Uridine phosphorylase activity of isolated plasma membranes of rat liver

1977 ◽  
Vol 55 (5) ◽  
pp. 528-533 ◽  
Author(s):  
Ratna Bose ◽  
Esther W. Yamada
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Enrichment Factors ◽  
Marker Enzyme ◽  
Differential Centrifugation ◽  
Uridine Phosphorylase ◽  
Phosphorylase Activity ◽  
Subcellular Organelles ◽  
Liver Homogenates ◽  
Triton X

Plasma membranes were isolated from rat liver homogenates either by differential centrifugation or by fractionation in discontinuous sucrose density gradients. Both membrane preparations contained about 17% of the total uridine phosphorylase (EC 2.4.2.3) activity and 44% of the total 5′-nucleotidase (EC 3.1.3.5). The enrichment factor for uridine phosphorylase in the fractions prepared by differential centrifugation was about 2.8 and by the gradient method, as much as 11.0; the respective enrichment factors for 5′-nucleotidase were 1.8 and 9.5. Uridine phosphorylase activity of isolated plasma membrane fractions was stimulated 2.5-fold by 0.1% Triton X-100. Unlike the cytosol enzyme, uridine phosphorylase of plasma membranes showed little or no deoxyuridine-cleaving activity. Contamination of the membrane fractions by thymidine phosphorylase (EC 2.4.2.4) of the cytosol was negligible.The other subcellular organelles obtained by either procedure and characterized by marker enzyme activities were found not to contain significant uridine phosphorylase activity; the cytosol fractions contained just over 70% of the total uridine phosphorylase activity with an enrichment of only about 2.8-fold. The activity of the cytosol enzyme was not stimulated by Triton X-100.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

Multivalent interaction between asialofetuin and plasma membrane preparations from the rat liver

1980 ◽  
Vol 58 (12) ◽  
pp. 1414-1420 ◽  
Author(s):  
Maria T. Debanne ◽  
Erwin Regoeczi ◽  
Mark W. C. Hatton
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Theoretical Models ◽  
Underlying Mechanism ◽  
Marker Enzyme ◽  
Electron Microscopic ◽  
Microscopic Appearance ◽  
Wide Range ◽  
Electron Microscopic Appearance

Binding of bovine asialofetuin by rat liver plasma membranes was studied using different techniques for the separation of the free and bound forms of the glycoprotein and also different approaches to measure nonspecific binding. The membrane preparations had the electron microscopic appearance of a mixture of lamellae and vesicles and their lipid:protein ratios and marker enzyme profiles fell within the range of values available from the literature. The binding capacity was approximately 15 pmol of asialofetuin per milligram of membrane protein.Scatchard plots of the values obtained over a wide range of concentrations (4.8–12.6 μg asialofetuin per 30 μg membrane protein) after incubation at 22 °C showed pronounced non-linearity which, in combination with evaluations according to other theoretical models, was referable to heterogeneity of binding. In sharp contrast, after incubation at 4 °C the Scatchard plot was linear. This difference is interpreted as the expression of a functional, rather than a chemical, heterogeneity in asialofetuin binding. The underlying mechanism is thought to be competition of galactose groups for binding sites with the result that the number of bonds varies between the galactose groups of a bound asialofetuin molecule and the hepatic lectin, depending on the concentration of the glycoprotein in the incubation mixture.

scholarly journals ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

1968 ◽  
Vol 37 (1) ◽  
pp. 163-181 ◽  
Author(s):  
Paul D. Sadowski ◽  
Janet Alcock Howden
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Orotic Acid ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Differential Centrifugation ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Triton X

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-14C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-14C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.

