Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation.
Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol-dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5'-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.
Abedinpour
Parisa
P
Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden.
Jergil
Bengt
B
D000199:Actins / Q000378:metabolism; D000818:Animals; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000494:pharmacology; D008168:Lung / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008854:Microscopy, Electron; D017830:Octoxynol / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000199:Actins; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D017830:Octoxynol