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

Abstract

We sought a rapid and non-ultracentrifugal method of recovering large amounts of highly pure rough endoplasmic reticulum (RER) membranes from livers. By substantially modifying a 20-year-old calcium precipitation technique, we 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 proteins (or enzyme activities) required for lipoprotein assembly: apolipoprotein B, microsomal triglyceride transfer protein, acyl CoA:diacylglycerol acyltransferase, and acyl CoA:cholesterol acyltransferase, when compared to two classical RER markers, RNA and glucose-6-phosphatase. From one 10–12 g rat liver, we recover ten to twelve RER pellets of 1.5–1.6 cm in diameter containing ∼110–125 mg of total protein, about half of which is sodium carbonate-releasable. By electron microscopy, these large RER pellets from rat livers are homogeneously comprised largely of non-vesiculated short strips of ribosome-rich membranes. This novel technique for isolating RER membranes from liver may provide a useful tool for future studies on the assembly of apolipoprotein B-containing lipoproteins as well as for research focused on mechanisms of secretory and membrane protein translation, translocation, and folding.—Hamilton, R. L., A. Moorehouse, S. R. Lear, J. S. Wong, and S. K. Erickson. A rapid calcium precipitation method of recovering large amounts of highly pure hepatocyte rough endoplasmic reticulum. J. Lipid Res. 1999. 40: 1140–1147.

Keywords

• apolipoprotein-B
• microsomal triglyceride transfer protein
• acyl coenzyme A:diacylglycerol acyltransferase
• acyl coenzyme A:cholesterol acyltransferase
• electron microscopy