scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1971 ◽  
Vol 49 (3) ◽  
pp. 899-905 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Carol Pannek ◽  
Daniel S. Friend
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Specific Activity ◽  
Total Activity ◽  
Thiamine Pyrophosphate ◽  
Transferase Activity ◽  
Galactosyl Transferase ◽  
Thiamine Pyrophosphatase ◽  
Cell Components

The thiamine pyrophosphatase (the enzyme [s] catalyzing the release of inorganic phosphate with thiamine pyrophosphate as the substrate) activities of Golgi apparatus-, plasma membrane-, endoplasmic reticulum-, and mitochondria-rich fractions from rat liver were compared at pH 8. Activity was concentrated in the Golgi apparatus fractions, which, on a protein basis, had a specific activity six to eight times that of the total homogenates or purified endoplasmic reticulum fractions. However, only 1–3% of the total activity was recovered in the Golgi apparatus fractions under conditions where 30–50% of the UDPgalactose:N-acetylglucosamine-galactosyl transferase activity was recovered. Considering both recovery of galactosyl transferase and fraction purity, we estimate that approximately 10% of the total thiamine pyrophosphatase activity of the liver was localized within the Golgi apparatus, with a specific activity of about ten times that of the total homogenate. Cytochemically, reaction product was found in the cisternae of the endoplasmic reticulum as well as in the Golgi apparatus. This is in contrast to results obtained in most other tissues, where reaction product was restricted to the Golgi apparatus. Thus, enzymes of rat liver catalyzing the hydrolysis of thiamine pyrophosphate, although concentrated in the Golgi apparatus, are widely distributed among other cell components in this tissue.

scholarly journals ISOLATION OF A GOLGI APPARATUS-RICH FRACTION FROM RAT LIVER

1970 ◽  
Vol 44 (3) ◽  
pp. 492-500 ◽  
Author(s):  
R. D. Cheetham ◽  
D. James Morré ◽  
Wayne N. Yunghans
Keyword(s):  
Endoplasmic Reticulum ◽  
Uric Acid ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Succinic Dehydrogenase ◽  
Acid Oxidase ◽  
Enzyme Substrate ◽  
Thiamine Pyrophosphatase ◽  
Cell Fractions

Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20–30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2–3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.

scholarly journals Hepatic endosome fractions contain an ATP-driven proton pump

1985 ◽  
Vol 225 (1) ◽  
pp. 51-58 ◽  
Author(s):  
T Saermark ◽  
N Flint ◽  
W H Evans
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Atpase Activity ◽  
Proton Pump ◽  
Subcellular Distribution ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Ph Gradients ◽  
Subcellular Organelle ◽  
Liver Homogenates

Endosome fractions were isolated from rat liver homogenates on the basis of the subcellular distribution of circulating ligands, e.g. 125I-asialotransferrin internalized by hepatocytes by a receptor-mediated process. The distribution of endocytosed 125I-asialotransferrin 1-2 min and 15 min after uptake by liver and a monensin-activated Mg2+-dependent ATPase activity coincided on linear gradients of sucrose and Nycodenz. The monensin-activated Mg2+-ATPase was enriched relative to the liver homogenates up to 60-fold in specific activity in the endosome fractions. Contamination of the endosome fractions by lysosomes, endoplasmic reticulum, mitochondria, plasma membranes and Golgi-apparatus components was low. By use of 9-aminoacridine, a probe for pH gradients, the endosome vesicles were shown to acidify on addition of ATP. Acidification was reversed by addition of monensin. The results indicate that endosome fractions contain an ATP-driven proton pump. The ionophore-activated Mg2+-ATPase in combination with the presence of undegraded ligands in the endosome fractions emerge as linked markers for this new subcellular organelle.

scholarly journals Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

1991 ◽  
Vol 266 (7) ◽  
pp. 4322-4328 ◽  
Author(s):  
P Moreau ◽  
M Rodriguez ◽  
C Cassagne ◽  
D M Morré ◽  
D J Morré
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Intracellular digestion and cellular defecation in a monogenean, Diclidophora merlangi

Parasitology ◽  
1975 ◽  
Vol 70 (3) ◽  
pp. 331-340 ◽  
Author(s):  
D. W. Halton
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Digestive Enzymes ◽  
Host Protein ◽  
Blood Proteins ◽  
Host Fish ◽  
Intracellular Digestion ◽  
Thiamine Pyrophosphatase ◽  
Lysosomal System ◽  
Pyrophosphatase Activity

The ultrastructural and cytochemical changes accompanying intracellular digestion and cellular defecation in the haematin cell of Diclidophora merlangi have been described. Blood proteins of the host-fish are sequestered by endocytosis and degraded within an interconnecting network of channels that form an integral, but changing, part of the cell. The digestive enzymes involved originate in the granular endoplasmic reticulum and are packaged in the Golgi apparatus and transferred to the channels in Golgi vesicles. The rate of haemoglobin absorption and the activity of the Golgi, as judged by vesicle counts and staining intensities for thiamine pyrophosphatase activity, are stimulated by the introduction of host protein into the gut lumen. The haematin residues of digestion are extruded periodically into the lumen by exocytosis involving membrane fusion. The process is a continuous one and, in worms starved of food, can result in the complete evacuation of pigment from the cell. It is suggested that a lysosomal system operates in the digestive cycle of the haematin cell.

scholarly journals Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes

1982 ◽  
Vol 201 (3) ◽  
pp. 653-656 ◽  
Author(s):  
B Burchell
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Specific Activity ◽  
Liver Microsomes ◽  
Wistar Rat ◽  
Transferase Activity ◽  
Rat Liver Microsomes ◽  
Phosphatidylcholine Liposomes ◽  
Gunn Rat ◽  
Rigid Environment

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

Early Effects of Aminonucleoside on Enzymes in the Golgi Apparatus of Rat Liver

1975 ◽  
Vol 53 (4) ◽  
pp. 549-554 ◽  
Author(s):  
E. Katona ◽  
M. A. Moscarello
Keyword(s):  
Alkaline Phosphatase ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Intravenous Dose ◽  
Arylsulfatase A ◽  
Single Intravenous Dose ◽  
Renal Lesions ◽  
Arylsulfatase B ◽  
Thiamine Pyrophosphatase ◽  
Early Effects

Rats were injected with a single intravenous dose of aminonucleoside (AMN) and sacrificed 1–48 h later. The activity of several enzymes was assayed in the Golgi apparatus isolated from the liver. Galactosyltransferase activity showed little changes after the AMN, but both acid (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) activities increased within the first hour and reached control levels only 5–24 h later. Thiamine pyrophosphatase and arylsulfatase A (EC 3.1.6.1) activities also increased and stayed at higher levels for the duration of the experiment. Arylsulfatase B (EC 3.1.6.1) activity decreased shortly after the AMN but later increased to above control levels. These findings support earlier results in which liver ultrastructural and biochemical changes were observed early before renal lesions and proteinuria.

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