ANALYTICAL SUBCELLULAR FRACTIONATION OF RAT LIVER HOMOGENATES BY COUNTERCURRENT PARTITION IN A TOROIDAL-COIL CENTRIFUGE

1981 ◽  
Vol 9 (2) ◽  
pp. 174P-174P ◽  
Author(s):  
I. A. Sutherland ◽  
J. Lai ◽  
W. B. Morris ◽  
T. J. Peters
Keyword(s):  
Rat Liver ◽  
Subcellular Fractionation ◽  
Liver Homogenates ◽  
Toroidal Coil

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 The mechanism of hepatic iron uptake from native and denatured transferrin and its subcellular metabolism in the liver cell

1979 ◽  
Vol 182 (1) ◽  
pp. 117-125 ◽  
Author(s):  
J P Milsom ◽  
R G Batey
Keyword(s):  
Rat Liver ◽  
Liver Cell ◽  
Iron Uptake ◽  
Subcellular Fractionation ◽  
Hepatic Iron ◽  
Lysosomal Fraction ◽  
Uptake Process ◽  
Liver Homogenates ◽  
Uptake And Metabolism

Hepatic iron uptake and metabolism were studied by subcellular fractionation of rat liver homogenates after injection of rats with a purified preparation of either native or denatured rat transferrin labelled with 125I and 59Fe. (1) With native transferrin, hepatic 125I content was maximal 5 min after injection and then fell. Hepatic 59Fe content reached maximum by 16 h after injection and remained constant for 14 days. Neither label appeared in the mitochondrial or lysosomal fractions. 59Fe appeared first in the supernatant and, with time, was detectable as ferritin in fractions sedimented with increasingly lower g forces. (2) With denatured transferrin, hepatic content of both 125I and 59Fe reached maximum by 30 min. Both appeared initially in the lysosomal fraction. With time, they passed into the supernatant and 59Fe became incorporated into ferritin. The study suggests that hepatic iron uptake from native transferrin does not involve endocytosis. However, endocytosis of denatured transferrin does occur. After the uptake process, iron is gradually incorporated into ferritin molecules, which subsequently polymerize; there is no incorporation into other structures over 14 days.

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 THE INCORPORATION OF THE CARBOXYL CARBON FROM ACETATE INTO CHOLESTEROL BY RAT LIVER HOMOGENATES

1954 ◽  
Vol 206 (1) ◽  
pp. 471-481 ◽  
Author(s):  
Ivan D. Frantz ◽  
Nancy L.R. Bucher ◽  
Henny S. Schneider ◽  
Naomi H. McGovern ◽  
Ruth Kingston
Keyword(s):  
Rat Liver ◽  
Liver Homogenates ◽  
Carboxyl Carbon

scholarly journals STIMULATION BY DINITROPHENOL OF FORMATION OF MELANIN-LIKE SUBSTANCE FROM TYROSINE BY RAT LIVER HOMOGENATES

1957 ◽  
Vol 225 (2) ◽  
pp. 735-744
Author(s):  
Henry Kamin ◽  
Mildred A. Koon ◽  
Philip Handler
Keyword(s):  
Rat Liver ◽  
Liver Homogenates
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