Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties

1989 ◽  
Vol 67 (11-12) ◽  
pp. 817-822 ◽  
Author(s):  
Patricia Berninsone ◽  
Eleonora Katz ◽  
Monica Napp ◽  
Julio Azcurra
Keyword(s):  
Rat Liver ◽  
Subcellular Distribution ◽  
Ache Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Subcellular Location ◽  
High Salt ◽  
Subcellular Fractions ◽  
Velocity Sedimentation ◽  
Molecular Forms ◽  
Extraction Properties

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.Key words: acetylcholinesterase, membrane association, molecular forms, nonspecific cholinesterase, rat liver, subcellular distribution.

scholarly journals Immuno-electron-microscopic studies on the subcellular distribution of rat liver epoxide hydrolase and the effect of phenobarbitone and 2-acetamidofluorene treatment

1982 ◽  
Vol 202 (3) ◽  
pp. 677-686 ◽  
Author(s):  
F Waechter ◽  
P Bentley ◽  
M Germann ◽  
F Oesch ◽  
W Stäubli
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Epoxide Hydrolase ◽  
Specific Binding ◽  
Subcellular Fractions ◽  
Electron Microscopic ◽  
Immuno Electron Microscopy ◽  
Microscopic Studies ◽  
The Individual

The distribution of rat liver epoxide hydrolase in various subcellular fractions was investigated by immuno-electron-microscopy. Ferritin-linked monospecific anti-(epoxide hydrolase) immunoglobulins bound specifically to the cytoplasmic surfaces of total microsomal preparations and smooth and rough microsomal fractions as well as the nuclear envelope. Specific binding was not observed when the ferritin conjugates were incubated with peroxisomes, lysosomes and mitochondria. The average specific ferritin load of the individual subcellular fractions correlated well with the measured epoxide hydrolase activities. This correlation was observed with fractions prepared from control, phenobarbitone-treated and 2-acetamidofluorene-treated rats.

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

1995 ◽  
Vol 1 (4) ◽  
pp. 151-161
Author(s):  
Kuixiong Gao ◽  
Emma Lou Cardell ◽  
Randal E. Morris ◽  
Bruce F. Giffin ◽  
Robert R. Cardell
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Smooth Endoplasmic Reticulum ◽  
Immunogold Electron Microscopy ◽  
Immunogold Staining ◽  
Thin Sections ◽  
Gold Particles

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 μm) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 μm) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles; hepatocytes in pericentral regions have a diffuse subcellular distribution of PEPCK and thus more scattered gold particles. When normal serum replaced the first antibody in the immunogold staining procedures, the background was very low.

scholarly journals Distribution of polypeptides binding guanosine 5′-[γ-[35S]thio]triphosphate and anti-(ras protein) antibodies in liver subcellular fractions. Evidence for endosome-specific components

1990 ◽  
Vol 271 (1) ◽  
pp. 179-183 ◽  
Author(s):  
N Ali ◽  
W H Evans
Keyword(s):  
Rat Liver ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Subcellular Fractions ◽  
Relative Distribution ◽  
Ras Proteins ◽  
Ras Protein ◽  
Gtp Binding Proteins ◽  
Gtp Binding

The subcellular distribution in rat liver of polypeptides binding guanosine 5′-[gamma-[35S]thio]triphosphate [( 35S]GTP[S]) and seven antibodies against ras oncoproteins was evaluated. Multiple low-Mr (21,000-28,000) GTP-binding proteins were detected, but their relative distribution among the membrane fractions varied. A more specific compartmentation of polypeptides which bind antibodies generated against ras proteins was evident, with an Mr-28,000 polypeptide and a probable Mr-56,000 dimer, identified by six of the antibodies tested, being confined mainly to endosomes. An Mr-23,000 polypeptide was detected by some of the antibodies in all of the membrane fractions, but especially in the plasma membranes.

scholarly journals Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification

1967 ◽  
Vol 105 (2) ◽  
pp. 427-442 ◽  
Author(s):  
N. F. González-Cadavid ◽  
P. N. Campbell
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Ammonium Phosphate ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Nuclear Fraction ◽  
Extraction And Purification

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4·0 with distilled water and the other extracted from the residues of the first extraction with 0·15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0·3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate–neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123μg. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24·4; mitochondrial fraction, 57·2; heavy microsomal fraction, 5·2; standard microsomal fraction, 10·6; cell sap, 2·7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate–neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4·0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75–80%.

scholarly journals Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

2000 ◽  
Vol 346 (1) ◽  
pp. 155-161 ◽  
Author(s):  
Paul BROOKS ◽  
Graciela FUERTES ◽  
Rachael Z. MURRAY ◽  
Suchira BOSE ◽  
Erwin KNECHT ◽  
...  
Keyword(s):  
Rat Liver ◽  
Subcellular Distribution ◽  
Mammalian Cells ◽  
Antigen Processing ◽  
Cultured Cells ◽  
26S Proteasome ◽  
Molecular Forms ◽  
20S Proteasome ◽  
Obvious Effect ◽  
Γ Interferon

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable γ-interferon-inducible catalytic β-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit specific antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core α-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some significant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immunofluorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following γ-interferon treatment of cultured cells but γ-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

scholarly journals The subcellular distribution of rat liver serine-pyruvate aminotransferase

