Isolation of caveolae using affinity two-phase partitioning

Detta är en avhandling från Biochemistry department, Box 124 221 00 Lund

Sammanfattning: The intention of this work was to establish alternative purification methods to obtain highly purified caveolae from various tissues. In order to isolate caveolae, sufficiently pure plasma membranes are needed. A method is presented for the isolation of plasma membranes from lung tissue using a combination of conventional polyethylene glycol/dextran two-phase partitioning and affinity partitioning using the lectin wheat germ agglutinin as affinity ligand. A caveolae-enriched fraction was purified from lung plasma membranes isolated by this procedure. Caveolae were released from the membranes by Triton X-100 treatment or by sonication. Highly purified caveolae were obtained at low buoyant density by sucrose gradient centrifugation. The affinity method is advantageous to other methods for the isolation of plasma membranes and should be useful for other tissues as well. A method to purify caveolae by immunoaffinity partitioning was developed exploiting the interaction between caveolin and anti-caveolin antibodies. A sandwich approach was used where primary antibodies directed against caveolin interacted with biotinylated secondary antibodies and NeutrAvidin coupled to dextran in a polyethylene glycol/dextran two-phase system. Caveolae were directed efficiently into the immunoaffinity bottom phase of the two-phase system by the anti-caveolin antibody. Immunoaffinity two-phase partitioning has wider applications potentially, as this technique should be useful to purify any type of membrane by selecting appropriate antibodies directed against surface components of the membrane of interest. Caveolae were isolated to a high degree of purity from apical and basolateral domains of liver plasma membranes. The caveolae from the two domains were quite homogeneous as studied by immunoaffinity partitioning and electron microscopy. Analysis showed that the caveolae differed in properties in several respects.

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