Protease inhibitors reduce lysosomal acid phospholipase A1 activity in cultured rat hepatocytes

When rat hepatocytes were cultured in the presence of various specific protease inhibitors, lysosomal acid phospholipase A1 activity decreased progressively. Exposure of the cultured cells to 0.1 micrograms/ml of pepstatin, E 64, leupeptin or chymostatin also reduced the catalytic activities of several lysosomal marker enzymes. Irrespective of the protease inhibitor type employed, acid phospholipase A1 activity reacted most sensitively, followed by acid phosphatase, acid beta-N-acetyl-D-hexosaminidase and acid beta-glucuronidase. Of the protease inhibitors studied, pepstatin appeared to be most potent in reducing lysosomal enzyme activities in cultured hepatocytes. These findings suggest that proteolytic processes at as yet unknown, possibly extralysosomal sites play an important role in the turnover rates of lysosomal enzymes.     

Induction of unscheduled DNA synthesis in the nuclear matrix of rat hepatocytes after whole-body gamma irradiation of the animals

DNA synthesis in hepatocytes was studied by incorporation of [3H]thymidine administered to portal vein of gamma-irradiated (80 Gy) rats. It was shown that the rate of replicative DNA synthesis decreased in hepatocytes of the regenerating liver and unscheduled DNA synthesis was induced at the nuclear matrix of resting cells of the intact liver. In addition to repair synthesis, DNA synthesis resembling replicative one ("aberrant" DNA synthesis) accounts for a considerable fraction of gamma-radiation-induced synthesis of DNA at the nuclear matrix.     

Characterization of an Mg2+-dependent endonucleolytic activity of the rat hepatocyte nuclear matrix

Initial degradation of chromatin into high-molecular mass DNA fragments during apoptosis reflects the periodicity of chromatin organization into nuclear matrix-attached loops. In this article, we put forward the hypothesis that this pattern of DNA cleavage is also a result of the localization of an endonuclease on the nuclear matrix. Namely, we observed an endonucleolytic activity of the isolated rat hepatocyte nuclear matrix. It was Mg2+-dependent, with an optimal activity at pH 7.2 in the absence of either Na+ or K+. It was fully active in the presence of Zn2+ and capable of introducing single-strand breaks into plasmid DNA. It did not display a sequence-specific activity. A 23 kDa DNA nuclease that was principally localized on the rat hepatocyte nuclear matrix was detected. The enzyme shared the biochemical requirements with the nuclear matrix endonucleolytic activity, thus we proposed that p23 could be responsible for the endonucleolytic activity of the nuclear matrix. In view of its properties and preferential localization on the nuclear matrix, the endonuclease described herein could be a possible candidate that brings about initial DNA cleavage during apoptosis.     

Receptor binding in the rat liver nuclear matrix

3H-Dexamethasone (Dex)-receptor complexes prepared from the rat liver cytosol efficiently bound to the nuclear matrix from the same tissue. The binding was increased with the concentration of the 3H-Dex-receptor complex added and reached a maximum plateau. However, when the partially purified 3H-Dex-receptor complex was used, saturation of the binding sites in the nuclear matrix was not observed in the range of concentration of 3H-Dex-receptor complex used. Therefore, it was considered that the apparent saturability observed in the binding of the unpurified receptor complexes is caused by the translocation inhibitor(s) in the cytosol. When the binding capacity was expressed on the basis of unit weight of DNA, the nuclear matrix exhibited 20 times more of that of the unfractionated nuclei. However, no line of evidence of enrichment of the binding sites in the DNA isolated from the nuclear matrix was observed. These observations show that the role of the nuclear matrix in the action of glucocorticoid is quite uncertain.     

Increased nucleoside triphosphatase activity of rat liver nuclear matrix following low dose carcinogen intoxication

Rats were treated with low doses of hepatocarcinogens and nuclear matrix was isolated 48 hours later. These treatments, which were associated with notable nuclear enlargement, produced a decrease in the proportion of large molecular weight nuclear matrix polypeptides in all of the treated preparations, in the absence of toxic sequelae. We also found that the control matrix preparations possessed a considerable Mg-dependent nucleoside triphosphatase; the specific activity was 1.77 μmol γ-Pi released per hour per mg protein, and the Michaelis constant was 0.5 mM ATP. The matrix activity hydrolyzed ATP, UTP, and TTP, but differed from the nuclear envelope enzyme in that it did not hydrolyze GTP, CTP, dATP, or ADP, and it lacked myokinase-like activity. Large increases in nuclear matrix nucleoside triphosphatase occurred following all of the carcinogen treatments. The increased activity may relate to nuclear envelope alterations and derangements in RNA transport associated with carcinogenesis.     

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.     