1982 ◽  
Vol 202 (2) ◽  
pp. 483-490 ◽  
Author(s):  
K V Rowsell ◽  
L M R Al-Naama ◽  
P Benett
Keyword(s):  
Rat Liver ◽  
Subcellular Distribution ◽  
Subcellular Location ◽  
Mitochondrial Activity ◽  
Transferase Activity ◽  
Mitochondrial Enzymes ◽  
Marker Enzymes ◽  
Aminotransferase Activity ◽  
Mitochondrial Marker ◽  
Differential Sedimentation

1. The subcellular distribution of L-serine-pyruvate aminotransferase activity in rat liver was investigated. About 80% was recovered from cell-free homogenates in a ‘total-particles’ fraction and the remainder in the cytosol. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients showed a distribution for serine-pyruvate aminotransferase activity closely matching that observed for mitochondrial marker enzymes. 3. A study of the solubilization of enzymes from combined subcellular particles by digitonin at various concentrations also indicated a common subcellular location for serine-pyruvate aminotransferase and established mitochondrial enzymes. 4. The increase in liver serine-pyruvate amino-transferase activity induced by glucagon injection was accounted for as an increased mitochondrial activity.

scholarly journals Electrophoretic mobility of γ-glutamyltransferase in rat liver subcellular fractions. Evidence for structure difference from the kidney enzyme

1989 ◽  
Vol 262 (2) ◽  
pp. 535-539 ◽  
Author(s):  
B Antoine ◽  
A Visvikis ◽  
C Thioudellet ◽  
A Rahimi-Pour ◽  
N Strazielle ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Liver Enzyme ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Kidney ◽  
Immunoblot Analysis ◽  
Subcellular Fractions ◽  
Golgi Membranes

Adult rat liver gamma-glutamyltransferase (GGT) has been poorly characterized because of its very low concentration in the tissue. In contrast with the kidney, the liver enzyme is inducible by some xenobiotics, and its relationship to hepatic ontogeny and carcinogenesis seems to be important. Liver GGT polypeptides were identified by immunoblot analysis in subcellular fractions (rough endoplasmic reticulum, smooth endoplasmic reticulum, Golgi membranes and plasma membranes). Rat liver GGT appeared as a series of polypeptides corresponding to different maturation steps. Polypeptides related to the heavy subunit of GGT were detected in rough endoplasmic reticulum at 49, 53 and 55 kDa, and in Golgi membranes at 55, 60 and 66 kDa. Two polypeptides related to the light subunit of GGT were also observed in Golgi membranes. In plasma membranes GGT was composed of 100 kDa, 66 kDa and 31 kDa polypeptides. The 66 kDa component could correspond to the heavy subunit of the rat liver enzyme, and if so has a molecular mass higher than that of the purified rat kidney form of GGT (papain-treated). These data suggest different peptide backbones for the heavy subunits of liver GGT and kidney GGT.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals Subcellular localization of the enzyme that forms mannosyl retinyl phosphate from guanosine diphosphate [14C]mannose and retinyl phosphate

1979 ◽  
Vol 180 (3) ◽  
pp. 449-453 ◽  
Author(s):  
M J Smith ◽  
J B Schreiber ◽  
G Wolf
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Subcellular Localization ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Subcellular Distribution ◽  
Smooth Endoplasmic Reticulum ◽  
Marker Enzymes ◽  
Guanosine Diphosphate

The subcellular distribution of the enzyme catalysing the conversion of retinyl phosphate and GDP-[14C]mannose into [14C]mannosyl retinyl phosphate was determined by using subcellular fractions of rat liver. Purity of fractions, as determined by marker enzymes, was 80% or better. The amount of mannosyl retinyl phosphate formed (pmol/min per mg of protein) for each fraction was: rough endoplasmic reticulum 0.48 +/- 0.09 (mean +/- S.D.); smooth membranes (consisting of 60% smooth endoplasmic reticulum and 40% Golgi apparatus), 0.18 +/- 0.03; Golgi apparatus, 0.13 +/- 0.03; and plasma membrane 0.02.

scholarly journals Endo-N-acetyl-β-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification

1985 ◽  
Vol 229 (2) ◽  
pp. 379-385 ◽  
Author(s):  
J J Lisman ◽  
C J van der Wal ◽  
B Overdijk
Keyword(s):  
Substrate Specificity ◽  
Subcellular Localization ◽  
Enzyme Inhibition ◽  
Rat Liver ◽  
Concanavalin A ◽  
Subcellular Distribution ◽  
Subcellular Fractions ◽  
Partial Purification ◽  
Cytoplasmic Localization

Endo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.96, endoglucosaminidase) has been partially purified (520-fold with respect to the cytoplasmic activity) by using concanavalin A-Sepharose, CM-Sephadex and Bio-Gel P-150 chromatography. From the influence of exogenous glycopeptides on the endoglucosaminidase activity it can be concluded that this activity consists of one enzyme hydrolysing both N-acetyl-lactosaminic-type and oligomannosidic-type substrates. Glycoproteins present in the homogenate inhibit the endoglucosaminidase activity. On re-examination of the subcellular distribution of endoglucosaminidase (after removal of inhibiting glycoproteins from the respective subcellular fractions), its cytoplasmic localization was confirmed.

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