Isolation of 125I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs.
A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213-222, 1986) were labeled with 125I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar alpha-methylmannoside (alpha MM) indicated that 125I-ConA bound specifically to the egg PM. Greater than 95% of 125I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M alpha MM, and incubation of eggs in alpha MM after 125I-ConA labeling caused release of 85-90% of bound label. Fractionation of 125I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of 125I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that 125I-ConA did not label other organelles. As a control, human erythrocytes were labeled with 125I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for 125I-ConA-labeled eggs. These results indicate that 125I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane; D003208:Concanavalin A; D005260:Female; D007457:Iodine Radioisotopes; D051379:Mice; D008815:Mice, Inbred Strains; D010063:Ovum / Q000648:ultrastructure*
D007457:Iodine Radioisotopes; D003208:Concanavalin A