A detergent-independent procedure for the isolation of gap junctions from rat liver.
In this paper, the isolation of rat liver gap junctions from alkali-extracted rat liver plasma membranes is described. The purification is significantly more rapid than the commonly used detergent-based approaches and is subject to less variability. The gap junctions isolated by this method are comprised of a 27,000-Da polypeptide previously identified as the major gap junction polypeptide. The isolated gap junctions have the characteristic double-membrane organization and subunit structure observed in vivo. The protein yield is from 8 to 10 micrograms/g of liver (wet weight), about a 10-fold increase in recovery over that of earlier isolation procedures. With the availability of increased amounts of material, antibodies were raised to the liver gap junction polypeptide. Immunofluorescence localization of these antibodies on rat liver sections revealed a distribution consistent with that expected from electron microscopic analysis of liver thin sections. Double diffusion of antibody against solubilized gap junctions in detergent-containing gels resulted in the formation of precipitin arcs, suggesting response to multiple determinants. Antibody binding to the 27,000-Da gap junction polypeptide was demonstrated by immunoblot analysis of sodium dodecyl sulfate-polyacrylamide gels containing rat liver plasma membranes and isolated gap junctions. These results confirm the identification of the 27,000-Da polypeptide as the major protein component of gap junctions.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000032:analysis; D017630:Connexins; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005779:Immunodiffusion; D008099:Liver / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008854:Microscopy, Electron; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D017630:Connexins; D003902:Detergents; D008565:Membrane Proteins