Analytical fractionation of human liver microsomal fractions: Localization of cholesterol and of the enzymes relevant to its metabolism

1981 ◽  
Vol 60 (4) ◽  
pp. 435-439 ◽  
Author(s):  
S. Balasubramaniam ◽  
K. A. Mitropoulos ◽  
S. Venkatesan ◽  
N. B. Myant ◽  
T. J. Peters ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Human Liver ◽  
Free Cholesterol ◽  
Cholesterol Metabolism ◽  
Distribution Profile ◽  
Analytical Fractionation ◽  
Coa Reductase ◽  
Isopycnic Centrifugation

1. The submicrosomal distribution of three enzymes concerned in cholesterol metabolism, and of free and esterified cholesterol, was determined in human liver by analytical isopycnic centrifugation on sucrose gradients. 2. The distribution profile and median density of acyl-CoA:cholesterol O-acyltransferase was similar to that of RNA, showing that this enzyme is confined largely to the ribosome-rich membranes of the endoplasmic reticulum. The distribution profiles and median densities of 3-hydroxy-3-methylglutaryl-CoA reductase and cholesterol 7α-mono-oxygenase showed that both enzymes are confined to the smooth, ribosome-poor, endoplasmic reticulum. 3. Most of the free cholesterol in the microsomal preparations was present in smooth membranes from the Golgi apparatus and in vesicles from plasma-membrane fragments. The distribution of esterified cholesterol was multimodal and extended throughout the whole gradient.

scholarly journals Analytical subcellular fractionation of needle-biopsy specimens from human liver

1978 ◽  
Vol 174 (2) ◽  
pp. 435-446 ◽  
Author(s):  
T J Peters ◽  
C A Seymour
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Needle Biopsy ◽  
Human Liver ◽  
Resolving Power ◽  
Equilibrium Density ◽  
Marker Enzyme ◽  
Acid Hydrolases ◽  
Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

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 Subcellular distribution of selenium in deficient mouse liver

1989 ◽  
Vol 258 (2) ◽  
pp. 541-545 ◽  
Author(s):  
R Reiter ◽  
R Otter ◽  
A Wendel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Trace Element ◽  
Fold Increase ◽  
Subcellular Fractionation ◽  
Fast Response ◽  
Cell Compartment ◽  
Deficient Mice

Selenium (Se)-deficient mice were labelled in vivo with single pulses of [75Se]selenite, and the intrahepatic distribution of the trace element was studied by subcellular fractionation. At 1 h after intraperitoneal injection of 3.3 or 10 micrograms of Se/kg body weight, 15% of the respective doses were found in the liver. Accumulation in the subcellular fractions followed the order: Golgi vesicular much greater than lysosomal greater than cytosolic = microsomal greater than mitochondrial, peroxisomal, nuclear and plasma-membrane fraction. At a dose of 3.3 micrograms/kg, more than 90% of the hepatic Se was protein-bound. When cross-contamination was accounted for, the following specific Se contents of the subcellular compartments were extrapolated: Golgi apparatus, 7.50 pmol/mg; cytosol, 0.90 pmol/mg; endoplasmic reticulum, 0.80 pmol/mg; mitochondria, 0.49 pmol/mg; nuclei, lysosomes, peroxisomes and plasma membrane, less than 0.4 pmol/mg. At 10 micrograms/kg, a roughly 2-3-fold increase in Se content of all fractions was found without major changes in the intrahepatic distribution pattern. An extraordinary rise in the cytosolic fraction was due to an apparently non-protein-bound Se pool. At 24 h after dosing, total hepatic Se had decreased to 6% of the initial dose and had become predominantly protein-bound. The 60% decrease in hepatic Se was reflected in a similar fall in the subcellular levels of the trace element. The Golgi apparatus still had the highest specific Se content, although accumulation was 5 times less than that after 1 h. The cytosolic pool accounted for 50% of the hepatic Se at both labelling times. After 1 h the Golgi apparatus was, with 19%, the second largest intrahepatic pool, followed by the endoplasmic reticulum with 16%. The high affinity and fast response of the Golgi apparatus to Se supplementation of deficient mice is interpreted in terms of a predominant function of this cell compartment in the processing and the export of Se-proteins from the liver.

scholarly journals Cytochemical demonstration of extraperoxisomal catalase. I. Sheep liver.

