scholarly journals Phospholipid asymmetry in rough- and smooth-endoplasmic-reticulum membranes of untreated and phenobarbital-treated rat liver

1980 ◽  
Vol 189 (3) ◽  
pp. 475-480 ◽  
Author(s):  
I C Bollen ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Smooth Endoplasmic Reticulum ◽  
Protein Composition ◽  
Asymmetric Distribution ◽  
Phospholipid Asymmetry ◽  
Outer Leaflet ◽  
Significant Difference ◽  
Small Change

Phospholipase C was used as a probe for the distribution of phospholipids about the membrane of rough and smooth microsomal fractions from normal and phenobarbital-treated rat liver. All membranes exhibited an asymmetric distribution, with phosphatidylethanolamine and phosphatidylserine concentrated in the inner leaflet of the bilayer and phosphatidylcholine and sphingomyelin concentrated in the outer leaflet. The only phospholipid showing a significant difference in distribution between fractions was phosphatidylcholine, which was shifted towards the outer leaflet in the smooth microsomal fraction compared with the rough microsomal fraction, and towards the outer leaflet in both rough and smooth microsomal fractions from phenobarbital-treated liver compared with the same preparations from untreated rat liver. Apart from this small change, the asymmetric distribution of phospholipids was conserved in microsomal fractions which had proliferated in response to phenobarbital and in which the protein composition had changed.

scholarly journals The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

1978 ◽  
Vol 174 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Santhirasegaram Balasubramaniam ◽  
Soundararajan Venkatesan ◽  
Konstantinos A. Mitropoulos ◽  
Timothy J. Peters
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Microsomal Fraction ◽  
Smooth Endoplasmic Reticulum ◽  
Cholesterol Acyltransferase ◽  
Specific Radioactivity ◽  
Cholesteryl Esters ◽  
Distribution Profile

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [14C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.

scholarly journals Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

1980 ◽  
Vol 190 (3) ◽  
pp. 737-746 ◽  
Author(s):  
Michael B. Cooper ◽  
John A. Craft ◽  
Margaret R. Estall ◽  
Brian R. Rabin
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Reductase Activity ◽  
Asymmetric Distribution ◽  
Trypsin Treatment ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).

scholarly journals The latency of rat liver microsomal protein disulphide-isomerase

1985 ◽  
Vol 228 (3) ◽  
pp. 635-645 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
High Speed ◽  
Microsomal Fraction ◽  
Membrane Integrity ◽  
Microsomal Protein ◽  
Permeability Barrier ◽  
Rat Liver Microsomes ◽  
Luminal Surface ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase (PDI) activity was not detectable in freshly prepared rat liver microsomes (microsomal fraction), but became detectable after treatments that damage membrane integrity, e.g. sonication, detergent treatment or freezing and thawing. Maximum activity was detectable after sonication. Identical latency was observed in microsomes prepared by gel filtration and in those prepared by high-speed centrifugation. PDI activity was latent in all particulate subcellular fractions, but not latent in the high-speed supernatant. When all fractions were sonicated to expose total PDI activity, PDI was found at highest specific activity in the microsomal fraction and co-distributed with marker enzymes of the endoplasmic reticulum. Washing of microsomes under various conditions that removed peripheral proteins and, in some cases, bound ribosomes did not remove significant quantities of PDI, nor did it affect the latency of PDI activity. Treatment of microsomes with proteinases, under conditions where the permeability barrier of the microsomal vesicles was maintained intact, did not inactivate PDI significantly or affect its latency. PDI was very readily solubilized from microsomal vesicles by low concentrations of detergents, which removed only a fraction of the total microsomal protein. In all these respects, PDI resembled nucleoside diphosphatase, a marker peripheral protein of the luminal surface of the endoplasmic reticulum, and differed from NADPH: cytochrome c reductase, a marker integral protein exposed at the cytoplasmic surface of the membrane. The data are compatible with a model in which PDI is loosely associated with the luminal surface of the endoplasmic reticulum, a location consistent with the proposed physiological role of the enzyme as catalyst of formation of native disulphide bonds in nascent and newly synthesized secretory proteins.

scholarly journals Oocytes with smooth endoplasmic reticulum aggregates do not impact blastocyst euploidy rate

2021 ◽  
Author(s):  
Jian Xu ◽  
Li Yang ◽  
Zhi-Heng Chen ◽  
Min-Na Yin ◽  
Juan Chen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Formation Rate ◽  
Tertiary Hospital ◽  
Fertilization Rate ◽  
Blastocyst Formation ◽  
Preimplantation Genetic Testing ◽  
Significant Difference ◽  
Normal Fertilization ◽  
Euploidy Rate

