Isolation of /sup 125/I-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.
- Research Organization:
- Medical College of Georgia, Augusta (USA)
- OSTI ID:
- 6174019
- Journal Information:
- Gamete Res.; (United States), Vol. 16:4
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL MEMBRANES
FRACTIONATION
CONCANAVALIN
BIOCHEMICAL REACTION KINETICS
EGGS
LABELLING
BIOLOGICAL MARKERS
IODINE 125
MICE
TRACER TECHNIQUES
ULTRACENTRIFUGATION
AGGLUTININS
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
CENTRIFUGATION
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
HEMAGGLUTININS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LECTINS
MAMMALS
MEMBRANES
NUCLEI
ODD-EVEN NUCLEI
RADIOISOTOPES
REACTION KINETICS
RODENTS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques