scholarly journals Two enzymes involved in the synthesis of O-linked oligosaccharides are localized on membranes of different densities in mouse lymphoma BW5147 cells.

1984 ◽  
Vol 99 (1) ◽  
pp. 327-331 ◽  
Author(s):  
A Elhammer ◽  
S Kornfeld
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Marker Enzymes ◽  
Continuous Sucrose Gradient ◽  
Microsomal Membranes ◽  
Intermediate Density ◽  
Mouse Lymphoma

Microsomal membranes from mouse lymphoma BW5147 cells were fractionated on a continuous sucrose gradient and assayed for two enzymes involved in the synthesis of O-linked oligosaccharides. Both enzymes were recovered in membranes that were less dense than the membranes containing the endoplasmic reticulum marker enzymes, glucosidase I and II. UDP-Gal:N-acetylgalactosamine-beta 1, 3-galactosyltransferase had a distribution that coincided with that of the galactosyltransferase that acts on asparagine-linked oligosaccharides. This latter enzyme has been immunolocalized to the trans Golgi elements. The UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase was recovered in a membrane fraction of intermediate density, between the endoplasmic reticulum and trans Golgi markers. These findings are consistent with the assembly of O-linked oligosaccharides occurring in at least two different Golgi compartments.

scholarly journals LYSOSOMAL PHOSPHOLIPASES A1 AND A2 OF NORMAL AND BACILLUS CALMETTE GUERIN-INDUCED ALVEOLAR MACROPHAGES

1973 ◽  
Vol 56 (3) ◽  
pp. 621-627 ◽  
Author(s):  
Richard C. Franson ◽  
Moseley Waite
Keyword(s):  
Acid Phosphatase ◽  
Alveolar Macrophages ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Marker Enzymes ◽  
Sucrose Gradient Centrifugation ◽  
Control Distribution ◽  
Specific Activities ◽  
Selective Increase ◽  
Phospholipases A

A single intravenous injection of 0.1 mg of heat-killed Bacillus Calmette Guérin (BCG) in 0.1 ml of Bayol F produced an accumulation of activated alveolar macrophages (BCG induced). Cells were collected 3.5–4.0 wk after injection. Phospholipases A and three lysosomal marker enzymes (acid phosphatase, ß-glucuronidase, and lysozyme) were measured in homogenates, and the distribution of the phospholipases A and lysosomal, mitochondrial, and microsomal marker enzymes were examined after sucrose gradient centrifugation of a postnuclear (1,000 g) supernatant. Homogenates of normal and BCG-induced macrophages contained phospholipases A1 and A2 which had optimal activity at pH 4.0 in the presence of 2.0 mM ethylenediaminetetraacetate (EDTA). These activities were inhibited 50–70% by 2.0 mM CaCl2. Homogenates of BCG-induced macrophages had specific activities of ß-glucuronidase, acid phosphatase, and lysozyme, which were increased 1.5- to 3.0-fold over the controls, whether expressed as activity per mg protein or activity per 107 cells. The specific activities of the phospholipases A, on the other hand, were consistently lower than those of the control. Distribution of the phospholipases A and the lysosomal marker enzymes after sucrose gradient centrifugation suggested that the phospholipases A active at pH 4.0 in the presence of EDTA are of lysosomal origin since: (a) BCG treatment caused a selective increase in the density of particles which contained both the phospholipases A and three lysosomal marker enzymes; and (b) since the density of mitochondria and microsomes were not affected by BCG treatment. The increase in the density of lysosomes seen here may be related to previously described morphologic changes of BCG-induced alveolar macrophages.

scholarly journals The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis

1967 ◽  
Vol 105 (2) ◽  
pp. 783-801 ◽  
Author(s):  
J. R. Tata
Keyword(s):  
Endoplasmic Reticulum ◽  
Ribosomal Proteins ◽  
Specific Activity ◽  
Tail Length ◽  
Similar Response ◽  
Carbamoyl Phosphate ◽  
Microsomal Membranes ◽  
Stimulation Of ◽  
Induction Of Metamorphosis

1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of 32P into the different phospholipid constituents of microsomal membranes. 7. Nascent 14C-labelled protein with the highest specific activity was recovered in the ‘heavy’ rough membrane fraction of microsomes, whereas little 14C was associated with ‘free’ polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7–8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis.

scholarly journals Subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase in rat liver

1989 ◽  
Vol 257 (1) ◽  
pp. 221-229 ◽  
Author(s):  
L Schepers ◽  
M Casteels ◽  
K Verheyden ◽  
G Parmentier ◽  
S Asselberghs ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholic Acid ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
The Other ◽  
Acid Activation ◽  
Long Chain Fatty Acids ◽  
Microsomal Membranes ◽  
Triton X ◽  
Long Chain

