Phospholipid composition of nuclear membranes and cell nuclei of rat liver and hepatoma 27]

Phospholipid composition of nuclei and nuclear membranes from rat liver and hepatoma-27 were investigated. Hepatoma nuclei and nuclear membranes were found to contain cardiolipin which was absent in the same fractions of rat liver. In the nuclei and nuclear membranes of hepatoma the content of sphingomyelin was higher and that of lecithin is lower than in the corresponding subcellular fractions of rat liver. The content of acid phospholipids was much higher in hepatoma nuclear membranes than those of the normal liver. The data obtained show that the general trend of lipid dedifferentiation which earlier was demonstrated for various membranes of different tumor cells is observed also in the case of nuclear membranes.     

Cytochrome oxidase of rat liver nuclear membranes and its interaction with mitochondrial cytochrome oxidase]

The percent of mitochondrial protein contamination in nuclei decreased 10-fold (from 18 to 1.8%) under purification of protein-labelled mitochondria before their introduction into nuclei-free homogenate, cytochromoxidase activity being unchanged. Thus, cytochromoxidase activity of nuclei does not correlate with the amount of nuclei-adsorbed mitochondrial protein, which demonstrates the presence of nuclear cytochromoxidase independent on mitochondrial protein. Radioactivity of protein-labelled mitochondria is proportially distributed between globuline, deoxyribonucleoprotein, acid and residual nuclear proteins, as it is shown under fractionation of nuclei isolated from protein-labeled mitochondria containing homogenate. The comparison of mitochondrial protein contamination of nuclear membranes and their possible contamination with cytochromoxidase and suecinate-cytochrome-c-reducatase activities revealed that cytochromoxidase activity of nuclear membranes is twice higher and succinate-cytochrome-c-reductase activity is considerably lower than it can be referred to mitochondrial protein contamination. The ratio of cytochrome-c-oxidase and succinate-cytochrome-c-reductase activities in isolated nuclear membranes is 4-7 times as high as that in mitochondrial membranes under the same isolation procedure. The data obtained make possible to consider the cytochromoxidase activity of nuclear membranes to be really nuclear enzyme, and not a contominant of nucleipreparation with mitochondrial membranes.     

Cryoresistance of rat liver endoplasmic reticulum membranes]

The effects of freezing of microsomes in liquid nitrogen and those of storage of microsomal suspensions at 2-4 degrees C and -3 - -5 degrees C for 24 hrs, on the enzymatic activities and hydrophobicity of membranes were studied. The hydrophobicity was determined by fluorescence of bound 1,8-anilino-naphthalene sulfonate. Rapid freezing of the microsomal suspension in liquid nitrogen followed by rapid warming did not change the hydrophobicity of the membranes, the rate of enzymatic lipid peroxidation, the level of cytochrome P-450 and the activity of NADH- and NADPH-cytochrome c reductase. A considerable decrease in the rate of enzymatic lipid peroxidation and membrane hydrophobicity was observed in the microsomes stored for 24 hrs at 2-4 degrees C. The 24-hr storage at -3 - -5 degrees C with subsequent thawing resulted in a rapid aggregation of the microsomes.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

Epoxide hydrase and mixed-function oxidase activities of rat liver nuclear membranes.

Rat liver nuclei have 2 to 12% of the corresponding microsomal aryl hydrocarbon hydroxylase, aminopyrine and benzphetamine N-demethylase, NADPH-cytochrome c reductase, and epoxide hydrase activities. Nuclear membranes were prepared from isolated liver nuclei by a sucrose density centrifugation technique. A 2.5- to 10.2-fold increase in the specific enzyme activities was observed in nuclear membrane as compared to intact nuclei. Several properties of the rat liver nuclear membrane and microsomal epoxide hydrase have been compared. Nuclear epoxide hydrase was similar to the corresponding microsomal enzyme in being induced by phenobarbital whereas 3-methylcholanthrene did not produce any effects. Nuclear membrane and microsomal epoxide hydrase were inhibited to a similar degree by 1,1,1-trichloropropene oxide, cyclohexene oxide, an trans-stilbene oxide. The apparent K_m value of nuclear membrane epoxide hydrase was 20 μm for benzo(a)pyrene 4,5-oxide, which is 5.5-fold lower than the corresponding microsomal K_m value (112 μm). Nuclear membranes were prepared from isolated nuclei of rat kidney, lung, spleen, and heart by the DNase digestion method. Epoxide hydrase activity in intact nuclei was in the following order: kidney > lung ? spleen, or heart. Increases of 2.2- and 2.5-fold in specific epoxide hydrase activity were observed in kidney and lung when nuclear membranes were compared to intact nuclei. DMSO, dimethylsulfoxide     

Age-related peculiarities of acetylation of rat liver nuclear histones]

The intensity of acetylation of different histone fraction is found to change in postnatal ontogenesis of albino rats. The intensity of acetylation of lisine-rich histone (F1) was maximal in 3-and 12-month old animals. The label incorporation into F3 (arginine-rich) histones considerably decreased in the time interval between 1st and 3d months while its incorporation increased into F2 histones furing all the period of postnatal development studied.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Neutral lipids of nuclear membranes and chromatin in rat liver after hydrocortisone administration

It is shown that over 80% of nuclear neutral lipids are found in the nuclear membrane preparations, whereas only 7%--in chromatin. The injection of hydrocortisone to animals significantly decreases the neutral lipids content in both fractions. These results and the data obtained previously by the authors indicate the possible participation of nuclear membrane and chromatin lipids in the process of hormonal regulation of the genetic activity.     

Oxidation and phosphorylation in rat liver nuclei and nuclear membranes in postnatal development and regeneration after partial hepatectomy]

Specific oxygen consumption by isolated nuclei of liver cells of newborn rats is higher and phosphorylation is lower as compared to adult animals. This is correlated with a higher free cytochrome oxidase activity as determined in the absence of detergents or tetramethyl-p-phenylenediamine. Correspondingly, oxygen consumption by isolated nuclear membranes of rat liver 44 hrs after partial hepatectomy is also increased and the P/O ratio is decreased 2.2-fold as compared to the controls.     

Phospholipids of outer and inner nuclear membranes in rat liver and BHK-21 cells

Isolated nuclei of rat liver and BHK-21 cells were treated with citric acid and the resulting outer nuclear membrane sheets were separated from the nuclear residues surrounded by the inner nuclear membrane. Both fractions contained approximately equal amounts of phospholipid in both cell types. The phospholipid compositions of the two fractions were remarkably similar. The results are in accordance with the notion of the structural continuity of the two nuclear membranes.