Organization of the lamin scaffold in the internal nuclear matrix of normal and transformed hepatocytes

Nuclear lamins are among the more abundant proteins making up the internal nuclear matrix, but very little is known about their structure in the nucleoplasm. Using immunoelectron microscopy, we demonstrate the organization of lamins in the nuclear matrix isolated from rat hepatocytes for the first time. Lamin epitopes are arrayed both in locally ordered clusters and in quasi-regular rows. Fourier filtering of the images demonstrates that the epitopes are placed at the nodes and halfway between the nodes of square or rhombic lattices that are about 50 nm on each side, as well as along rows at regular ∼25-nm intervals. In addition, we have compared this structure with that of the internal nuclear matrix isolated from persistent hepatocyte nodules. In transformed hepatocytes, the islands of lamin lattice are lost, and only a partial regularity in the rows of gold particles remains. We suggest that orthogonal lattice assembly might be an intrinsic property of lamin molecules, and that the disassembly may be triggered by simple molecular events such as phosphorylation.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

Characteristics of N-acetylglucosamine transfer to nuclear acceptors of rat hepatocytes.

In this report, we describe the main characteristics of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. The glycosylation pathway includes the presence of lipids which mediate the nuclear proteins glycosylation. The level of dolichylphosphate seems low and thus could be a regulation factor in the nuclear glycosylations. The discussion deals with the membranous character of the acceptors and the N-acetylglucosaminyltransferase, the N-linkage of the sugar moiety to nuclear proteins and the function of such glycosylation.     

Transfer of N-acetylglucosamine to nuclear endogenous acceptors of rat hepatocytes.

1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate.     

A protein-tyrosine kinase in the nuclear matrix from rat liver

Protein kinase activity in isolated nuclei from rat liver was detected in situ after electrophoresis on SDS-polyacrylamide gel containing no exogenous protein substrate. After renaturation of polypeptides, the gel was incubated with [gamma-32P]ATP and divalent cations. Among five major protein kinase activities observed as radioactive bands by autoradiography, a protein kinase autophosphorylating on tyrosine (Mr 30,000) was identified and found to be localized in the nucleus, particularly in the nuclear matrix. The intensity of the activity band representing the level of the protein-tyrosine kinase in rat liver nuclei did not appreciably change during 3-24 h after partial hepatectomy.     

Androgen-dependent nuclear proteins in rat ventral prostate are glycoproteins associated with the nuclear matrix

The major rat ventral prostate androgen-dependent nuclear proteins were studied using isolated nuclei, nuclear matrix and nuclear envelope fractions. Nuclear and subnuclear fractions obtained were characterized by electron microscopy and SDS-polyacrylamide gel electrophoresis. A group of approximately 20 kDa peptides is demonstrated to be present in nuclei, nuclear matrices and nuclear envelopes from normal prostate. Time course experiments indicate that the 20 kDa peptides become drastically reduced after 7 or 10 days following castration and are incompletely restored after 3 daily testosterone injections. Lectin binding studies demonstrate that the 20 kDa peptides bind both to Concanavalin A and Wheat Germ Agglutinin. These peptides represent the major nuclear Concanavalin A binding glycoproteins from normal prostate nuclei and nuclear matrices.     

DNA-polymerase activity and DNA synthesis in the hepatocyte nuclear matrix

The comparative analysis of DNA-synthetase activity of hepatocytes, isolated nuclei and nuclear matrix from normal and regenerating rat liver was performed. The highest enrichment with newly-synthesized DNA was registered in the DNA fraction associated with the nuclear matrix both in vivo and in vitro. The functioning of DNA polymerases alpha and beta in the matrix was shown. Our results indicate that DNA polymerase beta is more firmly bound with the nuclear matrix in the cells of normal liver but this enzyme is eluted almost completely from the nuclei of regenerating liver cells. At the first moment after gamma-irradiation of rats the preferential initiation of unscheduled DNA synthesis in vivo has been observed on the nuclear skeletal structures. This may serve as an indication on the possibility that DNA repair process occurs on the nuclear matrix.     

Expression of nuclear matrix proteins in rat liver tissue

We have studied the synthesis of nuclear matrix proteins as it occurs in the rat liver. To investigate their kinetics in tissue, nuclear matrix proteins were prepared from liver of rats injected with radioactive methionine. Synthesis of lamins was not observed in quiescent hepatocytes although they were the principal proteins of this subcellular fraction, suggesting that lamins are very stable in the liver. When hepatocytes were stimulated to divide by partial hepatectomy, only synthesis of lamin B was initiated. Many proteins not visible on Coomassie blue-stained gels were detectable by autoradiography. In the nuclear matrix extracts of quiescent hepatocytes, one of the most prominently labeled ones was a protein of 70 kDa. After hepatectomy, an additional protein of 62 kDa was detectable. These proteins were visible 1 h after the injection of radioactivity, but were no longer observed in nuclear matrices prepared 24 h after injection. These experiments indicate that in addition to lamins, two nuclear matrix proteins are present in the rat liver that were not detected previously, perhaps because of their rapid turnover.