scholarly journals Localization of fumarylacetoacetate fumarylhydrolase in rat liver

1978 ◽  
Vol 56 (9) ◽  
pp. 866-868 ◽  
Author(s):  
Kirit S. Doshi ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Marker Enzymes ◽  
Intracellular Location ◽  
Relative Specific Activity ◽  
Fractionation Procedure ◽  
Soluble Phase ◽  
Microscopic Studies

The intracellular location of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) has been demonstrated in rat liver tissue. Two fractionation procedures involving homogenization and differential centrifugation were adopted. The first fractionation procedure isolated the nuclear fraction while the second gave the mitochondrial, microsomal, and soluble phase fractions. The hydrolase is localized in the soluble phase of the rat liver tissue. The enzyme also showed a high relative specific activity in the soluble phase fraction. Fractionation efficiency was checked by microscopic studies and by determining the distribution of a number of marker enzymes.

Localization of oxalacetate keto–enol-tautomerase

1976 ◽  
Vol 54 (3) ◽  
pp. 233-237 ◽  
Author(s):  
James C. Wesenberg ◽  
Aravind Chaudhari ◽  
Robert G. Annett
Keyword(s):  
Enzymic Activity ◽  
The Other ◽  
Nuclear Fraction ◽  
Marker Enzymes ◽  
Animal Cells ◽  
Intracellular Location ◽  
Pig Kidney ◽  
Fractionation Procedure ◽  
Mitochondrial Fractions ◽  
Soluble Phase

The intracellular location of oxalacetate keto–enol-tautomerase (oxaloacetate keto–enol-isomerase) (EC 5.3.2.2) has been determined in two types of animal cells, rat liver and pig kidney. Two fractionation procedures were adopted and modified to suit each type of tissue. One fractionation procedure gave the soluble phase, microsomal and mitochondrial fractions, while the other isolated the nuclear fraction. The tautomerase is distributed among the soluble phase, microsomes and mitochondria in both tissues. Fractionation efficiency was checked by determining percentage recoveries of enzymic activity and total protein after each step, by microscopy studies and by determining the distribution of several marker enzymes.

scholarly journals ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

1968 ◽  
Vol 37 (1) ◽  
pp. 163-181 ◽  
Author(s):  
Paul D. Sadowski ◽  
Janet Alcock Howden
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Orotic Acid ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Differential Centrifugation ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Triton X

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-14C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-14C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.

scholarly journals LYSOSOMAL FRACTIONS FROM TRANSITIONAL EPITHELIUM

1965 ◽  
Vol 24 (2) ◽  
pp. 259-266 ◽  
Author(s):  
Norbert M. Kanczak ◽  
Joseph I. Krall ◽  
E. Russell Hayes ◽  
Willard B. Elliott
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Rat Kidney ◽  
Transitional Epithelium ◽  
Differential Centrifugation ◽  
Relative Specific Activity

Histochemical data suggested that the so called lipoid granules of transitional epithelium in some species are equivalent to lysosomes. Scrapings of bovine and canine transitional epithelium were subjected to differential centrifugation to confirm this identification biochemically. Fractions of rat liver, the classic source of lysosomes, were also prepared by the same methods to compare with the fractions obtained from urinary epithelium. In contrast to rat liver, uroepithelial fractions with a high relative specific activity for hydrolases were sedimented before the heavy mitochondria. Microscopically, these fractions contained the highest proportion of lipoid granules. The size and sedimentation characteristics of lysosomes from transitional epithelium more closely resembled those of lysosomes derived from rat kidney than those isolated from liver.

Structural and Metabolic Interrelationships Among Glycerophosphatides of Rat Liver In Vivo

1971 ◽  
Vol 49 (12) ◽  
pp. 1347-1356 ◽  
Author(s):  
B. J. Holub ◽  
A. Kuksis
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Specific Activity ◽  
Acyl Transfer ◽  
De Novo Synthesis ◽  
Individual Molecular Species ◽  
Relative Specific Activity ◽  
Proportional Contribution ◽  
Specific Activities

The specific activities of individual molecular species of rat liver diacylglycerylphosphorocholine (PC), diacylglycerylphosphoroethanolamine (PE), and diacylglycerophosphorylinositol (MPI) were determined and compared following intravenous injection of glycerol-14C. PC, PE, and MPI contained 41, 51, and 83%, respectively, tetraenoic species, and 40,17, and 9% combined mono-, di-, and trienoic species. The rest of the phosphatide mass of PC, PE, and MPI was contributed by 18, 32, and 8% penta- and hexaenoic species, respectively. The proportions of chemical classes of the glycerophosphatides differed by 1.1- to 18-fold while the fatty acid associations within the unsaturation classes common to these phosphatides varied 2.2- to 17-fold. After 5 min exposure to radioactive glycerol, the mono-, di-, and trienoic species of the PC, PE, and MPI possessed 13–18, 15–50, and 6–42 times, respectively, the specific activity of the tetraenes of the corresponding phosphatide classes. While the pentaenoic and hexaenoic species of PC and MPI had specific activities three to five times those of the respective tetraenes, the higher polyenes of PE were considerably more radioactive and approached the specific activity of the dienoic species of this phosphatide. With progressing time up to 60 min, the tetraenoic species of PC, PE, and MPI showed increases in relative specific activity of 50, 64, and 109%, respectively, in the three phosphatides. These results are consistent with an effective de novo synthesis of the oligoenoic species and a transacylation of the tetraenoic species of all liver glycerophosphatides tested. The proportional contribution of de novo synthesis in comparison to acyl transfer is apparently greater to the formation of PC and PE than to that of MPI.

