Abstract
<p>Plasma membranes (PMs) isolated from rat liver were analyzed by one- and two-dimensional polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (1D-SDS-PAGE and 2D- SDS-PAGE) in order to define the normal hepatocyte PM protein composition and to identify growth-related PM modifications in regenerating liver. 8.2-16% of the PM marker enzyme, 5'-nucleotidase, was recovered with a 19- to 21-fold enrichment relative to homogenate. Glucose-6-phosphatase (endoplasmic reticulum marker enzyme) recoveries were < 0.03-0.3% with an enrichment of < 0.06-0.5 relative to homogenate. Recovery of PM protein was 0.5-0.9mg/g wet weight liver. Electron microscopy demonstrated that the PM fraction comprised mainly membrane sheets and vesicles, including numerous junctional complexes and bile canaliculi. Tight junctions appeared in longitudinal section as typical focal membrane fusions with associated cytoplasmic densities, and in en face views as electron-dense networks possibly representing a cytoskeletal specialization. Gap junction, desmosome, and intermediate junction morphologies were typical of the same junctions in situ. 1D-SDS-PAGE of the normal, adult PM fractions resolved 49 polypeptides having apparent molecular weights (M(,r)) of 14K-240K. 2D-SDS-PAGE resolved the PM fraction into approximately 100 polypeptides, ranging in M(,r) from 14K-230K and in isoelectric point from 4.5-8.5. Asialoglycoprotein binding protein and actin were tentatively identified among the PM polypeptides detected. For the regeneration studies, weanling (24 day) male Sprague-Dawley rats were subjected either to 2/3-hepatectomy or to sham operations, and the PM fractions were isolated during the mitotic peak (28h following surgery). PMs from sham-operated livers were ultrastructurally indistinguishable from normal, adult PMs. Regenerating PMs were similar, except that gap junctions were greatly depleted. The PM fraction isolated from sham-operated livers is qualitatively identical by SDS-PAGE to the PM fraction prepared from normal, adult livers. 1D- and 2D-SDS-PAGE of regenerating PMs did not reveal the addition, loss, or modification of any PM proteins compared to sham-operated livers. These results suggest that gap junction protein is not lost from the PM concomitant with the disappearance of morphologically identifiable gap junctions during regeneration or, alternatively, that gap junction protein is not detectable by the methods used, even in normal liver.</p>