A procedure is described for isolating cell membranes from rat liver homogenates. 20 gm. of rat liver was homogenized in a Dounce homogenizer in ice cold water buffered to pH 7.5 with NaHCO3, rupturing all of the cells and most nuclei. The diluted homogenate was filtered through cheesecloth to remove precipitated nucleoprotein and centrifuged at 1500 g, 10 minutes, to sediment a crude membrane fraction. The membrane containing sediment was recentrifuged 3 times in conical tubes (1220 g, 10 minutes), the top layer of the 2-layered sediment being retained. Flotation in a sucrose solution d = 1.22 freed the preparation from contaminating cell fragments and nuclear membranes not previously disintegrated. The floating material ∼0.4 ml. was quite homogeneous and consisted of thin amorphous membranes. Electron micrographs revealed numerous double profiles similar in shape and dimensions to apposed liver cell membranes in intact tissue.
Enzymatic synthesis of Q*nucleoside containing mannose in the anticodon of tRNA: isolation of a novel mannosyltransferase from a cell-free extract of rat liver