1976 ◽  
Vol 24 (6) ◽  
pp. 713-724 ◽  
Author(s):  
F Roels
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Rough Endoplasmic Reticulum ◽  
Catalase Activity ◽  
Nonparenchymal Cells ◽  
Sheep Liver ◽  
Heat Stable ◽  
Cytochemical Demonstration ◽  
Electron Microscopes

In sheep hepatocytes catalase activity was demonstrated both within peroxisomes and within the cytosol. In the cytosol the catalase reaction product is contiguous to the plasma membrane and surrounds the nuclei, rough endoplasmic reticulum, cisternae, mitochondria and Golgi apparatus. This is the first cytochemical demonstration of guine extraperoxisomal catalase. No catalase reaction product was seen in the cytosol of nonparenchymal cells. To demonstrate catalase, both glutaraldehyde and formaldehyde fixation were used, followed by a diaminobenzidine technique modified from Novikoff and Goldfischer. Control reactions were performed to distinguish catalase reaction product from adsorption of oxidized diaminobenzidine and from precipitate due to oxidase-, peroxidase- or heat-stable peroxidatic activities. The results were evaluated in the light and electron microscopes.

Development of the lateral palatal brush in larvae of Aedes aegypti (L.) (Diptera: Culicidae)

1990 ◽  
Vol 68 (7) ◽  
pp. 1454-1467 ◽  
Author(s):  
K. M. Fry ◽  
S. B. McIver
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Aedes Aegypti ◽  
Rough Endoplasmic Reticulum ◽  
Light And Electron Microscopy ◽  
Fourth Instar ◽  
Muscle Fibres ◽  
Final Length

Light and electron microscopy were used to observe development of the lateral palatal brush in Aedes aegypti (L.) larvae. Development was sampled at 4-h intervals from second- to third-instar ecdyses. Immediately after second-instar ecdysis, the epidermis apolyses from newly deposited cuticle in the lateral palatal pennicular area to form an extensive extracellular cavity into which the fourth-instar lateral palatal brush filaments grow as cytoplasmic extensions. On reaching their final length, the filaments deposit cuticulin, inner epicuticle, and procuticle sequentially on their outer surfaces. The lateral palatal crossbars, on which the lateral palatal brush filaments insert, form after filament development is complete. At the beginning of development, the organelles involved in plasma membrane and cuticle production are located at the base and middle of the cells. As the filament rudiments grow, most rough endoplasmic reticulum, mitochondria, and Golgi apparatus move to the apex of the epidermal cells and into the filament rudiments. After formation of the lateral palatal brush filaments and lateral palatal crossbars, extensive organelle breakdown occurs. Lateral palatal brush formation is unusual in that no digestion and resorption of old endocuticle occurs prior to deposition of new cuticle. No mucopolysaccharide secretion by the lateral palatal brush epidermis was observed, nor were muscle fibres observed to attach to the lateral palatal crossbars, as has been suggested by other workers.

Effect of digitonin on rat myometrium subcellular membrane fractions

1981 ◽  
Vol 59 (11) ◽  
pp. 1128-1133 ◽  
Author(s):  
A. K. Grover ◽  
C. Y. Kwan ◽  
J. Crankshaw ◽  
E. E. Daniel
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cytochrome C ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
Mitochondrial Marker ◽  
Concentration Dependent Manner ◽  
Subcellular Membrane ◽  
Ca Uptake ◽  
Isopycnic Centrifugation

Isopycnic centrifugation experiments using sucrose density gradients showed that in digitonin-treated microsomes the distribution of the plasma membrane (PM) marker 5′-nucleotidase was shifted to higher densities. The treatment also caused similar but less pronounced changes in the distribution of protein, the putative endoplasmic reticulum (ER) marker NADPH-dependent cytochrome c reductase, and the inner mitochondrial marker cytochrome c oxidase. Similar experiments using more purified membrane fractions showed that the digitonin treatment led to a comparable increase in the densities of the fractions N1 and N2 previously described as subfractions of plasma membrane and to considerably less increase in the density of the fraction N3B which is enriched in the endoplasmic reticulum and the inner mitochondrial markers. Digitonin inhibited the ATP-dependent Ca uptake by the N1 fraction in a concentration-dependent manner (I50 = 0.3 mg/mL). Digitonin (0.5 mg/mL) inhibited the ATP-dependent azide-insensitive Ca uptake by all the fractions. The results support the hypothesis that (a) N1 and N2 are subfractions of plasma membrane, and (b) ATP-dependent azide-insensitive Ca uptake in rat myometrium is a property of plasma membranes.

scholarly journals Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane.

