scholarly journals Isolation of a matrix that binds medial Golgi enzymes

1994 ◽  
Vol 124 (4) ◽  
pp. 405-413 ◽  
Author(s):  
P Slusarewicz ◽  
T Nilsson ◽  
N Hui ◽  
R Watson ◽  
G Warren
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Dissociation Constant ◽  
Rat Liver ◽  
Proteinase K ◽  
Apparent Dissociation Constant ◽  
Low Salt ◽  
The Matrix ◽  
Matrix Removal ◽  
Triton X

Rat liver Golgi stacks were extracted with Triton X-100 at neutral pH. After centrifugation the low speed pellet contained two medial-Golgi enzymes, N-acetylglucosaminyltransferase I and mannosidase II, but no enzymes or markers from other parts of the Golgi apparatus. Both were present in the same structures which appeared, by electron microscopy, to be small remnants of cisternal membranes. The enzymes could be removed by treatment with low salt, leaving behind a salt pellet, which we term the matrix. Removal of salt caused specific re-binding of both enzymes to the matrix, with an apparent dissociation constant of 3 nM for mannosidase II. Re-binding was abolished by pretreatment of intact Golgi stacks with proteinase K, suggesting that the matrix was present between the cisternae.

scholarly journals The topographical location and unique nature of a glucokinase associated with the Golgi apparatus of rat liver

1976 ◽  
Vol 154 (1) ◽  
pp. 193-201 ◽  
Author(s):  
G Berthillier ◽  
R Coleman ◽  
D G Walker
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Freezing And Thawing ◽  
Golgi Membranes ◽  
Unique Nature ◽  
Triton X

A particulate glucokinase was recovered in the Golgi-rich fraction of rat liver prepared by the method of Morré [Methods Enzymol. (1971) 22, 130-148], thus extending the demonstration by Berthillier et al. [Biochim. Biophys. Acta (1973), 293, 370-378] of particulate glucokinase activity in a microsomal subfraction that showed enrichment in Golgi characteristics. The purity of this fraction was examined and it was then subjected to several treatments, the action of Triton X-100, freezing and thawing, and sonication to establish the topographical location of the glucokinase activity thus solubilized. The evidence suggests that the glucokinase activity is either soluble in the lumen of the Golgi apparatus or loosely associated with the inside of the Golgi membranes.

scholarly journals ELECTRON MICROSCOPY OF THE HUMAN SYNOVIAL MEMBRANE

1962 ◽  
Vol 14 (2) ◽  
pp. 207-220 ◽  
Author(s):  
Peter Barland ◽  
Alex B. Novikoff ◽  
David Hamerman
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Synovial Membrane ◽  
Current Knowledge ◽  
Cell Types ◽  
Type B ◽  
Joint Cavity ◽  
The Matrix ◽  
And Function ◽  
Cytoplasmic Processes

The structure of the lining cells at the surface of the synovial membrane facing the joint cavity has been studied by electron microscopy. The long cytoplasmic processes of these cells appear to be oriented toward the surface of the membrane, where they overlap and intertwine. The matrix of the lining cells contains dense material but no fibers with the periodicity of collagen. The lining cells are divided into two cell types or states of activity on the basis of their cytoplasmic contents. Type A is more numerous and contains a prominent Golgi apparatus, numerous vacuoles (0.4 to 1.5 microns in diameter) containing varying amounts of a dense granular material, many filopodia, mitochondria, intracellular fibrils, and micropinocytotic-like vesicles. Type B contains large amounts of ergastoplasm with fewer large vacuoles, micropinocytotic-like vesicles, and mitochondria. The probable functions of these cells are discussed in the light of current knowledge of the metabolism and function of the synovial membrane.

scholarly journals Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus.

