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 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 Determination of the intracellular sites and topology of glucosylceramide synthesis in rat liver

1991 ◽  
Vol 280 (2) ◽  
pp. 295-302 ◽  
Author(s):  
A H Futerman ◽  
R E Pagano
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Membrane Fraction ◽  
Marker Enzyme ◽  
Luminal Surface ◽  
Proteolytic Digestion ◽  
And Topology

We examined the intracellular site(s) and topology of glucosylceramide (GlcCer) synthesis in subcellular fractions from rat liver, using radioactive and fluorescent ceramide analogues as precursors, and compared these results with those obtained in our recent study of sphingomyelin (SM) synthesis in rat liver [Futerman, Stieger, Hubbard & Pagano (1990) J. Biol. Chem. 265, 8650-8657]. In contrast with SM synthesis, which occurs principally at the cis/medial Golgi apparatus, GlcCer synthesis was more widely distributed, with substantial amounts of synthesis detected in a heavy (cis/medial) Golgi-apparatus subfraction, a light smooth-vesicle fraction that is almost devoid of an endoplasmic-reticulum marker enzyme (glucose-6-phosphatase), and a heavy vesicle fraction. Furthermore, no GlcCer synthesis was detected in an enriched plasma-membrane fraction after accounting for contamination by Golgi-apparatus membranes. These results suggest that a significant amount of GlcCer may be synthesized in a pre- or early Golgi-apparatus compartment. Unlike SM synthesis, which occurs at the luminal surface of the Golgi apparatus, GlcCer synthesis appeared to occur at the cytosolic surface of intracellular membranes, since (i) limited proteolytic digestion of intact Golgi-apparatus vesicles almost completely inhibited GlcCer synthesis, and (ii) the extent of UDP-glucose translocation into the Golgi apparatus was insufficient to account for the amount of GlcCer synthesis measured. These findings imply that, after its synthesis, GlcCer must undergo transbilayer movement to the luminal surface to account for the known topology of higher-order glycosphingolipids within the Golgi apparatus and plasma membrane.

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 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

Formation of chlamydospores in Gilbertella persicaria

1981 ◽  
Vol 59 (5) ◽  
pp. 908-928 ◽  
Author(s):  
Martha J. Powell ◽  
Charles E. Bracker ◽  
David J. Sternshein
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Light And Electron Microscopy ◽  
Multivesicular Bodies ◽  
Marker Enzyme ◽  
Transparent Layer ◽  
Thiamine Pyrophosphatase ◽  
Gilbertella Persicaria

The cytological events involved in the transformation of vegetative hyphae of the zygomycete Gilbertella persicaria (Eddy) Hesseltine into chlamydospores were studied with light and electron microscopy. Thirty hours after sporangiospores were inoculated into YPG broth, swellings appeared along the aseptate hyphae. Later, septa, traversed by plasmodesmata, delimited each end of the hyphal swellings and compartmentalized these hyphal regions as they differentiated into chlamydospores. Nonswollen regions adjacent to chlamydospores remained as isthmuses. Two additional wall layers appeared within the vegetative wall of the developing chlamydospores. An alveolate, electron-dense wall formed first, and then an electron-transparent layer containing concentrically oriented fibers formed between this layer and the plasma membrane. Rather than a mere condensation of cytoplasm, development and maturation of the multinucleate chlamydospores involved extensive cytoplasmic changes such as an increase in reserve products, lipid and glycogen, an increase and then disappearance of vacuoles, and the breakdown of many mitochondria. Underlying the plasma membrane during chlamydospore wall formation were endoplasmic reticulum, multivesicular bodies, vesicles with fibrillar contents, vesicles with electron-transparent contents, and cisternal rings containing the Golgi apparatus marker enzyme, thiamine pyrophosphatase. Acid phosphatase activity was localized cytochemically in a cisterna which enclosed mitochondria and in vacuoles which contained membrane fragments. Tightly packed membrane whorls and single membrane bounded sacs with finely granular matrices surrounding vacuoles were unique during chlamydospore development. Microbodies were rare in the mature chlamydospore, but endoplasmic reticulum was closely associated with lipid globules. As chlamydospores developed, the cytoplasm in the isthmus became highly vacuolated, lipid globules were closely associated with vacuoles, mitochondria were broken down in vacuoles, unusual membrane configurations appeared, and eventually the membranes degenerated. Unlike chlamydospores, walls of the isthmus did not thicken, but irregularly shaped appositions containing numerous channels formed at intervals on the inside of these walls. The pattern of cytoplasmic transformations during chlamydospore development is similar to events leading to the formation of zygospores and sporangiospores.

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.

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