ISOLATION AND PARTIAL-PURIFICATION OF PLASMA-MEMBRANE FROM PORCINE OOCYTES

Egg plasma membrane (EPM) was isolated in comparatively large amounts from porcine slaughterhouse ovaries. Ovaries were minced, and the oocy te containing fluid was filtered to retrieve zona pellucidae-intact oo cytes. The oocytes were homogenized and filtered again to remove zona pellucidae. The egg filtrate was subjected to differential centrifugat ion to remove membrane bound organelles and the remaining plasma membr ane containing material was pelleted by ultracentrifugation. Plasma me mbranes were further separated from cellular material by sucrose densi ty gradient centrifugation and were collected from portions of the gra dient that correspond to the densities of plasma membrane. The purity of isolated plasma membranes was assessed by membrane marker enzyme an alysis and transmission electron microscopy. Activities of the plasma membrane marker enzymes 5' nucleotidase and alkaline phosphatase incre ased from nondetectable levers in the egg filtrate to relatively high levels in the plasma membrane preparation. Marker enzymes for mitochon drial and lysosomal membranes fell from detectable levels in the egg f iltrate to levels that were at the lower limits of the assays to detec t in the final preparation. Evidence provided by binding of biotin-lab eled EPM to capacitated sperm suggests that the isolated EPM retains i ts biological activity. The procedure presented here represents a nove l method of isolating porcine egg plasma membranes for further study i nvolving sperm-egg interaction. (C) 1994 Wiley-Liss, Inc.