Isolation and characterization of gap junctions from rat liver.

Abstract

Cell to cell communication, through the transmission of small molecules (Mr less than 1200), probably occurs via a membrane specialization, the gap junction. A procedure has been developed for the isolation of gap junctions from rat liver without the use of exogenous proteases. Plasma membranes were prepared from 45 to 50 rat livers (about 500 g) by a procedure which provided a 10% yield with a lo-fold enrichment, based upon the recovery of B’nucleotidase. Nonjunctional membrane was removed from this preparation by solubilization with Sarkosyl NL-97. Washing with 0.1 M Na&Os removed the uricase which, along with collagen, was the major contaminant. Gap junctions, with a density of 1.165 g/cm3, were then isolated by sedimentation in sucrose gradients containing 1 M urea. A typical yield was 50 to 100 pg of protein. Morphologically, the gap junctions were characterized as septilaminar structures comprised of two membranes with an intervening 2 to 4 nm gap. These junctional plaques contained 8.5 nm diameter particles with a 9.0 nm center to center spacing. When negatively stained, a circular electron dense 1.5 to 2.0 nm region was observed in the center of these particles, possibly the site of small molecule transmission. Electrophoretic analysis of the gap junction fraction in sodium dodecyl sulfate polyacrylamide gels revealed two protein bands. The larger was a broad band with an apparent molecular weight of 47,000, while the other, a sharper band, had an apparent molecular weight of 27,000. No glycosylation of these bands was detected. The lipid composition of the isolated gap junctions was similar to that of the plasma membrane fraction from which they were derived, except for the apparent absence of sphingomyelin.