, R.G. | Schneider, W.J.
The laying hen 95-kDa oocyte membrane receptor that transports hepatically synthesized very low density lipoprotein (VLDL) and vitellogenin (VTG) from the plasma to growing follicles was visualized by ligand blotting with biotinylated VLDL followed by enhanced chemiluminescence (ECL) detection. Plasma and egg yolk VLDL were isolated by ultracentrifugation and free epsilon-amino groups of lysines of apolipoprotein B (apo B), the protein constituent of VLDL that mediates binding to the 95-kDa oocyte membrane receptor, were biotinylated using D-biotin-N-hydroxysuccinimide ester. An apo B concentration of approximately 223 pM was sufficient to give a signal on 2.5 micrograms of protein from a chicken oocyte membrane detergent extract. Western blotting (immunoblotting) of the laying hen 95-kDa receptor with polyclonal rabbit anti-chicken oocyte VLDL receptor IgG resulted in an ECL signal with the same position of migration as that observed in ligand blots using biotinylated plasma and yolk VLDL. Binding of biotinylated plasma or yolk VLDL to the 95-kDa receptor was abolished by excess unlabeled plasma or yolk VLDL, respectively, as well as by EDTA. Receptor binding activity of biotinylated plasma and yolk VLDL was also demonstrated by a reverse ligand blotting procedure. Compared with conventional techniques involving the use of 125I-labeled ligands or antibodies, the laying hen 95-kDa oocyte membrane lipoprotein receptor can be safely and rapidly visualized with excellent sensitivity using the present nonradioactive method. In addition, it is suggested that ECL detection can be employed to further study the ligand-binding properties and specificity of this protein, which is essential to vitellogenesis in the chicken.
显示更多 [+] 显示较少 [-]