Characterization of intracisternal and membrane subfractions from sonically disrupted rough microsomal and Golgi-rich fractions of the rat liver

Abstract

1. 1. Rough microsomal and Golgi-rich fractions from rat liver were disrupted by sonication in 0.04 M Veronal buffer, pH 8.5, and centrifuged at 100 000 g for 60 min. The supernatant and pellet sub-fractions were characterized chemically and morphologically. 2. 2. In the rough microsomal fraction, the supernatant retained 5% of the glucose-6-phosphatase activity, 34% of the protein, 26% of the RNA and 14% of the lipid phosphorus of the total fraction. The pellet retained 109% of the glucose-6-phosphatase activity, 63% of the protein, 76% of the RNA and 86% of the lipid phosphorus. SDS-polyacrylamide slab gel electrophoresis revealed that the supernatant and pellet subfractions shared many proteins in common, but a few proteins were segregated either in the supernatant or in the pellet. Electron microscopic examination showed that the pellet consisted mainly of membrane fragments and vesicles whereas the supernatant consisted of amorphous granular material only. 3. 3. In the Golgi-rich fraction, the supernatant retained 24% of the galactosyltransferase, activity, 43% of the protein, 24% of the lipid phosphorus and 73% of the triglycerides present in the total fraction. The pellet retained 76% of the galactosyltransferase activity, 56% of the protein, 79% of the lipid phosphorus and 32% of the triglycerides. SDS-polyacrylamide slab gel electrophoresis revealed many proteins common to both subfractions, but a few pertained either to the supernatant or the pellet. Electron microscopic examination showed that the pellet consisted of membrane fragments and vesicles, whereas the supernatant contained lipoprotein particles, amorphous granular material and some membrane fragments. 4. 4. Thus, membrane subfractions can be successively prepared by sonic disruption of rough microsomal or Golgi organelles. They are chemically, enzymatically and morphologically distinct from the corresponding intracisternal subfractions. This should be a useful technique for studying subcellular membranes without interference from intracisternal contents.