Various enzymes of isolated nuclear membranes and cell nuclei of the liver and hepatoma 27 of rats]

A comparative study of glucose-6-phosphatase, alcaline RNase, ATPase, inosine diphosphatase and 5'-nucleotidase activities in isolated rat liver and hepatoma-27 nuclei and nuclear envelopes was performed. The tumor nuclear membranes were shown to be free from G-6-Pase activity in contrast to the liver nuclear membranes. The nuclear RNase activity was strongly inhibited in the hepatoma and could be unmasked in the presence of 3-10(-4) M pCMB. Hepatoma nuclear and nuclear envelopes ATP-ase activity was found to be moderately decreased as compared to those of the normal tissue. The values of inosine diphosphatase activity in hepatoma were similar to those in liver. The role of the nuclear envelope in nuclear-cytoplasmic interactions as well as nuclear location of G-6-Pase are discussed.     

Phospholipid exchange between mitochondria and microsomes of rat hepatoma 27]

The phospholipid exchange in vitro between mitochondria and microsomes from rat liver and rat hepatoma 27 was investigated. On incubation with a postmicrosomal protein fraction the phospholipid exchange between subcellular fractions of the tumor was found to proceed much faster and less specific than between mitochondria and microsomes from normal liver. These results indicate that the earlier demonstrated lipid dedifferentiation of tumor cell membranes may be connected with an altered transmembrane phospholipid exchange in vivo.     

Ribonucleases of rat liver nuclear membranes]

A comparative study of ribonuclease activity of isolated rat liver nuclei, nuclear membranes with buoyant density rho 1,19 and rho 1,22 and pH 8 nuclear membrane extract showed high nuclear membranes activity with different affinity to RNA and synthetic polyribonucleotides. Chromatographic analysis of poly-U degradation products demonstrates that the nuclear membrane extract contains at least two ribonucleases: a 3'-endonuclease and a 5'-endonuclease.     

Fatty acid composition and unsaturated of intracellular membrane phospholipids from rat liver and hepatoma 27]

The fatty acid composition of phospholipids of mitochondria and microsomes from rat liver and hepatoma 27 was investigated. Basing on the fatty acid and phospholipid composition the unsaturation of the lipid bilayer of the intracellular membranes was calculated. The unsaturation of the phospholipids of the hepatoma mitochondria and microsomes was found to be much lower than that of the corresponding rat liver membranes. The lipid bilayer of the rat liver and hepatoma plasma membranes was shown to be more saturated than that of the intracellular membranes.     

Investigation of lipid-exchange lipoproteins from rat liver and hepatoma 27]

A phospholipid exchange lipoprotein from the postmicrosomal supernatant of rat hepatoma 27, which stimulated in vitro the exchange of sphingomyelin between mitochondria and microsomes, was found. Sphingomyelin is incorporated into the mitochondria under incubation of this complex with rat liver mitochondria (in which sphingomyelin is absent) an microsomes. Under the same conditions the phospholipid exchange lipoproteins of rat liver do not transfer sphingomyelin form microsomes to mitochrondria.     

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.     

Mitochondrial thymidine kinase and ribonucleotide reductase from rat liver and rat hepatoma 27]

Fractions of heavy and light mitochondria are isolated from homogenates of homologous rat tissues (intact liver, regenerating liver within 24 hours after hepatectomy and 27 hepatoma) by means of differential centrifugation. It is found that tumour mitochondria have higher heterogeneity and lower buyoant density than mitochondria from normal hepatocytes. The activity of two enzymes of DNA precursors synthesis (ribonucleotide reductase and thymidine kinase) in subcellular fractions is demonstrated to correlate with the tissue growth rate. A single injection of cyclic AMP into hepatectomised rats resulted in the retardation of the regeneration process, and the activity of both enzymes reached its normal level in all the fractions studied after 24 hours after the operation. Thymidine kinase and ribonucleotide reductase are located mainly in the mitochondrial matrix, however, pronounced enzyme activity is observed also in membrane fractions. The activity of the enzymes in the fraction of external mitochondria membranes in rapidly growing tissues is 2--3 times as high as in the same fraction from normal rat liver.     

Phospholipid composition modifications influence phospholipase A2 activity in rat liver plasma membranes

The influence of the phospholipid composition and the physico-chemical properties of rat liver plasma membranes on the activity of membrane-bound phospholipase A2 has been investigated. The plasma membrane composition was modified by the aid of exogenous phospholipases A2, C and D, and by butanol treatment. The partially delipidated membranes thus obtained were enriched with different phospholipids. The steady-state fluorescent anisotropy of 1,6-diphenyl-1,3,5-hexatriene and the lipid order parameter-SDPH in the modified membranes were calculated. It was established that the activity of the membrane-bound phospholipase A2 was higher in rigid membranes and was decreased when the membrane lipid bilayer was fluidized.     

Study of insulin receptors on liver and hepatoma cell plasma membranes]

Plasma membranes (PM) of helathy rats as well as those hepatomas and PM of tumor-bearing rats were found (radioreceptor assay) to have apparently two groups of receptors for insulin with a high and low affinity for the hormone respectively and different insulin-binding capacities. Kass values of the receptors with a high affinity for insulin are drastically decreased in the PM of ascites Zajdela hepatoma (AZH) and of solid hepatoma 27 (SH-27) as well as in the liver of SH-27-bearing animals, but not in the liver of the AZH-bearing rats. Kass values of the receptors with a low affinity for insulin in the PM of AZH and in those of the liver of AZH-bearing rats appear nearly normal. In contrary, above Kass the receptors in the PM of SH-27 and SH-27-bearing animals are significantly decreased. The insulin-binding capacity of the receptors with a high affinity for the hormone in the PM of SH-27, AZH and the liver of both tumor-bearing rats is shown to be significantly higher than that in the PM of normal animals. The same property of the receptors with a low is affinity in the PM of the hepatomas and theliver of their hosts is also increased, especially in the PM of SH-27 and of the liver of SH-27-carring rats.     

Phospholipid interactions in rat liver nuclear matrix

Rat liver nuclear matrix has been isolated by salt extraction and nuclease digestion of nuclei. Under the electron microscope, the matrix appears as a spongelike network joined by thinner fibrils. Biochemical analysis shows a high protein content and low amounts of nucleic acid and phospholipid. Treatment of the matrix with phospholipase C results in a release of most of the nucleic acid, and a disappearance of the fibrils, however the appearance of the matrix is largely unaffected. It seems likely that phospholipids are responsible for the hydrophobic interactions between nucleic acids and matrix fibrils. From $\text{in vitro}$ labelling studies the released DNA is more recently synthesised than the bulk material, however the matrix bound RNA appears to label less rapidly than total nuclear RNA.     

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.