1981 ◽  
Vol 89 (2) ◽  
pp. 246-255 ◽  
Author(s):  
B Fleischer
Keyword(s):  
Golgi Apparatus ◽  
Rat Liver ◽  
Three Dimensional ◽  
Small Molecular Weight ◽  
Protein Substrates ◽  
Molar Ratios ◽  
Golgi Vesicles ◽  
Luminal Side ◽  
Triton X ◽  
Cis And Trans

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP-sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.

scholarly journals Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 541-547 ◽  
Author(s):  
K E Creek ◽  
D J Morré ◽  
C S Silverman-Jones ◽  
Y Shidoji ◽  
L M De Luca
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Subcellular Fractions ◽  
Transfer Activity ◽  
Dolichyl Phosphate ◽  
Endoplasmic Reticulum Protein ◽  
Triton X

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

scholarly journals COMBINED CYTOCHEMICAL AND ELECTRON MICROSCOPIC DEMONSTRATION OF ß-GLUCURONIDASE ACTIVITY IN RAT LIVER WITH THE USE OF A SIMULTANEOUS COUPLING AZO DYE TECHNIQUE

1968 ◽  
Vol 36 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Masando Hayashi ◽  
Tsuranobu Shirahama ◽  
Alan S. Cohen
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Dense Body ◽  
Electron Microscopic ◽  
Glucuronidase Activity ◽  
Dense Bodies ◽  
The Matrix ◽  
Matrix Control ◽  
Parenchymal Cells ◽  
Cytochemical Technique

Rat liver was fixed in formal-cacodylate-sucrose and frozen sections were incubated in a simultaneous-coupling medium containing naphthol AS-BI glucuronide as substrate and hexazonium pararosanilin as the diazo reagent. By light microscopy, the sections demonstrated ß-glucuronidase activity as red discrete granules in the pericanalicular cytoplasm and as a generalized cytoplasmic stain in the parenchymal cells. Brief treatment of sections in cold ethanol prior to incubation markedly enhanced the staining for the enzyme and made it possible to demonstrate sufficient amounts of the reaction product in sections embedded in epoxy resin following dehydration and propylene oxide treatment. Electron microscopy revealed that the reaction product was moderately electron opaque and deposited in greater amounts in the vacuolated dense bodies and occasionally in the dense bodies which did not show obvious vacuoles. In each dense body, the deposits occurred preferentially at the edge as well as in the area surrounding the vacuoles in the matrix. Control sections incubated in the presence of glucosaccharo-1:4-lactone were devoid of the reaction product. No deposits of the reaction product were found in the nucleus, mitochondria, or microbodies. The limitations of the present cytochemical technique for use in electron microscopy are briefly discussed.

Detergent Modification of Membranes: Fact or Artifact?

1985 ◽  
Vol 43 ◽  
pp. 558-559
Author(s):  
Margaret E. Hogan ◽  
Karen K. Baker ◽  
Stanley Flegler
Keyword(s):  
Electron Microscopy ◽  
Critical Micelle Concentration ◽  
Lipid Bilayer ◽  
Rat Liver ◽  
Tween 20 ◽  
Isolated Rat Liver ◽  
Critical Micelle Concentrations ◽  
Liver Nuclei ◽  
Triton X ◽  
Isolated Rat

Detergents have been used to modify biological membranes from nuclear envelopes and organelles to cellular bilayers of many cultured organisms. These modifications, usually designed to permeabilize or strip away the lipid bilayer, are as varied as the types of detergents used. The most commonly used agents are those with low critical micelle concentrations (Triton X-100, Tween-20, NP-40) making them difficult to completely remove from the reaction mixture. Octyl-POE (polydisperse octyloligooxyethylene), a detergent of high critical micelle concentration, is dialyzable and has been used in hopes of eliminating the possible masking effects of residual detergent in electron microscopy preparations. Through the use of a common treatment for all test solutions, a comparison of Triton X-100, NP-40, octyl-POE and a buffered solution as a control has been performed on isolated rat liver nuclei.

scholarly journals A microtubule-binding protein associated with membranes of the Golgi apparatus.

