The UDP-N-acetylglucosamine-asparagine-sequon N-acetyl-beta-D-glucosaminyltransferase activity in preparations of rough endoplasmic reticulum from regenerating rat liver.

The UDP-N-acetylglucosamine-asparagine-sequon N-acetylglucosaminyltransferase activity in preparations of rough endoplasmic reticulum from rat liver is less than 1% of that in preparations from rabbit liver. The activity of the enzyme is increased about 20-fold in preparations from regenerating rat liver within 48h after partial hepatectomy. A smaller, but still marked, increase (8-10-fold) occurs in preparations of rough endoplasmic reticulum from sham-operated rats.     

Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns.

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.     

Protein synthesis by microsomal particles from regenerating rat liver

1. Washed microsome particles from regenerating liver were shown to incorporate [(14)C]leucine into protein more actively than similar preparations from normal liver. 2. The total incorporation in the preparations from regenerating liver increased linearly with the amount of protein incubated, whereas this was not so with preparations from normal liver. 3. The greater activity of regenerating-liver microsomes appeared to be associated with the bound polysomes. 4. The size distribution of polysomes obtained after removal of membrane with deoxycholate was the same in normal and regenerating liver. 5. In general the activity of polysome preparations from normal and regenerating liver was similar. 6. It is concluded that the greater activity of the particles in the microsome fraction from regenerating liver is to be attributed to the ribosomes bound to membrane and that their activity is controlled by factors present in the membrane.     

Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3-4-fold in the first 24-48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50-250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2-3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. D-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [14C]nicotinamide as a precursor, was found to be 2-4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kr?ger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446-1452), support the view that nicotinamide methylase and its product, 1-methyl-nicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.     

Effect of estrogen administration on rat liver 2-5A synthetase activity

Interferon-induced 2-5A synthetase is also present in various cells and tissues in the absence of any interferon treatment. The activity of this enzyme, which synthesizes a series of oligoadenylates, ppp(A2'p)n5'A (collectively referred to as 2-5A), was previously shown to vary with the growth status of liver tissue i.e., it decreased before and during the peak of DNA synthesis activity induced in rat liver by a two third hepatectomy. In the course of studies aimed at testing the hypothesis that 2-5A synthetase activity might exert negative control on normal cell growth and multiplication, we show here that a treatment of ovariectomized rats with a single dose of estradiol-17beta (100 micrograms/100 g body weight) induced a transient increase in the [3H]thymidine labelling index in the liver after 24 h and markedly decreased the 2-5A synthetase activity. A time course study revealed that 2-5A synthetase activity started to decrease after 3 h, reaching a minimal value (10% of the control level) after 12 h, then slowly increased to come back to control level at 48 h. These results, together with our similar data on regenerating liver, suggest that low 2-5A synthetase activity is permissive for acquisition of proliferative 'competence' by G0 cells.     

Induction of rat liver glycine methyltransferase by high methionine diet

Dietary experiments were carried out to evaluate the physiological role of glycine methyltransferase. When rats received a 18% casein diet containing excess methionine, the activity of the enzyme in liver extracts increased with increasing methionine content in the diet. Adenosylmethionine synthetase and adenosylhomocysteinase activities were also elevated, while guanidoacetate methyltransferase activity showed no significant change. The glycine methyltransferase activity reached a maximal level after 4–6 days on the 3% methionine diet. Immunological titration showed that the increase in activity was associated with the increase in amount of the enzyme.     

Effect of hydrocortisone and glucagon on glycogen metabolism in the fetal rat liver.

1. Hydrocortisone increases in vivo incorporation of [14C] glucose into fetal liver glycogen in the last days of gestation, whereas in glucagon-treated fetuses, a slight decrease in the incorporation rate was found. 2. Hydrocortisone increases total synthetase activity as that of synthetase a but was without effect on fetal liver glycogen phosphorylase. 3. Glucagon causes a slight increase in phosphorylase a activity on days 19-21, and was without effect on the activities of synthetase a and total synthetase. 4. Dibutyryl cyclic AMP had no effect on the key enzymes of glycogen metabolism 1 h after injection in utero, whereas after 6 h an increase in phosphorylase a activity was found without any change in synthetase a activity.     

Neoplastic transformation-linked alterations in adenylosuccinate synthetase activity.

Adenylosuccinate synthetase has been measured in rats in normal, differentiating, and regenerating liver, transplantable hepatomas of different growth rates, kidney cortex, and a transplantable kidney tumor. The activity was increased 1.6 to 3.7-fold in all the tumors. The activity showed no correlation with the degree of histological or biochemical differentiation of the tumors, nor with their growth rate. Adenylosuccinate synthetase activity in regenerating liver was unchanged and in neonatal liver it was much lower than in adult liver. It is concluded that the ubiquitous increase in the tumors of liver and kidney was linked with the neoplastic transformation.     

