scholarly journals ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVER

1974 ◽  
Vol 62 (3) ◽  
pp. 717-745 ◽  
Author(s):  
Alain Amar-Costesec ◽  
Maurice Wibo ◽  
Denise Thinès-Sempoux ◽  
Henri Beaufay ◽  
Jacques Berthet
Keyword(s):  
Electron Microscope ◽  
Cytochrome C ◽  
Rat Liver ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Equilibrium Density ◽  
Substantial Part ◽  
Rat Liver Microsomes ◽  
Cytochrome C Reductase ◽  
Nucleoside Diphosphatase

Isopycnic equilibration and sedimentation rate studies of rat liver microsomes led previously to the assignment of microsomal constituents into group a1 (monoamine oxidase), group a2 (5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and cholesterol), group a3 (galactosyltransferase), group b (NADH cytochrome c reductase, NADPH cytochrome c reductase, aminopyrine demethylase, cytochrome b5 and P 450), and group c (glucose 6-phosphatase, esterase, nucleoside diphosphatase, ß-glucuronidase and glucuronyltransferase). Confirmation and extension of the assignment into groups has been obtained by studying the differential effect of the reagents digitonin, EDTA, and PPi. Digitonin specifically affected the equilibrium density only of the group a2 and (to a lesser extent) group a3, and not of groups b and c under conditions which preserved the structure-linked latency of nucleoside diphosphatase and galactosyltransferase. Within experimental error the rate of sedimentation of all microsomal constituents was unaffected. The morphological appearance under the electron microscope was indistinguishable from that of nondigitonin-treated microsomes, except that a few smooth membranes (< 10%) exhibited broken-looking profiles. Treatment of microsomes with EDTA or PPi detached a substantial part of RNA and released protein in excess over the amount accountable for by detachment of ribosome constituents. This detachment was confirmed by electron microscopy. EDTA and PPi decreased markedly the equilibrium density and the density dispersion of groups b and c, due mainly to the uncoating of rough elements. EDTA and PPi shifted slightly the distribution profiles of groups a towards lower densities, possibly as a result of the release of adsorbed proteins. The combination of EDTA and digitonin, used subsequently, rendered the average equilibrium density of group a2 higher than that of groups b and c. Dense subfractions were thus enriched in constituents of group a2 and showed mainly broken-looking vesicles under the electron microscope. The import of our results on the biochemical and enzymic properties of the subcellular components of the microsome fractions is discussed.

scholarly journals Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody.

1976 ◽  
Vol 71 (2) ◽  
pp. 551-564 ◽  
Author(s):  
J Remacle ◽  
S Fowler ◽  
H Beaufay ◽  
A Amarcostesec ◽  
J Berthet
Keyword(s):  
Electron Microscope ◽  
Rat Liver ◽  
Sucrose Gradient ◽  
Analytical Study ◽  
Cytochrome B5 ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Microscope Examination ◽  
Group B ◽  
Isopycnic Centrifugation

The distribution of cytochrome b5 in rat liver microsomes, and in two microsomal subfractions isolated by density equilibration in a linear sucrose gradient, was studied under the electron microscope by means of a ferritin-labeled hybrid anti-cytochrome b5/anti-ferritin antibody. Results of this study show that cytochrome b5 is present in essentially all microsomal vesicles derived from endoplasmic reticulum (ER), whether rough or smooth. Thus, the dissociation of ER constituents into two groups (b and c), achieved by subfractionating microsomes by isopycnic centrifugation (Beaufay, H., A. Amar-Costesec, D. Thines-Sempoux, M. Wibo, M. Robbi, and J. Berthet. 1974. J. Cell Biol. 61:213-231), does not reflect the association of each group with distinct microsomal particles but reflects rather an enzymatic heterogeneity of the ER: the ratio of group c to group b enzymes increasing with the density and ribosome load of the particles.

scholarly journals ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

1962 ◽  
Vol 12 (1) ◽  
pp. 17-29 ◽  
Author(s):  
J. Chauveau ◽  
Y. Moulé ◽  
C. Rouiller ◽  
J. Schneebeli
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Sucrose Solution ◽  
Liver Microsomes ◽  
Liver Homogenate ◽  
Enzymatic Activities ◽  
Rat Liver Microsomes ◽  
Differential Centrifugation ◽  
Cytochrome C Reductase

Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry.

scholarly journals Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes.

1976 ◽  
Vol 68 (2) ◽  
pp. 189-201 ◽  
Author(s):  
T Morimoto ◽  
S Matsuura ◽  
S Sasaki ◽  
Y Yashiro ◽  
T Omura
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Morphological Data ◽  
Rat Liver Microsomes ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Antibody Conjugate ◽  
Enzyme Molecules ◽  
Nadph Cytochrome C Reductase

By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules.

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