scholarly journals Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver

1974 ◽  
Vol 142 (3) ◽  
pp. 667-671 ◽  
Author(s):  
Kenneth A. Norris ◽  
Miloslav Dobrota ◽  
Faiz S. Issa ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Heterogeneous Distribution ◽  
Similar Product ◽  
Liver Homogenates

Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5′-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product.

scholarly journals Subcellular fractionation of rat liver homogenates using two-polymer phase systems in a toroidal-coil centrifuge

1984 ◽  
Vol 217 (3) ◽  
pp. 751-759 ◽  
Author(s):  
D Heywood-Waddington ◽  
I A Sutherland ◽  
W B Morris ◽  
T J Peters
Keyword(s):  
Plasma Membrane ◽  
Ethylene Glycol ◽  
Rat Liver ◽  
Flow Rate ◽  
Marker Enzyme ◽  
Poly Ethylene Glycol ◽  
Lower Phase ◽  
Liver Homogenates ◽  
Toroidal Coil ◽  
Poly Ethylene

The principal organelles of rat liver homogenates were fractionated by two-phase partition chromatography using toroidal-coil centrifugation with a mixture of dextran T 500 and poly(ethylene glycol) 6000 in 0.26 M-sucrose containing 10 mM-sodium phosphate/phosphoric acid buffer, pH 7.4. The effects of varying the following parameters on organelle elution profiles, as reflected by their marker-enzyme activities, were studied: centrifuge speed; the composition and relative proportion of dextran-rich and poly(ethylene glycol)-rich phases in the eluent; flow rate; sample volume; homogenate concentration; helix diameter; tubing bore and the number of loops in the coil. Optimal resolution of the organelles was achieved with a toroidal coil of internal diameter 1.07 mm with a 4.55 mm helix diameter on a 0.42 m-diameter rotor running at 1000 rev./min. The eluent was prepared by combining, in a ratio of 93:7 (v/v), the poly(ethylene glycol)-rich upper phase and dextran-rich lower phase obtained from a phase mixture containing 3.3% (w/w) dextran and 5.4% (w/w) poly(ethylene glycol). The flow rate of the eluent was 14ml/h. Optimal conditions for separation of the organelles were evaluated. Resolution of plasma membrane and lysosomes was achieved. Separation of endoplasmic reticulum, which showed marked heterogeneity, from plasma membrane was also demonstrated. DNA and marker enzymes for peroxisomes, mitochondria and cytosol showed distinct elution profiles.

scholarly journals The heterogeneous distribution of acid hydrolases within a homogeneous population of cultured mammalian cells

1973 ◽  
Vol 132 (3) ◽  
pp. 493-500 ◽  
Author(s):  
Judith P. Milsom ◽  
C. H. Wynn
Keyword(s):  
Rat Liver ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Sucrose Density Gradient ◽  
Acid Hydrolases ◽  
Heterogeneous Distribution ◽  
Homogeneous Population ◽  
Homogeneous Cell ◽  
Liver Homogenates ◽  
Isopycnic Centrifugation

1. Chinese-hamster ovary fibroblasts were cultured to provide a homogeneous cell population. Homogenates obtained from these cells were fractionated by centrifugation techniques and the resulting fractions were analysed for protein and for enzymes representative of certain subcellular particles. 2. Unlike those in rat liver homogenates, the mitochondrial and lysosomal populations proved impossible to separate by differential centrifugation owing to the similarity of their sedimentation properties. Their resolution was possible by using isopycnic centrifugation in a continuous sucrose density gradient. 3. The mitochondrial population equilibrated at a density of 1.17g·cm−3 as in rat liver homogenates. However, the lysosomal population equilibrated at a lower rather than a higher density position than the mitochondria and the probable reasons for this are discussed. 4. The lysosomal population subdivided into two groups characterized by differences in acid hydrolase content and equilibrium densities. The fraction with a density of 1.15g·cm−3 contained the majority of arylsulphatases A and B, of cathepsin and of β-acetylglucosaminidase activities, whereas that with a density of 1.09g·cm−3 contained the majority of the acid phosphatase and acid ribonuclease activities. The probable division of the lysosomal population of a single cell into a number of distinguishable subgroups is suggested.

