N-acetylneuraminic acid, phosphate and thiol groups of pyruvate kinase isoenzymes from Morris hepatoma 7777 and normal rat liver.

The highest amount of N-acetylneuraminic acid (AcNeu) was found in pyruvate kinase isoenzyme L from normal rat liver (24 moles/mole of enzyme tetramer), with the highest electrophoretic mobility. On the other hand, isoenzyme M2 from Morris hepatoma 7777, with the lowest electrophoretic mobility, had the lowest AcNeu content (5 moles/mole of enzyme tetramer). This tumour isoenzyme M2 of pyruvate kinase was, however, characterised by the highest phosphate content (12 moles/mole protein), in comparison to isoenzyme L (3 moles/mole protein) or normal liver isoenzyme M2 (6 moles/mole protein). This could indicate a regulatory change caused by reversible enzyme phosphorylation and dephosphorylation or sialization and desialization. Despite these differences, the sum of the two negatively charged residues was lower in tumour pyruvate kinase isoenzyme M2, with the slowest migration rate, than in normal rat liver isoenzyme M2. Moreover, isoenzyme M2 from tumour material, in comparison with isoenzyme M2 from normal rat liver, had a twice as high content of thiol groups (20 moles/mole protein), especially of free and superficially located ones, than the isoenzyme M2 from normal liver (10 moles/mole protein). This may explain abnormal susceptibility of tumour isoenzyme M2 to stereospecific inhibition by exogenous L-cysteine, and indicate genetically dependent changes in amino-acid content of tumour enzyme which take place during cell tumourigenic transformation.

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Amino-acid composition of pyruvate kinase M2 isoenzyme variants from rat liver and Morris hepatoma 7777.

Acta Biochimica Polonica ◽

10.18388/abp.1998_4271 ◽

1998 ◽

Vol 45 (3) ◽

pp. 775-780 ◽

Cited By ~ 1

Author(s):

J Ignacak ◽

M Gumińska ◽

J Steczko

Keyword(s):

Amino Acid ◽

Pyruvate Kinase ◽

Rat Liver ◽

Acid Content ◽

Polyacrylamide Gel Electrophoresis ◽

Amino Acid Content ◽

Normal Rat ◽

Morris Hepatoma ◽

Amino Acid Analyser ◽

Blue Sepharose

Cytosolic fractions B (salted out between 51-70% ammonium sulphate saturation) from rat liver and Morris hepatoma 7777, containing pyruvate kinase (EC 2.7.1.40) M2 isoenzymes, were purified by affinity chromatography on Blue Sepharose CL-6B. When compared by polyacrylamide gel electrophoresis at pH 8.3, all three M2 pyruvate kinase variants from Morris hepatoma 7777 had lower mobilities (alpha2, beta2, gamma3) than the three corresponding variants (alpha1, beta1, gamma2) from normal rat liver. Using an automatic amino-acid analyser, significant differences in selected amino-acid content have been found in corresponding highly purified gamma3 and gamma2 variants from Morris hepatoma and normal rat liver, respectively. The gamma3-variant of the Morris hepatoma M2 isoenzyme had twice the amount of L-tyrosine and L-cysteine, and a content of L-serine higher by 20% than the corresponding gamma2 variant of the normal rat liver M2 isoenzyme. It contained, however, significantly less dicarboxylic amino acids which explains its lower electrophoretic mobility. It showed also a decrease (by about 10%) in several other amino-acid content, corresponding to a 10% decrease in the tumour enzyme molecular mass.

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Electrophoretic pattern of cytosolic pyruvate kinase fractions A and B (type L and M2) from normal rat liver and Morris hepatoma 7777

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology ◽

10.1016/0167-4838(95)00165-4 ◽

1996 ◽

Vol 1292 (1) ◽

pp. 99-105 ◽

Cited By ~ 4

Author(s):

Maria Gumińska ◽

Jan Ignacak

Keyword(s):

Pyruvate Kinase ◽

Rat Liver ◽

Electrophoretic Pattern ◽

Normal Rat ◽

Morris Hepatoma

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Comparison of pyruvate kinase variants from rat liver and Morris hepatoma 7777, obtained by an affinity chromatography on blue sepharose CL-6B.