Abstract Objective: To investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum (SERa) positive cycles and oocytes are impacted.Design: Retrospective cohort study.Setting: A tertiary hospital-based reproductive medicine center.Patient(s): A total of 601 preimplantation genetic testing (PGT) cycles with obtained oocytes in our center between April 2017 and May 2021 were included in the study. Intervention(s): Women＞35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared between SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.Main Outcome Measure(s): Embryological outcomes and blastocyst euploidy rate.Results: No significant difference was observed in the normal fertilization rate (82.1 % vs. 77.8 % vs. 83.1 %, respectively, P=0.061), blastocyst formation rate (71.0 % vs. 72.5 % vs. 68.4 %, respectively, P=0.393), good quality blastocyst formation rate (46.4 % vs. 48.3 % vs. 42.6 %, respectively, P=0.198) between the SERa+ oocyte group, sibling SERa- oocyte group and SERa- oocyte group. No significant difference was observed in the euploidy rate (50.0 % vs. 62.5 % vs. 63.3 %, respectively, P=0.324), mosaic rate (12.5 % vs. 9.7 % vs. 13.4 %, respectively, P=0.506) and aneuploidy rate (37.5 % vs. 27.8% vs. 23.2 %, respectively, P=0.137) between the three groups.Conclusion: Our results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes are not impacted.

Studies on the site of addition of sialic acid and glucosamine to rat α1-acid glycoprotein

1977 ◽  
Vol 55 (4) ◽  
pp. 408-414 ◽  
Author(s):  
J. C. Jamieson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sialic Acid ◽  
Rat Liver ◽  
Golgi Complex ◽  
Smooth Endoplasmic Reticulum ◽  
Main Site ◽  
Acid Glycoprotein ◽  
Membrane Bound ◽  
Polypeptide Chains

Ultrasonic extracts of rough and smooth endoplasmic reticulum fractions and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to α1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free α1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing α1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to α1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of α1-acid glycoprotein and albumin were assembled mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of α1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

scholarly journals The distribution of the components of mixed-function oxidase between the rough and the smooth endoplasmic reticulum of liver cells

1968 ◽  
Vol 110 (3) ◽  
pp. 407-412 ◽  
Author(s):  
J. L. Holtzman ◽  
T. E. Gram ◽  
P. L. Gigon ◽  
J. R. Gillette
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Endoplasmic Reticulum ◽  
Reductase Activity ◽  
Mixed Function Oxidase ◽  
Protein Ratio ◽  
Nadph Cytochrome ◽  
Rate Limiting Step ◽  
Significant Difference ◽  
The Difference ◽  
Cytochrome P 450

Mixed-function oxidase activity, when measured by the N-demethylation of ethylmorphine or the hydroxylation of aniline, is significantly higher in the smooth hepatic endoplasmic reticulum than in the rough. In the rabbit the smooth membrane/rough membrane activity ratios are significantly greater than 1 whether the activities are expressed per g. of liver (ratio 5), per mg. of protein (ratio 3–5), per μg. of phospholipid phosphorus (ratio 2), per unit of cytochrome P-450 (ratio 1·7) or per unit of NADPH–cytochrome c reductase activity (ratio 2). On the other hand, if the activities are normalized to the NADPH–cytochrome P-450 reductase, there is no significant difference between the rough and smooth membranes. These results suggest that, in the rabbit, the rate-limiting step is the reduction of cytochrome P-450. In contrast, in the rat the difference in activities can be explained by differences in the concentration of cytochrome P-450.

scholarly journals Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

1983 ◽  
Vol 97 (5) ◽  
pp. 1559-1565 ◽  
Author(s):  
P M Novikoff ◽  
N F La Russo ◽  
A B Novikoff ◽  
R J Stockert ◽  
A Yam ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Intracellular Sorting ◽  
Specialized Region ◽  
Beta Galactosidase ◽  
Specific Polyclonal Antibody ◽  
Galactosyl Residues

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

scholarly journals Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver

1989 ◽  
Vol 259 (3) ◽  
pp. 659-663 ◽  
Author(s):  
F Vanstapel ◽  
L Hammaker ◽  
K Pua ◽  
N Blanckaert
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane System ◽  
Permeability Barrier ◽  
Membrane Bound ◽  
Marker Studies ◽  
High Degree ◽  
Regulatory Properties

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.

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