1984 ◽  
Vol 99 (3) ◽  
pp. 1101-1109 ◽  
Author(s):  
A A Rogalski ◽  
J E Bergmann ◽  
S J Singer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
G Protein ◽  
Rough Endoplasmic Reticulum ◽  
Intracellular Transport ◽  
Temperature Shift ◽  
Surface Expression ◽  
Microtubule Assembly ◽  
In Cells

We studied the effects of changes in microtubule assembly status upon the intracellular transport of an integral membrane protein from the rough endoplasmic reticulum to the plasma membrane. The protein was the G glycoprotein of vesicular stomatitis virus in cells infected with the Orsay-45 temperature-sensitive mutant of the virus; the synchronous intracellular transport of the G protein could be initiated by a temperature shift-down protocol. The intracellular and surface-expressed G protein were separately detected and localized in the same cells at different times after the temperature shift, by double-immunofluorescence microscopic measurements, and the extent of sialylation of the G protein at different times was quantitated by immunoprecipitation and SDS PAGE of [35S]methionine-labeled cell extracts. Neither complete disassembly of the cytoplasmic microtubules by nocodazole treatment, nor the radical reorganization of microtubules upon taxol treatment, led to any perceptible changes in the rate or extent of G protein sialylation, nor to any marked changes in the rate or extent of surface appearance of the G protein. However, whereas in control cells the surface expression of G was polarized, at membrane regions in juxtaposition to the perinuclear compact Golgi apparatus, in cells with disassembled microtubules the surface expression of the G protein was uniform, corresponding to the intracellular dispersal of the elements of the Golgi apparatus. The mechanisms of transfer of integral proteins from the rough endoplasmic reticulum to the Golgi apparatus, and from the Golgi apparatus to the plasma membrane, are discussed in the light of these observations, and compared with earlier studies of the intracellular transport of secretory proteins.

scholarly journals Possible role for gp160 in constitutive but not insulin-stimulated GLUT4 trafficking: dissociation of gp160 and GLUT4 localization

1998 ◽  
Vol 330 (1) ◽  
pp. 405-411 ◽  
Author(s):  
Anthony FILIPPIS ◽  
Stella CLARK ◽  
Joseph PROIETTO
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Golgi Apparatus ◽  
High Glucose ◽  
Marked Decrease ◽  
High Density ◽  
Major Protein ◽  
Low Density ◽  
Concomitant Increase ◽  
First Time

GLUT4-containing vesicles are constantly cycling in both basal and insulin-stimulated states. Our previous studies have shown that basal cycling of GLUT4 is impaired under conditions of high glucose or glucosamine and, as a consequence, GLUT4 is retained intracellularly in low-density microsomes [Filippis A., Clark, S., and Proietto, J. (1997) Biochem. J. 324, 981-985]. In addition to GLUT4 itself, a major protein component of GLUT4-containing vesicles is a glycoprotein of Mr 160000 (gp160). In all studies so far published gp160 has been co-localized with GLUT4 under all conditions. In this study, we show that retention of GLUT4 in low-density microsomes (enriched in Golgi apparatus) is associated with a decrease in gp160 levels in this compartment. A concomitant increase of gp160 in high-density microsomes (enriched in endoplasmic reticulum), demonstrates for the first time a dissociation in the localization of gp160 and GLUT4. Despite the marked decrease in gp160 levels in the GLUT4-containing compartment, insulin-stimulated translocation was normal, while little gp160 appeared in the plasma membrane in response to insulin. The retention of gp160 in the high-density microsomes is apparently not due to a change in the glycosylation state of gp160 as measured by [3H]mannose incorporation. It is concluded that, in rat adipocytes, gp160 is not required for insulin-stimulated translocation, but may be necessary for constitutive trafficking of the GLUT4-containing vesicle.

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