The Purification and Biochemical Characterization of Bovine Liver Nuclear Membranes

Abstract Bovine liver nuclear membranes were purified by means of a high salt (0.5 m MgCl2) discontinuous sucrose gradient. The purified nuclear membranes were isolated at a density of ρ = 1.05 to 1.23 and contained 62% of the protein recovered from the gradient. The presence of nuclear pore complexes identified the fraction as nuclear membrane. Twenty-six per cent of the protein was recovered at a density of ρ = 1.23 to 1.30. Electron microscopy demonstrated both nuclear membranes and nonmembranous nucleolar components with a predominance of the latter. A fraction sedimenting through the most dense high salt sucrose solution (ρ ≥ 1.30) made up 12% of the total recovered protein. Ultrastructural analysis revealed nucleolar constituents in both a granular-fibrillar network and in the more condensed form characteristic of nucleoli in intact nuclei. In the absence of high salt, the nuclear membranes and nucleolar material aggregated and sedimented at a density intermediate between the lighter membranes and the heavier nonmembranous components. Purified nuclear membranes consisted of 70.4% protein, 5.8% RNA, 1.1% DNA, and 22.6% phospholipid. The comparatively nonmembranous nature of the heavier fractions was indicated by the much lower phospholipid contents of 9.7% and 9.8%, respectively. Since nucleoli have a characteristically high RNA content, the much higher RNA levels of the heavier fractions (8.0% and 11.4%), as compared to the lighter nuclear membranes, were consistent with the ultrastructural demonstration of nucleolar components in these fractions. Eighty to 90% of the endoplasmic reticulum-type enzymes NADH-cytochrome c reductase, glucose 6-phosphatase, and Mg++-stimulated ATPase, and the characteristic inner mitochondrial membrane activity, cytochrome c oxidase, were not due to microsomal and mitochondrial contamination. The low succinate dehydrogenase activity in purified nuclear membranes is correlated with the absence of acid nonextractable, trypsin-releasable flavin, which is associated with mitochondrial succinate dehydrogenase flavoprotein. In addition, coenzyme Q, a characteristic component of inner mitochondrial membranes, was not detected. The above enzyme activities were increased 1.2- to 1.9-fold in purified nuclear membranes compared to the unpurified membranes. In contrast, the high salt sucrose gradient fractions of higher density demonstrated progressive decreases in specific activities from 0.32 to 0.61 in the intermediate density fraction to 0.15 to 0.41 in the heaviest fraction. This was paralleled by the progressive decreases in nuclear membranes in these two fractions. Moreover, 84 to 90% of the total enzyme activities were found in the purified nuclear membranes, whereas only 7 to 15% and 1 to 4% were present in the intermediate and heaviest fractions, respectively. The membranes contained 0.322 nmole of cytochrome b5 per mg of protein and 0.045 nmole of cytochrome a, a3 per mg of protein, which were 33% and 30% of values in microsomes and mitochondria. The cytochrome P-450 content of nuclear membranes was only 4.4% of microsomes and can be attributed to microsomal contamination. Direct enzymatic comparison of nuclear and microsomal membranes demonstrated a strong endoplasmic reticulumtype character in nuclear membranes. Nuclear membranes differed from endoplasmic reticulum, however, by the presence of cytochrome c oxidase activity and cytochrome a, a3 and the only trace amounts of NADPH-cytochrome c reductase and cytochrome P-450.