A rapid calcium precipitation method of recovering large amounts of highlypure hepatocyte rough endoplasmic reticulum

We sought a rapid and non-ultracentrifugal method of recovering large amoun ts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, w e obtained a RER fraction from rat liver and established its high degree of purity by, quantitating classic membrane markers for different subcellular organelles. This RER fraction is highly enriched in four known, n proteins (Or enzyme activities) required for lipoprotein assembly: apolipoprotein B , microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltra nsferase and acyl CoA:cholesterol acyltransferase, when compared to two cla ssical RER markers, RNA and glucose-6-phosphatase. From one 10-12 g rat liv er, we recover ten to twelve RER pellets of 1.5-1.6 cm in diameter containi ng similar to 110-125 mg of total protein, about half of which is sodium ca rbonate-releasable. Ey electron microscopy these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. j/r This novel technique for isolating RER mem branes from liver may provide a useful tool for future studies on the assem bly of apolipoprotein B-containing lipoproteins as Hell as for research foc used on mechanisms of secretory and membrane protein translation, transloca tion, and folding.-A rapid calcium precipitation method of re covering larg e amounts of highly pare hepatocyte rough endoplasmic reticulum.