Acta Biochimica Polonica ◽

10.18388/abp.1993_4827 ◽

1993 ◽

Vol 40 (2) ◽

pp. 261-267 ◽

Cited By ~ 3

Author(s):

J Ignacak ◽

M Gumińska

Keyword(s):

Affinity Chromatography ◽

Pyruvate Kinase ◽

Rat Liver ◽

Kinase Activity ◽

Signal Molecules ◽

Pyruvate Kinase Activity ◽

Normal Rat ◽

Morris Hepatoma ◽

Blue Sepharose ◽

Main Substrate

Fractions A (salted out by ammonium sulphate between 21-30% saturation), and fractions B (salted out between 51-70% saturation) of pyruvate kinase (EC 2.7.1.40.) corresponding respectively to pyruvate kinase types L and M2 from rat liver and Morris hepatoma 7777 were purified by an affinity chromatography on Blue Sepharose CL-6B. Peaks of inactive proteins were eliminated and the enzyme fractions bound biospecifically to the gels were eluted by free ADP. The molecular mass of purified hepatoma pyruvate kinase fraction B was smaller than that of liver pyruvate kinase fraction B. Morris hepatoma pyruvate kinase fraction B represented a variant of type M2, characterised by greatest affinity to 2-phosphoenolpyruvate as a main substrate and different sensitivity to low-molecular effectors in comparison with types L from both liver and hepatoma and in comparison with type M2 from normal rat liver. Only this hepatoma fraction B showed a tumour specific sensitivity to L-cysteine and was insensitive to normal signal molecules i.e. to ATP and fructose-1,6-diphosphate which influence liver pyruvate kinase activity. L-Cysteine inhibited the tumour fraction B of pyruvate kinase by decreasing its Vmax and increasing the Km values in relation to 2-phosphoenolpyruvate.

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Different Expression of Esterase Variants in Rat Hepatic and Hepatom a-Derived Cell Lines Detected by Electrophoresis

Zeitschrift für Naturforschung C ◽

10.1515/znc-1995-9-1011 ◽

1995 ◽

Vol 50 (9-10) ◽

pp. 664-668 ◽

Cited By ~ 2

Author(s):

Adel S. Afify ◽

Yoshimitsu Yamazaki ◽

Yu-ichi Kageyama ◽

Shiro Yusa ◽

Yoshikatsu Ogawa ◽

...

Keyword(s):

Cell Lines ◽

Rat Liver ◽

Hepatoma Cell ◽

Polyacrylamide Gel Electrophoresis ◽

Liver Homogenate ◽

Normal Liver ◽

Liver Parenchyma ◽

Hepatoma Cell Lines ◽

Normal Rat ◽

Transformed Cell Lines

Abstract Esterases in nine rat hepatic and hepatoma-derived cell lines and normal rat liver homogenate were detected by polyacrylamide gel electrophoresis coupled with active staining with a-naphthyl acetate or butyrate as a substrate. The esterase band patterns of the non-cancerous and oncogene-transformed cell lines were alike, but different from those of hepatoma cell lines and normal rat liver homogenate. The former groups of cells might have completely lost the characteristics of rat liver parenchymal cells, or else they might have their origin at cells other than liver parenchyma. The esterase patterns of the hepatoma cell lines (e.g., McA-RH7777) and the normal rat liver highly resembled with each other, exemplifying the slight biochemical deviation of cancer from normal cells. However, two-dimensional electrophoretogram for the McA-RH7777 cell line showed a prominent esterase spot {p/ 6.0-Mr 110 kDa) that was lacking in the normal liver. This result indicates that there is invariably some change in esterase expression between the cancer cells and the normal liver cells

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Mechanism of 67Ga Accumulation in Normal Rat Liver Lysosomes

Nuklearmedizin ◽

10.1055/s-0038-1624962 ◽

1976 ◽

Vol 15 (04) ◽

pp. 185-194 ◽

Cited By ~ 7

Author(s):

A. Gebhardt ◽

E. Schulz ◽

U. Haubold ◽

M. Hristowa ◽

B. Zeggel ◽

...

