Plasma membranes of liver cells of the chick embryo. I. Isolation procedures

Abstract

The technique of isolation consists of a combination of homogenization, differential centrifugation, gradient centrifugation and the multiple poolings of isolated fractions. Large numbers of embryos are used to provide 5–10 g of accumulated liver tissue from the younger embryos (7, 11 days). Contamination by components of red blood cells or nuclear envelopes is avoided. The tissues are homogenized as dilute suspensions in either isotonic Krebs-Ringer solution or isotonic sucrose solutions supplemented with magnesium. Intact red blood cells are separated from the homogenate. Initial fractions are separated by multiple differential centrifugations. Fluffy coats and supernatants are pooled sequentially to increase the final yield of membranes. Intact nuclei can be separated by centrifugal rotation at 100 000 × g for 2 h over appropriate sucrose solutions. The separation of the plasma membrane fraction is carried out by density gradient centrifugation at 150 000 × g for 2 h. Fractions isolated in either isotonic sucrose or saline appear to be similar. The cytochrome oxidase and glucose-6-phosphatase activities are negligible. β-Glucuronidase activity is exceedingly low. Mg 2+-dependent ATPase activity is enhanced by the presence of Na 2+. The isolation of plasma membrane of embryonic cells allows for a more direct assessment of membrane development, cell-contact phenomena and membrane function.