Mechanism of 67Ga Accumulation in Normal Rat Liver Lysosomes

1976 ◽  
Vol 15 (04) ◽  
pp. 185-194 ◽  
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.

scholarly journals STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES

1966 ◽  
Vol 29 (3) ◽  
pp. 395-403 ◽  
Author(s):  
Takeshi Utsunomiya ◽  
Jay S. Roth
Keyword(s):  
Natural Products ◽  
Sodium Chloride ◽  
Rat Liver ◽  
Messenger Rna ◽  
Rnase Activity ◽  
Optimum Ph ◽  
Normal Rat ◽  
Pancreatic Rnase ◽  
The Stability ◽  
Normal Constituent

The RNase activity and properties of ribosome and polysome preparations from normal rat liver and some hepatomas have been examined. Polysome and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hepatomas had considerably higher specific RNase activity than corresponding preparations from normal rat liver, Novikoff ascites, or Morris 5123 hepatomas. The optimum pH of the RNase was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromercuribenzenesulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg++ ions, subunits could reaggregate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with spermine. In the hepatomas, evidence was obtained for the preexistence of considerable amounts of the smaller, RNase-containing subunits in the cell. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was taken up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may become associated with these particulates if dissociation and reassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown are discussed.

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

1995 ◽  
Vol 50 (9-10) ◽  
pp. 664-668 ◽  
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

Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

1976 ◽  
Vol 54 (5) ◽  
pp. 423-431 ◽  
Author(s):  
Kun-Tsan Lin ◽  
John C. Crawhall
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Protein Band ◽  
Final Concentration ◽  
Assay Method ◽  
Enzyme Extract ◽  
Crude Enzyme Extract ◽  
Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

scholarly journals Purification of NADPH-dependent dehydroascorbate reductase from rat liver and its identification with 3α-hydroxysteroid dehydrogenase

1994 ◽  
Vol 304 (2) ◽  
pp. 385-390 ◽  
Author(s):  
B Del Bello ◽  
E Maellaro ◽  
L Sugherini ◽  
A Santucci ◽  
M Comporti ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gel Filtration ◽  
Dehydroascorbic Acid ◽  
Hydroxysteroid Dehydrogenase ◽  
Protein Band ◽  
Sds Page ◽  
Single Protein ◽  
Ammonium Sulphate Fractionation ◽  
Inflammatory Drugs ◽  
Rat Liver Cytosol

Rat liver cytosol has been found to reduce dehydroascorbic acid (DHAA) to ascorbic acid in the presence of NADPH. The enzyme responsible for such activity has been purified by ammonium sulphate fractionation, DEAE-Sepharose, Sephadex G-100 SF and Reactive Red column chromatography, with an overall recovery of 27%. SDS/PAGE of the purified enzyme showed one single protein band with an M(r) of 37,500. A similar value (36,800) was found by gel filtration on a Sephadex G-100 SF column. The results indicate that the enzyme is a homogeneous monomer. The Km for DHAA was 4.6 mM and the Vmax. was 1.55 units/mg of protein; for NADPH Km and Vmax. were 4.3 microM and 1.10 units/mg of protein respectively. The optimum pH was around 6.2. Several typical substrates and inhibitors of the aldo-keto reductase superfamily have been tested. The strong inhibition of DHAA reductase effected by steroidal and non-steroidal anti-inflammatory drugs, together with the ability to reduce 5 alpha-androstane-3,17-dione strongly, suggest the possibility that DHAA reductase corresponds to 3 alpha-hydroxysteroid dehydrogenase. Microsequence analysis performed on the electro-transferred enzyme band shows that the N-terminus is blocked. Internal primary structure data were obtained from CNBr-derived fragments and definitely proved the identity of NADPH-dependent DHAA reductase with 3 alpha-hydroxysteroid dehydrogenase.

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

Nutrients ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 1612 ◽  
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.

scholarly journals Purification and characterization of glutathione-dependent dehydroascorbate reductase from rat liver

1994 ◽  
Vol 301 (2) ◽  
pp. 471-476 ◽  
Author(s):  
E Maellaro ◽  
B Del Bello ◽  
L Sugherini ◽  
A Santucci ◽  
M Comporti ◽  
...  
Keyword(s):  
Rat Liver ◽  
Protein Identification ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Dehydroascorbic Acid ◽  
Protein Band ◽  
Dehydroascorbate Reductase ◽  
Apparent Homogeneity ◽  
Sds Page

GSH-dependent enzymic reduction of dehydroascorbic acid to ascorbic acid has been studied in rat liver cytosol. After gel filtration of cytosol on Sephadex G-100 SF, dehydroascorbate reductase activity was recovered in two distinct peaks, one corresponding to glutaredoxin (an enzyme already known for its dehydroascorbate reductase activity) and another, much larger one, corresponding to a novel enzyme different from glutaredoxin. The latter was purified to apparent homogeneity. The purification process involved (NH4)2SO4 fractionation, followed by DEAE-Sepharose, Sephadex G-100 SF and Reactive Red chromatography. SDS/PAGE of the purified enzyme in either the presence or absence of 2-mercaptoethanol demonstrated a single protein band of M(r) 31,000. The M(r) determined by both Sephadex G-100 SF chromatography and h.p.l.c. was found to be approx. 48,000. H.p.l.c. of the denatured enzyme gave an M(r) value identical with that obtained by SDS/PAGE (31,000). The apparent Km for dehydroascorbate was 245 microM and the Vmax. was 1.9 mumol/min per mg of protein; for GSH they were 2.8 mM and 4.5 mumol/min per mg of protein respectively. The optimal pH range was 7.5-8.0. Microsequence analysis of the electro-transferred enzyme band showed that the N-terminus is blocked. Data on internal primary structure were obtained from CNBr-and N-chlorosuccinimide-derived fragments. No significative sequence similarity was found to any of the protein sequences contained in the Protein Identification Resource database.

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

1997 ◽  
Vol 44 (2) ◽  
pp. 201-208 ◽  
Author(s):  
J Ignacak ◽  
M Gumińska
Keyword(s):  
Pyruvate Kinase ◽  
Electrophoretic Mobility ◽  
Rat Liver ◽  
Amino Acid Content ◽  
Normal Liver ◽  
Regulatory Change ◽  
Thiol Groups ◽  
Acetylneuraminic Acid ◽  
Normal Rat ◽  
Morris Hepatoma

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.

scholarly journals STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS

1972 ◽  
Vol 55 (2) ◽  
pp. 282-298 ◽  
Author(s):  
Joan A. Higgins ◽  
Russell J. Barrnett
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Normal Liver ◽  
Subsequent Treatment ◽  
Cell Fractionation ◽  
Acyltransferase Activity ◽  
Normal Rat

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.

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