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.

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 Fractionation of rat liver plasma-membrane regions by two-phase partitioning

1986 ◽  
Vol 235 (3) ◽  
pp. 685-691 ◽  
Author(s):  
P Gierow ◽  
M Sommarin ◽  
C Larsson ◽  
B Jergil
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Phase System ◽  
Two Phase ◽  
Phase Partitioning ◽  
Aqueous Polymer ◽  
Counter Current

Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5′-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
Author(s):  
A. R. EASTMAN ◽  
A. M. NEVILLE
Keyword(s):  
Molecular Weight ◽  
Adrenal Glands ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Molecular Sizes ◽  
Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

The Differentiation of Acid Phosphatases of Human Blood Platelets

1971 ◽  
Vol 26 (02) ◽  
pp. 353-361 ◽  
Author(s):  
H. D Kaulen ◽  
R Gross
Keyword(s):  
Rat Liver ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Human Platelets ◽  
Acid Phosphatases ◽  
Sucrose Density Gradient Centrifugation ◽  
Lysosomal Fraction ◽  
Hydrolysis Of ◽  
Human Blood Platelets

SummaryThe Acid Phosphatase was tested in human platelets and in the rat liver mitochondrial-lysosomal fraction with p-nitrophenylphosphate and β-glycerophosphate as substrates. In the platelets the following differences were found between the hydrolysis of these two substrates, whereas in rat liver no such differences were observed. 1. The relative rates of hydrolysis and the pH optima for both substrates are different (pH 4.6 for the β-glycerophosphatase, pH 6.0 for the p-nitrophenylphosphatase in the platelets). 2. The p-nitrophenylphosphatase of the platelets is inhibited by p-chlormercuribenzoate and N-ethylmaleimide, but not by fluoride or L + tartrate, whereas the contrary is true for the platelet β-glycerophosphatase and the rat liver activities. 3. The platelet p-nitrophenylphosphatase is rapidly inactivated by preincubation at 40-45° C for 15 min, the other phosphatases are much more heat-resistant. 4. Sucrose density gradient centrifugation of platelet homgenates showed a separation of the two platelet phosphatase activities, the p-nitrophenylphosphatase with its maximum at lower densities than the β-glycerophosphatase.It is concluded that in human platelets there are at least two different Acid Phosphatases. The β-glycerophosphatase probably represents the lysosomal (as compared to the rat liver enzyme) phosphatase whereas the p-nitrophenylphosphatase of the platelets is a different enzyme whose subcellular localization and functions are as yet unknown.

scholarly journals Purification, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1973 ◽  
Vol 58 (2) ◽  
pp. 436-462 ◽  
Author(s):  
Gert Kreibich ◽  
Pascale Debey ◽  
David D. Sabatini
Keyword(s):  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Transport Systems ◽  
Secretory Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Detergent Concentration ◽  
Doc Concentration ◽  
Microsomal Membranes

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

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.

PLASMA MEMBRANE ATPASE ISOLATED FROM GREEN BELL PEPPER FRUIT BY TWO-PHASE PARTITIONING OR SUCROSE DENSITY GRADIENT

1994 ◽  
Vol 42 (1) ◽  
pp. 15-27
Author(s):  
R. Ronen ◽  
Z. Lipsker ◽  
L. Sonego ◽  
Susan Lurie
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Bell Pepper ◽  
Phase System ◽  
Plasma Membrane Atpase ◽  
Two Phase ◽  
Phase Partitioning ◽  
Pepper Fruit ◽  
Membrane Atpase

Plasma membrane was isolated from mature green bell pepper fruit by two-phase partitioning or by sucrose density gradient. The yield of plasma membrane was higher from the sucrose density gradient, but the two-phase system was less contaminated by other membranes, particularly those from chloroplasts and mitochondria. In the two-phase partitioned membranes, ATPase activity was stimulated by Triton X-100 by 100% and in sucrose density gradient membranes by 40%. Plasma membranes from two-phase partitioning exhibited simultaneous proton pumping and ATP hydrolysis, while the sucrose density purified membranes did not. Immunoblotting with ATPase antibody showed enrichment of plasma membrane ATPase in both the U3 phase of the two-phase system, and the 34% sucrose fraction of the sucrose gradient. However, the two-phase partitioned membranes were superior to those prepared by sucrose density for investigating functions of the ATPase.

scholarly journals Fractionation of microsomal membranes on the basis of their surface properties

1978 ◽  
Vol 172 (1) ◽  
pp. 189-192 ◽  
Author(s):  
R Ohlsson ◽  
B Jergil ◽  
H Walter
Keyword(s):  
New York ◽  
Surface Properties ◽  
Gradient Centrifugation ◽  
Membrane Surface ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Membrane Surfaces ◽  
Microsomal Membranes ◽  
Poly Ethylene ◽  
Rat Liver Microsome

Partition in dextran-poly(ethylene glycol) aqueous-phase systems can be used for both membrane subfractionation and gaining information on membrane surface properties [H. Walter (1977) in Methods of Cell Separation (Catsimpoolas, N., ed.), vol. 1, pp. 307-354, Plenum, New York]. Smooth, light rough and heavy rough rat liver microsome (obtained by sucrose-density-gradient centrifugation) were subjected to countercurrent distribution in such a system. Smooth microsomal membranes had the highest, heavy rough microsomal membranes the lowest and light rough microsomal membranes an intermediate partition coefficient. The separation is based primarily on hydrophobic differences in the membrane surfaces of the three preparations and is thus due to microsomal properties not previously utilized in their fractionation. The method permits additional subfractionations of microsomes.

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