Analytical Subcellular Fractionation Studies on Rat Liver and on Isolated Jejunal Enterocytes with Special Reference to the Separation of Lysosomes, Peroxisomes and Mitochondria

1976 ◽  
Vol 50 (5) ◽  
pp. 355-366 ◽  
Author(s):  
T. J. Peters ◽  
H. Shio
Keyword(s):  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Low Molecular Weight ◽  
Rat Jejunum ◽  
Marker Enzymes ◽  
Good Separation ◽  
Subcellular Organelles ◽  
Rat Liver Cells ◽  
Isopycnic Centrifugation

1. Enterocytes were isolated from rat jejunum and characterized morphologically. 2. Attempts to separate the enterocyte subcellular organelles, characterized by their marker enzymes, with isopycnic centrifugation were unsuccessful but good separation of peroxisomes, lysosomes and mitochondria was achieved by sedimentation through a shallow sucrose density gradient with a superimposed inverse gradient of low-molecular-weight dextran. 3. The properties and enzyme activities of the principal subcellular organelles in rat liver cells and enterocytes were compared.

Analytical Subcellular Fractionation Studies on Different Cell Types Isolated from Normal Rat Liver

1978 ◽  
Vol 55 (5) ◽  
pp. 423-427
Author(s):  
Clare Selden ◽  
A. M. Wootton ◽  
D. W. Moss ◽  
T. J. Peters
Keyword(s):  
Biliary Tract ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Marker Enzymes ◽  
Sucrose Density Gradient Centrifugation ◽  
Normal Rat ◽  
Parenchymal Cells

1. Parenchymal, Kupffer and biliary tract cells were isolated from normal rat liver by perfusion with collagenase solution. 2. The specific activities (munits of enzyme activity/mg of protein) of marker enzymes for the principal subcellular organelles were determined in the isolated cell homogenates and compared with whole liver homogenates. 3. The cells were disrupted and the extracts subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation. Lysosomal integrity was determined by assaying latent β-N-acetylglucosaminidase in the extracts. 4. Similar subcellular distributions were found for lysosomal, endoplasmic reticulum and plasma membrane marker enzymes in the whole liver and in parenchymal and biliary tract cells. In Kupffer cells, the proportion of these enzymes in the cytosol was significantly increased compared with the other fractions. In addition the equilibrium densities of the various organelles in these cells were lower than those from parenchymal cells.

scholarly journals Analytical subcellular fractionation of alveolar macrophages from normal and BCG-vaccinated rabbits with particular reference to heterogeneity of hydrolase-containing granules

1979 ◽  
Vol 178 (3) ◽  
pp. 761-767 ◽  
Author(s):  
D B Lowrie ◽  
P W Andrew ◽  
T J Peters
Keyword(s):  
Acid Phosphatase ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Equilibrium Density ◽  
Type B ◽  
Type C ◽  
Pulmonary Granulomas ◽  
B Granules ◽  
Isopycnic Centrifugation

Macrophages were obtained by pulmonary lavage from normal rabbits or rabbits that had developed pulmonary granulomas after receiving intravenous BCG vaccine 2-3 weeks earlier. The cells were disrupted in iso-osmotic sucrose and a low-speed supernatant was fractionated by isopycnic centrifugation on a linear sucrose density gradient. Three populations of hydrolase-containing granules (putative lysosomes) were found in both normal and BCG-induced macrophages. They were distinguished by their different distributions in the gradient and different sensitivities to disruption by digitonin and were termed:type A, containing lysozyme; type B, containing N-acetyl-beta-glucosaminidase, beta-glactosidase, beta-glucuronidase and possibly some lysozyme; type C, containing cathepsin D. Acid phosphatase appeared to be about equally distributed between type B and C granules. Type A and B granules from BCG-induced macrophages showed markedly greater equilibrium density than did those from normal macrophages. Beta-glucuronidase and acid phosphatase had greater specific activity in the induced cells.

Pyrimidine reducing enzymes of rat liver

1976 ◽  
Vol 54 (2) ◽  
pp. 178-184 ◽  
Author(s):  
Ronald O. Hallock ◽  
Esther W. Yamada
Keyword(s):  
Carbon Dioxide ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Marker Enzyme ◽  
Good Separation ◽  
Cytosol Fraction ◽  
Radioactive Carbon ◽  
Mitochondrial Fractions ◽  
Optimum Ph ◽  
Rat Liver Cells

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4.Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH-dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH.Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.

scholarly journals Subcellular localization of ornithine decarboxylase in liver of control and growth-hormone-treated rats