Keyword(s):

Rat Liver ◽

Malignant Tumors ◽

Gel Filtration ◽

Normal Liver ◽

Protein Band ◽

Lysosomal Fraction ◽

Coomassie Blue ◽

Normal Rat ◽

The Stability ◽

Rat Liver Lysosomes

Summary 67Ga accumulates in various malignant tumors and parenchymatous tissues. It was found to be associated with the soluble fraction of lysosomes (11). The present work investigates the mechanism of 67Ga accumulation in normal liver cells.Lysosomes were isolated from rat liver after intravenous injection of carrier free 67Ga. The soluble lysosomal fraction was obtained by sonication followed by centrifugation at 105,000 xg for 2 hrs. Gel filtration on Sephadex G 25 superfine was carried out on the soluble lysosomal fraction in order to investigate the stability of the 67Ga-protein complex within the lysosomes under EDTA treatment. After treatment with 1 mM/1 EDTA a considerable amount of the protein bound radioactivity was found to be liberated. In further experiments the 67Ga binding lysosomal proteins were fractionated by electrophoresis on 7% Polyacrylamide gels (0.5 cm x 5.5 cm). After staining with Coomassie blue 18 separated protein bands were apparent. 67Ga distribution within the gels was assessed by direct counting of radioactivity in gel slices. A considerable amount of the intralysosomal protein bound radioactivity migrated with a relative mobility of 0.36 corresponding to a protein band of molecular weight 85,000 — 90,000. This peak corresponded to the peak of 67Ga labeled purified transferrin in control gels. These data were confirmed by Immunoelectrophoresis combined with autoradiography: within the soluble lysosomal fraction a slight transferrin line could be identified.We conclude that 67Ga which is transported in the blood by transferrin (23) and taken up by the hepatic cell through endocytosis (32) is accumulated in the lysosomes associated with transferrin and its degraded fragments.

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A discoordinate increase in the cellular amount of 3-hydroxy-3-methylglutaryl-CoA reductase results in the loss of rate-limiting control over cholesterogenesis in a tumour cell-free system

Biochemical Journal ◽

10.1042/bj2580421 ◽

1989 ◽

Vol 258 (2) ◽

pp. 421-425 ◽

Cited By ~ 26

Author(s):

N I Azrolan ◽

P S Coleman

Keyword(s):

Cholesterol Biosynthesis ◽

Normal Liver ◽

Preliminary Evidence ◽

Free System ◽

Normal Rat ◽

Morris Hepatoma ◽

Coa Reductase ◽

Rate Limiting ◽

Hydroxymethylglutaryl Coa Reductase ◽

Tumour System

Cholesterol biosynthesis was characterized in cell-free post-mitochondrial supernatant systems prepared from both normal rat liver and Morris hepatoma 3924A. The rate of cholesterol synthesis per cell was 9-fold greater in the tumour system than in that from normal liver, and the tumour systems showed the loss of rate-limiting control at the hydroxymethylglutaryl-CoA reductase (HMGR)-catalysed step. The apparent absence of rate-limiting control over cell-free tumour cholesterogenesis was traced primarily to a discoordinate and dramatic increase in the amount of HMGR in the tumour relative to the liver system. Preliminary evidence for an altered control of the post-lanosterol portion of the pathway was also obtained with the tumour system.

