Composition and synthesis of plasma membranes and smooth endoplasmic reticulum in isolated cells of new-born rat brain

Abstract

Plasma membranes and smooth endoplasmic reticulum were isolated from a suspension of cells prepared from newborn rat brain. The patterns of proteins and glycoproteins of the membrane fractions were studied by sodium dodecyl sulphate gel electrophoresis. Phospholipid classes were analyzed by thin-layer chromatography. Biosynthesis of membrane proteins was studied by incubating the cells with labelled leucine in vitro. 1. 1. Sodium dodecyl sulphate gel electrophoresis revealed three major polypeptides in plasma membranes corresponding to 45 000, 50 000 and 58 000 daltons. Smoth endoplasmic reticulum had prominent polypeptides of 45 000 and 53 000 daltons. 2. 2. Plasma membranes were enriched in Schiff-positive material as compared to smooth endoplasmic reticulum. Both fractions contained lipid-extractable material of low molecular weight in sodium dodecyl sulphate gels, while only plasma membranes showed glycoprotein bands ranging in apparent molecular weights from 40 000 to over 100 000. 3. 3. Both membrane types contained phosphatidylcholine as their major phospholipid. Plasma membranes were enriched in phosphatidylcholine and phosphatidylethanolamine while smooth endoplasmic reticulum showed higher concentration of phosphatidylserine. No sphingomyelin was detected in plasma membranes. 4. 4. Incorporation of [ 3H]leucine into plasma membranes and smooth endoplasmic reticulum was only 6 and 14%, respectively, of the radioactivity in the cell homogenate in a 60-min incubation. A 2-h chase with nonradioactive leucine increased the specific radioactivity of proteins in plasma membranes while decreased the labelling of smooth endoplasmic reticulum.