1976 ◽  
Vol 157 (1) ◽  
pp. 33-39 ◽  
Author(s):  
B J Murphy ◽  
M E Brosnan
Keyword(s):  
Growth Hormone ◽  
Ornithine Decarboxylase ◽  
Rat Liver ◽  
Subcellular Fractionation ◽  
Decarboxylase Activity ◽  
Differential Centrifugation ◽  
Marker Enzymes ◽  
Aminotransferase Activity ◽  
Apparent Affinity ◽  
Hormone Administration

1. Ornithine-2-oxo acid aminotransferase activity was inhibited by amino-oxyacetate (10(-5) M). This permitted the measurement of ornithine decarboxylase in the presence of mitochondria by using the 14CO2-trapping technique. 2. Subcellular fractionation of rat liver by differential centrifugation, followed by the assay of ornithine decarboxylase in the presence of amino oxyacetate and of marker enzymes for each fraction, demonstrated that ornithine decarboxylase was located in the cytosol. 3. The greatly increased ornithine decarboxylase activity observed after growth-hormone administration was also found to be localized in the cytosol. 4. The Km of ornithine decarboxylase from rat liver for ornithine was 28 muM. Administration of growth hormone 4 h before death did not affect the apparent affinity of ornithine decarboxylase for ornithine.

scholarly journals Subcellular localization of transglutaminase. Effect of collagen

1988 ◽  
Vol 250 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Juprelle-Soret ◽  
S Wattiaux-De Coninck ◽  
R Wattiaux
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Collagen Type I ◽  
Type I ◽  
Nuclear Fraction ◽  
Binding Studies ◽  
Isopycnic Centrifugation

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.

scholarly journals Subcellular localization and properties of rat liver adenosine diphosphatase

1980 ◽  
Vol 192 (2) ◽  
pp. 527-535 ◽  
Author(s):  
G P Smith ◽  
G D Smith ◽  
T J Peters
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Single Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Outer Membranes ◽  
Selective Membrane

ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme.

Evidence for the presence of a cell-surface ribonuclease in mechanically prepared rat-liver cells in suspension

1982 ◽  
Vol 2 (10) ◽  
pp. 751-760 ◽  
Author(s):  
R. Sirdeshmukh ◽  
P. M. Bhargava
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Rat Liver ◽  
Degradation Products ◽  
Sucrose Density Gradient ◽  
Liver Parenchymal ◽  
Rat Liver Cells ◽  
A Cell ◽  
Parenchymal Cells ◽  
Soluble Material

Rat-liver parenchymal cells obtained in suspension by a mecahnical method are shown to contain a cell-surface nuclease(s) that rapidly degrades exogenously added total Escherichia coli RNA. However, no acid-soluble products are formed; all the degradation products in the incubation medium sediment in the 4–55 RNA region on a sucrose density gradient. A part of the degraded RNA seems to be taken up by the cells; the uptake of the degradation products, presumably derived from rRNAs, is more than that of purified 4–55 RNA. Most of the RNA taken up by the cell sediments in the 4–55 region; only a small proportion is degraded to acid-soluble material within the cell.

scholarly journals Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning

1989 ◽  
Vol 262 (1) ◽  
pp. 55-61 ◽  
Author(s):  
P Gierow ◽  
B Jergil
Keyword(s):  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Phase System ◽  
Marker Enzymes ◽  
Two Phase ◽  
Sucrose Density Gradient Centrifugation ◽  
Phase Partitioning ◽  
Microsomal Membranes

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.

scholarly journals The characterization of rat kidney plasma membranes prepared by zonal centrifugation

1972 ◽  
Vol 129 (4) ◽  
pp. 919-928 ◽  
Author(s):  
R. G. Price ◽  
D. G. Taylor ◽  
D. Robinson
Keyword(s):  
Alkaline Phosphatase ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Rat Kidney ◽  
Marker Enzymes ◽  
Subcellular Organelles ◽  
High Degree ◽  
Isopycnic Centrifugation ◽  
Zonal Rotor

1. Plasma membranes were isolated from a 10000g-min pellet prepared from a renal cortical homogenate in 20mm-NaHCO3 by isopycnic centrifugation in a linear sucrose gradient in an ‘A’-type zonal rotor. 2. The preparation was characterized by electron microscopy, and alkaline phosphatase, 5′-nucleotidase, l-leucine β-naphthylamidase and l-leucine p-nitroanilidase activities were found to be selectively associated with the renal plasma membrane. 3. The preparation had a high degree of purity, as indicated by the presence of low activities of marker enzymes associated with subcellular organelles. A preliminary chemical analysis indicated that the chemical composition resembled that of plasma membranes of other tissues. 4. Plasma membranes were also prepared from tubular fragments and their enzyme contents were found to be similar to those of plasma membranes prepared from cortical homogenates. 5. l-Leucine β-naphthylamidase, l-leucine p-nitroanilidase and 5′-nucleotidase were not enriched to the same extent as alkaline phosphatase in the preparation of plasma membranes from tubular fragments. A possible explanation for this finding is discussed.

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