Low-density caveolae-like membrane from Xenopus laevis oocytes is enriched in Ras.
Detergent-free discontinuous sucrose density gradient centrifugation was used to resolve low- and high-density membrane fractions from Xenopus laevis oocytes. Compared to high-density membrane, low-density oocyte membrane is enriched two-fold in cholesterol and highly enriched in ganglioside GM1. Protein immunoblotting of membrane fractions from whole cells with polyclonal anti-human caveolin antibody detected multiple bands, including a distinctive triad with apparent molecular weights of 21, 33, and 48 kDa. To more clearly determine which of these caveolin-like protein(s) is associated with the oocyte plasma membrane, microdissection was used to separate external membrane (cortical preparations containing plasma membrane) from intracellular membrane. Cortical membrane preparations displayed a single 21-kDa caveolin-like protein in low-density membrane. Internal oocyte membrane displayed the higher molecular weight bands of 33 and 48 kDa and a lesser amount of the 21-kDa protein in low-density membrane fractions. Monoclonal anti-human Ras antibody detected a single 23-kDa immunoblot band that is enriched an average of eight-fold in low-density membrane fractions prepared from whole cells. This is the first report of caveolin-associated, low-density membrane in amphibian oocytes, and is consistent with a role for caveolin and caveolae-like microdomains in oocyte signal transduction.
Sadler
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Department of Biological Sciences, University of Denver, University Park, Denver, CO 80208, USA. ssadler@du.edu
D000818:Animals; D021941:Caveolae / Q000737:chemistry* / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D015151:Immunoblotting; D009865:Oocytes / Q000166:cytology*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D018631:ras Proteins / Q000378:metabolism*
C495539:CAV1 protein, Xenopus; D051242:Caveolin 1; D022461:Caveolins; D029867:Xenopus Proteins; D005677:G(M1) Ganglioside; D002784:Cholesterol; D018631:ras Proteins