scholarly journals Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

1978 ◽  
Vol 78 (2) ◽  
pp. 349-368 ◽  
Author(s):  
R Wattiaux ◽  
S Wattiaux-De Coninck ◽  
M F Ronveaux-dupal ◽  
F Dubois
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Specific Activity ◽  
Mitochondrial Fraction ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Isopycnic Centrifugation

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

scholarly journals CYTOCHEMISTRY OF GOLGI FRACTIONS PREPARED FROM RAT LIVER

1974 ◽  
Vol 60 (1) ◽  
pp. 8-25 ◽  
Author(s):  
Marilyn G. Farquhar ◽  
J. J. M. Bergeron ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Liver Tissue ◽  
Marker Enzymes ◽  
Reliable Identification ◽  
Fractionation Procedure ◽  
Thiamine Pyrophosphatase ◽  
Opposite Face ◽  
Liver Homogenates

Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF1, GF2, GF3) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF1 and GF2, and along the outside of the cisternal membranes in GF3. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF1 and within many VLDL-filled vacuoles in GF1 and GF2, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF3 and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF1 and GF2, and was not found in the cisternae in GF3. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF1 and GF3, representing primarily trans-Golgi elements from the secretory Golgi face, and GF3 consisting largely of cis-Golgi components from the opposite face.

scholarly journals Liver monoamine oxidase in the obese–hyperglycaemic (ob/ob) mouse

1979 ◽  
Vol 177 (3) ◽  
pp. 943-949 ◽  
Author(s):  
J H Tong ◽  
M Limson-Zamora ◽  
A D'Iorio ◽  
N Bégin-Heick
Keyword(s):  
Enzyme Activity ◽  
Monoamine Oxidase ◽  
Rat Liver ◽  
Mouse Liver ◽  
Specific Activity ◽  
Residual Activity ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Marker Enzymes ◽  
Acetone Water

1. The specific activity of monoamine oxidase was found to be greater in liver mitochondria from ob/ob mice than from lean mice. The activities of marker enzymes were similar in both tissues. 2. Experiments with various substrates (5-hydroxytryptamine, benzylamine and tyramine) and inhibitors (clorgyline and deprenyl) indicated that, unlike rat liver mitochondria, mouse liver mitochondria contain a predominance of the B-form of monoamine oxidase. 3. The Km values for lean and ob/ob mice were the same for any given substrate and were in the increasing order 5-hydroxytryptamine less than tyramine less than benzylamine. Vmax. was approximately 50% greater in obese than in lean mice. 4. Extraction of liver mitochondria with acetone/water or acetone/water/NH3 to remove lipids decreased the enzyme activity relatively more in obese- than in lean-mice preparations, but residual activity was the same in both preparations.

scholarly journals Postprandial Glycogen Content Is Increased in the Hepatocytes of Human and Rat Cirrhotic Liver

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 976
Author(s):  
Natalia N. Bezborodkina ◽  
Sergey V. Okovityi ◽  
Boris N. Kudryavtsev
Keyword(s):  
Rat Liver ◽  
Liver Tissue ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Fibrous Tissue ◽  
Glycogen Metabolism ◽  
Liver Parenchyma ◽  
Dry Mass ◽  
Cirrhotic Liver ◽  
Liver Biopsies

Chronic hepatitises of various etiologies are widespread liver diseases in humans. Their final stage, liver cirrhosis (LC), is considered to be one of the main causes of hepatocellular carcinoma (HCC). About 80–90% of all HCC cases develop in LC patients, which suggests that cirrhotic conditions play a crucial role in the process of hepatocarcinogenesis. Carbohydrate metabolism in LC undergoes profound disturbances characterized by altered glycogen metabolism. Unfortunately, data on the glycogen content in LC are few and contradictory. In this study, the material was obtained from liver biopsies of patients with LC of viral and alcohol etiology and from the liver tissue of rats with CCl4-induced LC. The activity of glycogen phosphorylase (GP), glycogen synthase (GS), and glucose-6-phosphatase (G6Pase) was investigated in human and rat liver tissue by biochemical methods. Total glycogen and its labile and stable fractions were measured in isolated individual hepatocytes, using the cytofluorometry technique of PAS reaction in situ. The development of LC in human and rat liver was accompanied by an increase in fibrous tissue (20- and 8.8-fold), an increase in the dry mass of hepatocytes (by 25.6% and 23.7%), and a decrease in the number of hepatocytes (by 50% and 28%), respectively. The rearrangement of the liver parenchyma was combined with changes in glycogen metabolism. The present study showed a significant increase in the glycogen content in the hepatocytes of the human and the rat cirrhotic liver, by 255% and 210%, respectively. An increased glycogen content in cells of the cirrhotic liver can be explained by a decrease in glycogenolysis due to a decreased activity of G6Pase and GP.

Analytical Subcellular Fractionation Studies on Rat Liver and on Isolated Jejunal Enterocytes with Special Reference to the Separation of Lysosomes, Peroxisomes and Mitochondria

1976 ◽  
Vol 50 (5) ◽  
pp. 355-366 ◽  
Author(s):  
T. J. Peters ◽  
H. Shio
Keyword(s):  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Low Molecular Weight ◽  
Rat Jejunum ◽  
Marker Enzymes ◽  
Good Separation ◽  
Subcellular Organelles ◽  
Rat Liver Cells ◽  
Isopycnic Centrifugation

1. Enterocytes were isolated from rat jejunum and characterized morphologically. 2. Attempts to separate the enterocyte subcellular organelles, characterized by their marker enzymes, with isopycnic centrifugation were unsuccessful but good separation of peroxisomes, lysosomes and mitochondria was achieved by sedimentation through a shallow sucrose density gradient with a superimposed inverse gradient of low-molecular-weight dextran. 3. The properties and enzyme activities of the principal subcellular organelles in rat liver cells and enterocytes were compared.

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