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Evidence for Toxic Advanced Glycation End-Products Generated in the Normal Rat Liver

Nutrients ◽

10.3390/nu11071612 ◽

2019 ◽

Vol 11 (7) ◽

pp. 1612 ◽

Cited By ~ 2

Author(s):

Takanobu Takata ◽

Akiko Sakasai-Sakai ◽

Jun-ichi Takino ◽

Masayoshi Takeuchi

Keyword(s):

Rat Liver ◽

Advanced Glycation End Products ◽

Normal Liver ◽

Advanced Glycation ◽

Alcoholic Fatty Liver ◽

Healthy Humans ◽

High Fructose ◽

Glycation End Products ◽

End Products ◽

Normal Rat

Glucose/fructose in beverages/foods containing high-fructose corn syrup (HFCS) are metabolized to glyceraldehyde (GA) in the liver. We previously reported that GA-derived advanced glycation end-products (toxic AGEs, TAGE) are generated and may induce the onset/progression of non-alcoholic fatty liver disease (NAFLD). We revealed that the generation of TAGE in the liver and serum TAGE levels were higher in NAFLD patients than in healthy humans. Although we propose the intracellular generation of TAGE in the normal liver, there is currently no evidence to support this, and the levels of TAGE produced have not yet been measured. In the present study, male Wister/ST rats that drank normal water or 10% HFCS 55 (HFCS beverage) were maintained for 13 weeks, and serum TAGE levels and intracellular TAGE levels in the liver were analyzed. Rats in the HFCS group drank 127.4 mL of the HFCS beverage each day. Serum TAGE levels and intracellular TAGE levels in the liver both increased in the HFCS group. A positive correlation was observed between intracellular TAGE levels in the liver and serum TAGE levels. On the other hand, in male Wister/ST rats that drank Lactobacillus beverage for 12 weeks—a commercial drink that contains glucose, fructose, and sucrose— no increases were observed in intracellular TAGE or serum TAGE levels. Intracellular TAGE were generated in the normal rat liver, and their production was promoted by HFCS, which may increase the risk of NAFLD.

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The M1 subunit of rat liver ribonucleotide reductase appears to be modified by ubiquitination

Biochemistry and Cell Biology ◽

10.1139/o92-032 ◽

1992 ◽

Vol 70 (3-4) ◽

pp. 215-223 ◽

Cited By ~ 5

Author(s):

Marianna Sikorska ◽

Joanna Kwast-Welfeld ◽

Tony Youdale ◽

Robert Richards ◽

James F. Whitfield ◽

...

Keyword(s):

Rat Liver ◽

Ribonucleotide Reductase ◽

Reductase Activity ◽

Normal Liver ◽

Liver Cells ◽

Synthetic Activity ◽

M1 Protein ◽

Rat Liver Cells ◽

Molecular Masses ◽

Morris Hepatoma

Using a combination of immunoblotting, double immunoprecipitation, immunoglobulin-affinity chromatography, and isoelectrofocusing, we have been able to identify a group of proteins that display CDP-reductase activity and contain antigenic epitopes recognized by anti-ribonucleotide reductase M1 subunit and anti-ubiquitin antibodies. In the cytoplasm of rat liver cells, we could detect a total of five proteins with molecular masses of 92, 89, 56, 45, and 37 kilodaltons which reacted with the anti-M1 subunit serum. All of them, except the 89-kilodalton protein (the nascent unmodified M1), were also recognized by the anti-ubiquitin antibody. In normal liver cells, all of the apparently ubiquitinated species of the M1 protein were found in the cytoplasm, but not in the nuclear envelope associated pool of the enzyme. However, we did not detect ubiquitinated M1 protein fragments in the cytoplasm of Morris hepatoma 5123tc. The level of the apparently ubiquitinated fragments of the M1 subunit increased in parallel to the DNA-synthetic activity of normal liver cells, suggesting that ubiquitination plays a key role in the regulation of the activity of the enzyme during the cell cycle.Key words: ribonucleotide reductase turnover, proteolysis, ubiquitin, DNA replication.

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