scholarly journals GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

1973 ◽  
Vol 59 (1) ◽  
pp. 73-88 ◽  
Author(s):  
J. J. M. Bergeron ◽  
J. H. Ehrenreich ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Companion Paper ◽  
Transferase Activity ◽  
Ethanol Treatment ◽  
Liver Homogenates ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6–7% of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction (GF1) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF3) is contaminated by endoplasmic reticulum membranes to the extent of ∼15% of its protein. The three fractions taken together account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for ∼70% of the activity of the original homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.

scholarly journals Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

1971 ◽  
Vol 125 (2) ◽  
pp. 545-555 ◽  
Author(s):  
Patience C. Barrow ◽  
S. J. Holt
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Intact Cell ◽  
Cellular Damage ◽  
Phase System ◽  
Two Phase ◽  
Liver Homogenates ◽  
Cell Fractions ◽  
Standard Fractionation

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113–0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.

scholarly journals Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups

1976 ◽  
Vol 157 (2) ◽  
pp. 479-482 ◽  
Author(s):  
T J Visser ◽  
I Does-Tobé ◽  
R Docter ◽  
G Hennemann
Keyword(s):  
Rat Liver ◽  
Essential Role ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Enzymic Activity ◽  
Marker Enzyme ◽  
Converting Enzyme ◽  
Thiol Groups ◽  
Liver Homogenates

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

scholarly journals Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1969 ◽  
Vol 113 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Elizabeth H. Shephard ◽  
G. Hübscher
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Microsomal Fraction ◽  
Mitochondrial Fraction ◽  
Urate Oxidase ◽  
Outer Mitochondrial Membrane ◽  
Short Term ◽  
Conventional Fractionation ◽  
Liver Homogenates ◽  
Nadh Cytochrome

1. After conventional fractionation of rat liver homogenates in 0·88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.

scholarly journals Biosynthesis of liver membranes. Incorporation of [3H]leucine into proteins and of [14C]glucosamine into proteins and lipids of liver microsomal and plasma-membrane fractions

1971 ◽  
Vol 125 (2) ◽  
pp. 615-624 ◽  
Author(s):  
W. H. Evans ◽  
J. W. Gurd
Keyword(s):  
Plasma Membrane ◽  
Mouse Liver ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Protein Ratio ◽  
Glycolipid Synthesis ◽  
Liver Membranes ◽  
Liver Homogenates ◽  
Chloroform Methanol ◽  
Soluble Components

1. The smooth-and rough-microsomal and the light and heavy plasma-membrane fractions of mouse liver homogenates were prepared and characterized by using biochemical markers. 2. The hexosamine/protein ratio was threefold higher in the plasma membranes than in the smooth-microsomal fraction. Glucosamine was bound only to protein, and galactosamine was attached mainly to lipids. 3. [3H]-Leucine and [14C]glucosamine were injected into animals and the rates of incorporation of radioactivity into the fractions were determined. Both precursors were rapidly incorporated into the microsomal fractions, but plasma membranes showed a slower rate of synthesis which reached a maximum at 2–4h after intravenous administration. 4. The light- and heavy-plasma-membrane fractions showed similar patterns of incorporation, and therefore a precursor–product relationship appears unlikely. 5. Plasma membranes, especially the light subfraction, showed appreciable incorporation of hexosamine into chloroform–methanol-soluble components which were shown to be mainly glycolipids. 6. The results indicate that liver plasma-membrane proteins and glycoproteins are synthesized at similar rates. However, glycolipid synthesis in plasma membranes occurred more rapidly.

scholarly journals GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

1973 ◽  
Vol 59 (1) ◽  
pp. 45-72 ◽  
Author(s):  
J. H. Ehrenreich ◽  
J. J. M. Bergeron ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Liver Homogenates ◽  
Main Component

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ∼90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ∼13 x 106 g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.

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