Purification of rabbit endometrial plasma membranes from receptive and non-receptive uteri.
We have developed a method for isolation of plasma membranes from rabbit endometrium, with high yield and purification. Endometrial homogenates are precipitated with calcium chloride and the resulting supernatant is fractionated by centrifugation in a self-forming gradient of 20% Percoll. Before fractionation, the intact luminal epithelial surface was labelled with 125I-labelled soyabean agglutinin. Between buoyant densities of 1.015 and 1.017 g/ml, a discrete peak of surface label was obtained, which coincided with activities for 5'-nucleotidase and alkaline phosphatase, enzyme markers for the plasma membrane. This peak was well separated from the majority of cellular protein, and from marker enzyme activities for mitochondria and microsomes (NADH cytochrome C reductase) and lysosomes (acid phosphatase). Electron microscopy of the purified membranes showed membrane sheets and vesicles free from other cellular organelles. Analysis of detergent-soluble membrane proteins, fractionated by concanavalin A-affinity chromatography, revealed differences in the protein pattern of membranes from uteri of rabbits receptive (Day 6 of pregnancy) and non-receptive (Day 3) for implantation. The method will be useful for generation of immunological and affinity probes for surface antigens involved in ovoimplantation.
D015720:5'-Nucleotidase; D000135:Acid Phosphatase / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D010064:Embryo Implantation; D004717:Endometrium / Q000648:ultrastructure*; D005260:Female; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009245:NADH Dehydrogenase / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D011247:Pregnancy; D011817:Rabbits
D008565:Membrane Proteins; D009245:NADH Dehydrogenase; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D000135:Acid Phosphatase; D015720:5'-Nucleotidase