A new immunoblotting assay for thymidylate synthetase and its application to the regulation of enzyme activity in regenerating rat liver

A highly sensitive and specific immunoblot assay has been developed to quantitate the content of rat liver thymidylate synthetase (EC 2.1.1.45). Applying the method, it is demonstrated that the increase of the activity of thymidylate synthetase in liver regeneration after partial hepatectomy is due to the de novo synthesis of the enzyme protein. Administration of cycloheximide, phenoxybenzamine, phorbol 12-myristate 13-acetate, nifedipine, dexamethasone or indomethacin to partially hepatectomized rats prevented the synthesis of thymidylate synthetase in regenerating liver. Thyroparathyroidectomy also inhibited the increase of the enzyme in liver regeneration. These observations are discussed in relation to the signal transduction concerning the alpha 1-receptor, which was shown to regulate liver regeneration in our previous papers.     

Methylation of nicotinamide in rat liver cytosol and its correlation with hepatocellular proliferation

The changes in the activity of nicotinamide: S-adenosylmethionine methyltransferase (nicotinamide methylase) were studied in rat liver which was subjected to different rates of cellular proliferation. The cytosolic enzyme activity increased 3–4-fold in the first 24–48 h after partial hepatectomy and decreased again to the basal levels until 4 days post-operatively, whereas it remained unchanged in the livers of sham-operated animals. A single administration of thioacetamide at a dose of 50–250 mg/kg body weight, a treatment which induces hepatocellular proliferation as well, also enhanced the enzyme activity 2–3-fold 24 h after drug administration. This activity increase was associated with a marked lowering of intracellular NAD content of as much as 50% of the control levels. d-Galactosamine, a known hepatotoxic agent causing acute hepatitis in experimental animals and preventing DNA synthesis in regenerating liver, blocked the activity increase in regenerating rat liver. The rate of 1-methylnicotinamide synthesis, as measured by incubating liver slices in the culture medium supplemented with [¹⁴C]nicotinamide as a precursor, was found to be 2–4 times higher in the slices from regenerating liver and thioacetamide-treated rat liver than those from non-proliferating control liver. These results, together with our previous finding on the enhancement by 1-methylnicotinamide of the growth of cultured rat liver cells (Hoshino, J., Kühne, U. and Kröger, H. (1982) Biochem. Biophys. Res. Commun. 105, 1446–1452), support the view that nicotinamide methylase and its product, 1-methylnicotinamide, are involved in the control of hepatocellular DNA synthesis and proliferation.     

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.     

Changes in DNA methyltransferase induced by treatment with N-2-acetylaminofluorene

We have compared the levels of DNA methyltransferases from rat liver and spleen in both sexes following a single injection of N-2-acetylaminofluorene (AAF). Enzyme extracts from treated animals were obtained at different intervals (2-34 days) after treatment. The extracts were assayed in the presence of chicken erythrocyte DNA and S-adenosyl-L-[Me-3H]methionine. A 55% increase in male rat-liver methyltransferase activity measured by Me-3H incorporation into DNA occurred on day 14. By contrast, female methyltransferase after a similar period revealed a 33% decrease in activity. Between days 21 and 34, there is a progressive return to normal methyltransferase levels. Spleen-derived enzyme studied between days 7 and 14, showed a decrease in methylating activity in both sexes. After replacing corn seed oil by ethanol as the vehicle for AAF injection, we observed a change in liver methyltransferase 48 h after injection. Quantification of radioactive eluates in m5C fractions together with the increase in the integrated area identified as m5C in HPLC chromatograms allowed positive identification of methylated products.     

Adenosine aminohydrolase activity in the regenerating rat liver

The specific activity of adenosine aminohydrolase in the regenerating rat liver is significantly increased 12 h after partial hepatectomy. There is a twofold increase in enzyme activity at 48 h, after which the activity begins to decline. However, increased values still persist 7 days postsurgery. The enzyme is located mainly in the soluble supernatant (90-95%) of the cell. The purified enzyme from 48-h regenerating liver and control liver has similar kinetic properties (Km 54-58 microM for adenosine), similar molecular weights (30,000-35,000), and are equally inhibited by an irreversible transition-state analog and a reversible competitive inhibitor. It is concluded that adenosine aminohydrolase in regenerating liver is an integral component of a salvage pathway designed for the reutilization of nucleotides, and thus helps maintain a "growth state" for the regenerating liver.     