The subcellular distribution and characteristics of trihydroxycoprostanoyl-CoA synthetase were studied in rat liver and were compared with those of palmitoyl-CoA synthetase and choloyl-CoA synthetase. Trihydroxycoprostanoyl-CoA synthetase and choloyl-CoA synthetase were localized almost completely in the endoplasmic reticulum. A quantitatively insignificant part of trihydroxycoprostanoyl-CoA synthetase was perhaps present in mitochondria. Peroxisomes, which convert trihydroxycoprostanoyl-CoA into choloyl-CoA, were devoid of trihydroxycoprostanoyl-CoA synthetase. As already known, palmitoyl-CoA synthetase was distributed among mitochondria, peroxisomes and endoplasmic reticulum. Substrate- and cofactor- (ATP, CoASH) dependence of the three synthesis activities were also studied. Cholic acid and trihydroxycoprostanic acid did not inhibit palmitoyl-CoA synthetase; palmitate inhibited the other synthetases non-competitively. Likewise, cholic acid inhibited trihydroxycoprostanic acid activation non-competitively and vice versa. The pH curves of the synthetases did not coincide. Triton X-100 affected the activity of each of the synthetases differently. Trihydroxycoprostanoyl-CoA synthetase was less sensitive towards inhibition by pyrophosphate than choloyl-CoA synthetase. The synthetases could not be solubilized from microsomal membranes by treatment with 1 M-NaCl, but could be solubilized with Triton X-100 or Triton X-100 plus NaCl. The detergent-solubilized trihydroxycoprostanoyl-CoA synthetase could be separated from the solubilized choloyl-CoA synthetase and palmitoyl-CoA synthetase by affinity chromatograpy on Sepharose to which trihydroxycoprostanic acid was bound. Choloyl-CoA synthetase and trihydroxycoprostanoyl-CoA synthetase could not be detected in homogenates from kidney or intestinal mucosa. The results indicate that long-chain fatty acids, cholic acid and trihydroxycoprostanic acid are activated by three separate enzymes.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Fractionation of isolated liver cells after disruption with a nitrogen bomb and sonication

1982 ◽  
Vol 57 (1) ◽  
pp. 1-13
Author(s):  
F. Autuori ◽  
U. Brunk ◽  
E. Peterson ◽  
G. Dallner
Keyword(s):  
Rat Liver ◽  
Sucrose Gradient ◽  
Divalent Cations ◽  
Liver Cells ◽  
Cell Disruption ◽  
Isolated Liver Cells ◽  
Discontinuous Sucrose Gradient ◽  
Microsomal Membranes ◽  
Isolated Liver

Hepatocytes from rat liver were prepared by perfusion with collagenase, and rough and smooth microsomes and mitochondria were prepared after cell disruption. By applying 1000 lb/in2 (1 lb/in2 = 6.9 kPa) in a nitrogen bomb followed by decompression, 75% of the cells were disrupted after four consecutive treatments. Intact mitochondria, and rough and smooth microsomes with little contamination were prepared from the homogenate. A more rapid disruption was attained by a short sonication with a low output, thus increasing the efficiency of homogenization. The microsomal subfractions prepared from this homogenate were comparable to those obtained after decompression. Sonication resulted in smooth microsomes, which exhibited a higher contamination with non-microsomal membranes. These, however, were partly removed by additional centrifugation with a discontinuous sucrose gradient containing divalent cations.

Gel filtration of marker enzymes of plasma membrane and endoplasmic reticulum from rat liver

1969 ◽  
Vol 193 (2) ◽  
pp. 468-471 ◽  
Author(s):  
Kenji Nakai ◽  
Shigehide Takemitsu ◽  
Toshisuke Kawasaki ◽  
Ikuo Yamashina
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gel Filtration ◽  
Marker Enzymes

scholarly journals ADENOSINE TRIPHOSPHATE-LINKED CONCENTRATION OF CALCIUM IONS IN A PARTICULATE FRACTION OF RABBIT MUSCLE

1962 ◽  
Vol 14 (3) ◽  
pp. 389-400 ◽  
Author(s):  
Setsuro Ebashi ◽  
Fritz Lipmann
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Electron Micrographs ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Membrane Fraction ◽  
Ion Concentration ◽  
Rabbit Muscle ◽  
Differential Centrifugation ◽  
Calcium Ion Concentration

ATPase and ATP-dependent calcium ion concentration was studied with a membrane fraction isolated from homogenized rabbit skeletal muscle by differential centrifugation. Electron micrographs of the fraction indicate that it consists mainly of resealed tubules and vesicles of the endoplasmic reticulum. The up-to-1400-fold concentration of calcium in this fraction might be explained by proposing the existence of an energy-requiring system for the transport of calcium ions into the tubules or vesicles.

Biosynthesis of microsomal phospholipids and mitochondrial polyglycerophosphatides in rapidly sedimenting endoplasmic reticulum

1982 ◽  
Vol 60 (9) ◽  
pp. 877-881 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Neutral Lipids ◽  
Sucrose Density Gradient ◽  
Marker Enzymes ◽  
Cytochrome C Reductase ◽  
Level Of Activity ◽  
Succinate Cytochrome C Reductase ◽  
Experimental Findings

Rapidly sedimenting endoplasmic reticulum (RSER), which is known to be a complex between endoplasmic reticulum and mitochondria, was isolated from rat liver and purified through a sucrose density gradient by centrifugation according to a well established procedure previously published by G. C. Shore and J. R. Tata. This complex was characterized by microsomal (NADPH–cytochrome c reductase) and mitochondrial (succinate–cytochrome c reductase and NADP–isocitrate dehydrogenase) marker enzymes and was examined for the ability to synthesize microsomal lipids and mitochondrial polyglycerophosphatides. Results of these experiments showed that the RSER is capable of synthesizing key microsomal lipids, i.e., phosphatide acid, phosphatidylcholine, and neutral lipids, as well as mitochondrial phosphatidylglycerophosphate and phosphatidyiglycerol. Furthermore, the level of synthesis of these lipids paralleled the level of activity of microsomal and mitochondrial marker enzymes found in the RSER preparation. Details of these experimental findings and some implications are discussed in view of the possible functional role of RSER.

scholarly journals Immunoelectron microscope localization of cytochrome P-450 on microsomes and other membrane structures of rat hepatocytes.

1978 ◽  
Vol 78 (2) ◽  
pp. 503-519 ◽  
Author(s):  
S Matsuura ◽  
Y Fujii-Kuriyama ◽  
Y Tashiro
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membranes ◽  
Control Level ◽  
Rat Hepatocytes ◽  
Present Observation ◽  
Membrane Structures ◽  
Rat Liver Cells ◽  
Microsomal Membranes ◽  
Almost All ◽  
Cytochrome P 450

Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.

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