1986 ◽  
Vol 103 (6) ◽  
pp. 2229-2239 ◽  
Author(s):  
V J Allan ◽  
T E Kreis
Keyword(s):  
Golgi Apparatus ◽  
Culture Cell ◽  
Proteinase K ◽  
Microtubule Network ◽  
Microtubule Binding ◽  
Golgi Stack ◽  
Cell Extracts ◽  
Triton X

A monoclonal antibody (M3A5), raised against microtubule-associated protein 2 (MAP-2), recognized an antigen associated with the Golgi complex in a variety of non-neuronal tissue culture cells. In double immunofluorescence studies M3A5 staining was very similar to that of specific Golgi markers, even after disruption of the Golgi apparatus organization with monensin or nocodazole. M3A5 recognized one band of Mr approximately 110,000 in immunoblots of culture cell extracts; this protein, designated 110K, was enriched in Golgi stack fractions prepared from rat liver. The 110K protein has been shown to partition into the aqueous phase by Triton X-114 extraction of a Golgi-enriched fraction and was eluted after pH 11.0 carbonate washing. It is therefore likely to be a peripheral membrane protein. Proteinase K treatment of an isolated Golgi stack fraction resulted in complete digestion of the 110K protein, both in the presence and absence of Triton X-100. A the 110K protein is accessible to protease in intact vesicles in vitro, it is presumably located on the cytoplasmic face of the Golgi membrane in vivo. The 110K protein was able to interact specifically with taxol-polymerized microtubules in vitro. These results suggest that the 110K protein may serve to link the Golgi apparatus to the microtubule network and so may belong to a novel class of proteins: the microtubule-binding proteins.

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

1970 ◽  
Vol 44 (3) ◽  
pp. 484-491 ◽  
Author(s):  
D. James Morré ◽  
R. L. Hamilton ◽  
H. H. Mollenhauer ◽  
R. W. Mahley ◽  
W. P. Cunningham ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Complex Network ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Rat Hepatocytes ◽  
Secretory Vesicles ◽  
Differential Centrifugation

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.

Electron microscopy and diffraction studies of polyimides

1983 ◽  
Vol 41 ◽  
pp. 28-29
Author(s):  
O.C. de Hodgins ◽  
K. R. Lawless ◽  
R. Anderson
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Structural Features ◽  
Inert Atmosphere ◽  
Polyimide Films ◽  
Resolution Electron ◽  
The Matrix ◽  
High Resolution Electron Microscope ◽  
Diffraction Patterns ◽  
Polymeric Samples

Commercial polyimide films have shown to be homogeneous on a scale of 5 to 200 nm. The observation of Skybond (SKB) 705 and PI5878 was carried out by using a Philips 400, 120 KeV STEM. The objective was to elucidate the structural features of the polymeric samples. The specimens were spun and cured at stepped temperatures in an inert atmosphere and cooled slowly for eight hours. TEM micrographs showed heterogeneities (or nodular structures) generally on a scale of 100 nm for PI5878 and approximately 40 nm for SKB 705, present in large volume fractions of both specimens. See Figures 1 and 2. It is possible that the nodulus observed may be associated with surface effects and the structure of the polymers be regarded as random amorphous arrays. Diffraction patterns of the matrix and the nodular areas showed different amorphous ring patterns in both materials. The specimens were viewed in both bright and dark fields using a high resolution electron microscope which provided magnifications of 100,000X or more on the photographic plates if desired.

Site of Lysosome Production During Hepatic Injury

1969 ◽  
Vol 27 ◽  
pp. 348-349
Author(s):  
Nalin J. Unakar
Keyword(s):  
Electron Microscopy ◽  
Golgi Apparatus ◽  
Mouse Liver ◽  
Hepatic Injury ◽  
Close Approximation ◽  
Experimental Conditions ◽  
Toxic Injury

The increased number of lysosomes as well as the close approximation of lysosomes to the Golgi apparatus in tissue under variety of experimental conditions is commonly observed. These observations suggest Golgi involvement in lysosomal production. The role of the Golgi apparatus in the production of lysosomes in mouse liver was studied by electron microscopy of liver following toxic injury by CCI4.

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