Studies on the preparation of rat liver plasma membrane fractions and on their polypeptide patterns

Abstract

Plasma membrane and bile canalicular membrane fractions were prepared from rat liver using NaHCO3, NaHCO3–CaCl2, and K2HPO4–KH2PO4 buffers (all at pH 7.4). The amount (expressed as milligrams protein per gram liver) of plasma membrane fraction exceeded the amount of bile canalicular membrane fraction using each of these three media; the use of NaHCO3–CaCl2 afforded a substantially higher yield of both types of membranes. The two membrane fractions exhibited complex patterns of polypeptides (> 30) on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Several reproducible differences in polypeptide patterns were observable between the two membrane fractions; in particular, components possibly corresponding to the heavy chain of myosin and to actin were prominent in the bile canalicular membrane fraction. The effects of incubation in the above three buffers and in Tris–HCl (pH 7.4) on the polypeptide patterns of both types of membrane were studied. Many polypeptides were released from each type of membrane in all of these media. Differential effects on the polypeptide patterns of either type of membrane fraction were observed among the various buffers. In terms of minimizing loss of polypeptides, in general, NaHCO3–CaCl2 appeared to be the best buffer and Tris–HCl the worst buffer. The significance of these results for the preparation and storage of liver cell plasma membrane fractions is briefly discussed.