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

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 approximately 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.

Isolation and properties of nascent lipoproteins from highly purified rat hepatocytic Golgi fractions.

Two procedures were used to isolate hepatocytic Golgi fractions from rat liver. One procedure yields a light Golgi fraction (GF1 + 2) and the other "intact" stacks of cisternae. Triglyceride fatty acids in nascent very low density lipoproteins (VLDL) were labeled by injection of [3H]palmitate intravenously, and radiolabeled lipoproteins were injected as markers of potentially contaminating endosomes. GF1 + 2 fractions were enriched manyfold in the endosomal markers, indicative of substantial endosomal contamination, whereas intact Golgi fractions from the same livers were about 7% as contaminated. By electron microscopy, GF1 + 2 fractions contained mainly multivesicular bodies (MVBs), together with some Golgi-derived secretory vesicles. The small endosomal contamination of intact Golgi fractions was further reduced by a simple modification of the procedure, which removed most entrained endosomes. The surface constituents of Golgi VLDL (d less than 1.010 g/ml) released from these highly purified intact Golgi fractions differed from those of plasma VLDL. Golgi VLDL contained fivefold less unesterified cholesterol than plasma VLDL, but twofold more phospholipids. Golgi VLDL and plasma VLDL contained similar amounts of cholesteryl esters and triglycerides. The protein content of Golgi VLDL was substantially lower than that of plasma VLDL. ApoB-100 and apoB-48 were similarly represented, but nascent VLDL contained less of the C apolipoproteins. ApoA-I was present mainly as the proprotein in Golgi VLDL, but was virtually lacking in plasma VLDL. ApoE comprised about 22% of the protein mass of Golgi VLDL as well as plasma VLDL; the distribution of apoE isoforms was also similar. Apolipoproteins E and pro A-I released from ruptured Golgi cisternae were largely bound to the Golgi VLDL or were associated with Golgi membranes. Particles resembling low density lipoproteins (LDL) and high density lipoproteins (HDL) were not seen by electron microscopy in contents of intact Golgi fractions. These observations indicate that nascent Golgi VLDL are the primary particulate precursors of rat plasma lipoproteins of hepatocytic origin, and suggest that particles with the density of plasma HDL and LDL do not exist within the secretory pathway of normal hepatocytes. Thus, the results of this research on the properties of nascent plasma lipoprotein precursors contained within uncontaminated hepatocytic Golgi fractions differ substantially from previous published work.