Distribution of aminoacyl-t-RNA-synthetase activity in rabbit liver cells during disruption of protein biosynthesis in experimental myocardia infarct

Distribution of the aminoacyl-tRNA synthetase activity has been studied in the normal rabbit liver cells and in the model of protein synthesis damage, i.e. under experimental myocardial infarction (EMI). The activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal extracts from rabbit liver homogenate has been determined to increase 12 h after EMI. Gel filtration of the postribosomal extract on Sepharose 6B shows that the activity of aminoacyl-tRNA synthetases is distributed among the fractions with Mr 1.82 x 10(6), 0.84 x 10(6) and 0.12 = 0.35 x 10(6). The first two fractions (high-molecular-weight aminoacyl-tRNA synthetase complexes) contain arginyl-, glutamyl-, isoleucyl-, leucyl-, lysyl- and valyl-tRNA synthetases, whereas the low-molecular-weight fraction contains alanyl-, arginyl-, glycyl-, phenylalanyl-, seryl-, threonyl-, tryptophanyl- and tyrosyl-tRNA synthetases. In a case of EMI all the aminoacyl-tRNA synthetases translocate from the complexes with Mr 1.82 x 10(6) into the complexes with Mr 0.84 x 10(6), what provided evidence for the possibility to regulate protein synthesis by changes in compartmentalization of aminoacyl-tRNA synthetases.     

tRNA sulfurtransferase: a member of the aminoacyl-tRNA synthetase complex in rat liver

Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

II. The influence of 17-beta-oestradiol on aminoacyl-tRNA synthetases from mouse uterus and mouse liver.

The activities of 17 aminoacyl-tRNA synthetases have been determined in liver and uterus preparations of mice. One group of mice were castrated and seven days later given 5 mug 17-beta-oestradiol in olive oil; a similar dose was given after 24 h. The animals were sacrificed one day later. A control group of mice which were also castrated, received olive oil without 17-beta-oestradiol. As the aminoacyl-tRNA synthetases may be found both in high molecular weight and low molecular weight forms, the forms present in the preparations are discussed. The activities of the aminoacyl-tRNA synthetases from uterus augmented under the influence of 17-beta-oestradiol, but to different degrees. The increase in the activities of isoleucyl- and prolyl-tRNA synthetases was not significant. In liver, only the activity of lysyl-tRNA synthetase augmented significantly.     

Postnatal developmental profile of 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthetase and cholesterol 7 alpha-hydroxylase activities in the liver of domestic swine

Activities of 3-hydroxy-3-methylglutaryl-CoA reductase, squalene synthetase and cholesterol 7 alpha-hydroxylase, measured in liver microsomal preparations from domestic swine between birth and adolescence, correlated strongly in individual animals. A synchronous increase was observed between 4 and 6 weeks after birth, i.e., immediately after weaning. Rise in activity was highest for HMG-CoA reductase (30-fold), and smallest for squalene synthetase (5-fold). In pubertal pigs (16 to 30 weeks old), activities of these enzymes had the same low values as in suckling piglets. The increase of both HMG-CoA reductase and squalene synthetase activities may be caused by the shift from high-cholesterol milk intake to a chow diet with low-cholesterol content. The rise in cholesterol 7 alpha-hydroxylase activity might be due to other dietary or hormonal factors.     

Studies on the mechanism of the effects of experimental inflammation on adaptive synthesis of rat liver fatty acid synthetase

The mechanism of suppression, by experimental inflammation of the usual increase in hepatic fatty acid synthetase activity resulting from fat-free feeding following starvation (adaptive synthesis), was investigated immunochemically. That suppression results from changes in amount of hepatic fatty acid synthetase was shown by the observation that fatty acid synthetase preparations from inflamed and uninflamed animals, exhibiting a wide variety of specific enzyme activities, had identical immunochemical equivalence points. In confirmation of this, the amounts of fatty acid synthetase, determined by radial immunodiffusion in gels containing anti-fatty acid synthetase serum, varied concomitantly with changes in enzyme activity regardless of the relative times of inflammation and onset of adaptive synthesis. Serum insulin levels were not dramatically elevated during the first 48 h of fat-free feeding, but rose markedly thereafter. Inflammation, either alone or combined with fat-free feeding, resulted in increased serum glucose levels, followed by a similar pattern of increased serum insulin levels some 12 h later. Fat-free feeding did not affect serum cortisol levels, but depressed liver cyclic AMP. Inflammation invariably resulted in a marked increase in serum cortisol within 12 h and a concomitant elevation of hepatic cyclic AMP, indicating possible roles for cortisol and cyclic AMP in suppression of hepatic fatty acid synthetase synthesis.