The formation of bilirubin diglucuronide by rat liver microsomal preparations

1980 ◽  
Vol 58 (11) ◽  
pp. 1302-1310 ◽  
Author(s):  
Ellen R. Gordon ◽  
Carl A. Goresky
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Assay System ◽  
Microsomal Membrane ◽  
Liver Plasma Membranes ◽  
Triton X ◽  
Nonionic Detergents

Bilirubin transformation in vitro to bilirubin conjugates in the presence of activated rat liver microsomal preparations and UDPglucuronate was assessed with a method involving isolation of the products as tetrapyrroles. The proportions of bilirubin monoglucuronide and diglucuronide formed by the microsomal bilirubin UDPglucuronosyltransferase were found to be governed by the concentration of bilirubin present and the nature of the activation of the microsomal membrane. Activation of the microsomal preparations with the nonionic detergents Triton X-100 or Emulgen 911, or with digitonin for 24 h, produced bilirubin monoglucuronide as the only product at all concentrations of bilirubin investigated. In contrast, bilirubin diglucuronide was the only conjugate formed when hepatic microsomal preparations were activated with digitonin for periods of less than 2 h and the concentration of bilirubin was 20 μM. Increasing the concentration of bilirubin utilized in this assay system changed the relative amounts of bilirubin monoglucuronide and diglucuronide formed. As the level of bilirubin was increased from 20 to 166 μM, the proportion of bilirubin diglucuronide decreased and that of bilirubin monoglucuronide increased, until at levels of 108 and 166 μM bilirubin only bilirubin monoglucuronide was formed. No evidence was found with liver plasma membranes that transglucuronidation plays a major role in the formation of bilirubin diglucuronide from bilirubin monoglucuronide.

Deacylation of bis(monoacylglycero)phosphate by lysosomal and microsomal lysophospholipases from rat liver

1982 ◽  
Vol 60 (6) ◽  
pp. 599-607 ◽  
Author(s):  
Srebrenka Huterer ◽  
John R. Wherrett
Keyword(s):  
Arachidonic Acid ◽  
Rat Liver ◽  
Ethylenediaminetetraacetic Acid ◽  
Subcellular Fractionation ◽  
Alkaline Ph ◽  
Phosphodiesterase Activity ◽  
Triton Wr 1339 ◽  
Liver Homogenates ◽  
Triton X ◽  
Catalytic Exchange

The degradation of bis(monoacylglycero)phosphate by subcellular fractions of rat liver, using substrates labelled biosynthetically with [14C]arachidonic acid and [4C]oleic acid and chemically by catalytic exchange with tritium, was studied. Liver homogenates catalyzed maximum degradation at alkaline pH and subcellular fractionation localized this activity to microsomes. The degradation by microsomes was found to be a deacylation to lysophosphatidylglycerol and was without phosphodiesterase activity. The deacylation was maximal at pH 8.3 and did not require Ca2+ or Mg2+ but was stimulated by ethylenediaminetetraacetic acid and inhibited by Fe2+ and Hg2+. It was also inhibited by p-chloromercuribenzoate, deoxycholate, Triton X-100, and Triton WR-1339. The apparent Km was determined to be 5.5 × 10−5 M and the corresponding Vmax was 4.1 nmol product released/min per milligram protein. The three labelled substrates were degraded by microsomes to give the same products in similar relative proportions. Degradation of bis(monoacylglycero)phosphate by lysosomes was maximal at acid pH as previously described by Y. Matsuzawa and K. Y. Hosteller. Contrary to their finding, deacylase activity in lysosomes was much greater than phosphodiesterase activity. The lysosomal deacylase but not the phosphodiesterase activity was inhibited reversibly by n-butanol. Sphingomyelin inhibited the microsomal deacylase but not the lysosomal deacylase.

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 Metabolism of d-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate

1987 ◽  
Vol 246 (1) ◽  
pp. 139-147 ◽  
Author(s):  
S B Shears ◽  
J B Parry ◽  
E K Tang ◽  
R F Irvine ◽  
R H Michell ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Dependent Manner ◽  
The Novel ◽  
Metabolic Fate ◽  
Inositol Tetrakisphosphate ◽  
Liver Homogenates ◽  
And Function

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5′-[beta gamma-imido]triphosphate, adenosine 5′-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5′-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.

scholarly journals Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions.

1976 ◽  
Vol 71 (2) ◽  
pp. 535-550 ◽  
Author(s):  
S Fowler ◽  
J Remacle ◽  
A Trouet ◽  
H Beaufay ◽  
J Berthet ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rat Liver ◽  
Analytical Study ◽  
Plasma Membranes ◽  
Cytochrome B5 ◽  
Subcellular Fractions ◽  
Mitochondrial Membranes ◽  
Subcellular Organelles ◽  
Peptic Digestion

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.

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