Ronald Berezney

The isolation of nuclear membrane from a large-scale preparation of bovine liver nuclei.

Abstract 1. 1. A procedure for the large-scale preparation of bovine liver nuclei is described. 2. 2. Nuclear membrane was prepared by treatment of nuclei with deoxyribonuclease followed by extraction with a 0.5 M MgCl2 solution. 3. 3. An evaluation of mitochondrial and microsomal contamination involving chemical, enzymatic and ultrastructural analysis indicated only slight contamination in both nuclear and nuclear membrane fractions. 4. 4. The total contaminating protein averaged 2.65% for nuclei and 10.6% for nuclear membrane. 5. 5. Electron microscopy showed that the membrane fraction contained both outer and inner nuclear membranes. 6. 6. The nuclear membrane fraction contained 74.6% protein, 8.9% RNA, 0.92% DNA, and 13.7% phospholipid. Recovery studies showed an average of 10.3% of the nuclear protein, 23.9% of the nuclear RNA, 0.27% of the nuclear DNA, and 47.1% of the nuclear phospholipid recovered in the membrane fraction.

Lipid composition of nuclear membranes isolated from bovine liver

Abstract 1. 1. Nuclear membrane fractions were prepared from rat-liver nuclei by treatment with deoxyribonuclease and MgCl2. 2. 2. The lipid distribution and fatty acid composition of total lipids and individual phospholipids was determined. 3. 3. The ratio of protein to total lipid was 2.96. Phospholipids accounted for 62% of the total lipid fraction. The remainder was accounted for as neutral lipids. Non-phosphorus-containing polar lipids were not detected. Based on lipid phosphorus distribution the major phospholipids were phosphatidylcholine (55%), phosphatidylethanolamine (23%), phosphatidylinositol (8%), phosphatidylserine (6%) and sphingomyelin (6%). Cholesterol and free fatty acids were the major constituents of the neutral lipid fraction, accounting for t6 and t3%, respectively, of the total membrane lipids. 4. 4. Phosphatidylcholine was the least saturated and sphingomyelin the most saturated of the major phospholipids. Eighteen-carbon chain-length fatty acids accounted for 70% of the acyl residues of the total lipid fraction. The palmitic acid to oleic acid ratio of the total lipid fraction was 0.55.

Lipid composition of further purified bovine liver nuclear membranes

The lipid content, distribution and fatty acid composition of highly purified bovine liver nuclear membranes was determined and compared to those of microsomes prepared in parallel. Contrasted with microsomes, nuclear membranes while containing nearly the same levels of lipid had more cholesterol and total neutral lipid and less phospholipid. Phospholipid and neutral lipid patterns generally were similar for the two types of membranes. The same fatty acids, in similar proportions, were observed in respective total lipid, total polar lipid, phosphatidyl choline and phosphatidyl ethanolamine fractions of the two membrane types. The microsomal lipid fractions contained slightly greater percentages of unsaturated fatty acids. With respect to previous results from preparations contaminated with nonmenbranous nuclear material, purified fractions contained more total lipid on a protein basis and more total unsaturated fatty acids. Only minor differences in levels and distribution of phospholipids and neutral lipids were observed between the crude and highly purified fractions.

Nuclear electron transport I electron transport enzymes in bovine liver nuclei and nuclear membrane

Abstract The electron transport enzymes NADH-cytochrome c reductase, NADH oxidase and cytochrome c oxidase are present in isolated bovine liver nuclei and highly concentrated in a nuclear membrane fraction. Evaluation of contamination indicates that the enzymes are of nuclear origin. Nuclear NADH oxidase is distinguished from mitochondrial NADH oxidase by the effects of exogenous cytochrome c, DNase and histone on enzyme activity. We suggest that the nuclear electron transport enzymes are located in the nuclear membrane.

Cytochromes in bovine liver nuclear membranes

Abstract Cytochrome a,a3 has been detected in highly purified nuclear membrane preparations. Nuclear membranes resemble microsomal membranes in cytochrome b5 content but differ by the absence of the microsomal cytochrome P-450. Cytochrome a,a3 content in nuclear membranes is 30% that of mitochondria, and cytochrome b5 content is 33% of control microsomes.

Electron transport in mammalian nuclei II. Oxidative enzymes in a large-scale preparation of bovine liver nuclei

1. A large-scale preparation of highly purified bovine liver nuclei is studied enzymatically. 2. Trace activities of succinate oxidase and NADPH-cytochrome c reductase are ascribed to slight mitochondrial and microsomal contamination, respectively. 3. Rotenone, antimycin A, piericidin A-insensitive NADH-cytochrome c reductase, NADH oxidase, cytochrome c oxidase, glucose-6-phosphatase, and Mg2+-stimulated ATPase are shown to be endogenous to nuclei. 4. The O2 consuming nuclear NADH oxidase differs from its mitochondrial counterpart in its greater resistence to histone inhibition, its susceptibility to inhibition by deoxyribonuclease, its dependency on cytochrome c for activity, and the 10-fold higher concentration of exogenous cytochrome c needed for maximum O2 consumption. 5. The nuclear cytochrome c oxidase system cannot oxidize tetrachlorohydroquinone in contrast to the mitochondrial cytochrome c oxidase system. 6. The nuclear preparations lack coenzyme Q, a characteristic component of the electron transport system of inner mitochondria membrane. 7. The NADH/cytochrome c oxidase ratio is 1.3 for the nuclear oxidases and 0.59 for mitochondrial oxidases. 8. The NADH oxidases of nuclei, outer mitochondrial membrane and microsomes are discussed.