scholarly journals STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN HEPATOCYTES OF PHENOBARBITAL-TREATED RATS

1974 ◽  
Vol 62 (3) ◽  
pp. 635-646 ◽  
Author(s):  
Joan A. Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Actinomycin D ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Control Level ◽  
Phospholipid Synthesis ◽  
Protein Ratio ◽  
Nadph Cytochrome C Reductase

The specific activity of the acyltransferases of smooth microsomes of rat liver rose threefold by 12 h after injection of phenobarbital, while the activity of the acyltransferases of the rough microsomes rose slightly to peak at 3–4 h, and subsequently fell. The latter rise was abolished by treatment of the animal with actinomycin D or puromycin, while that of the smooth microsomes was unaffected. Incorporation of [14C]glycerol into phospholipid of smooth microsomes was elevated 100% by phenobarbital, while that of the rough microsomes was elevated 15%, and this could be accounted for by exchange between the microsomal phospholipids. The phospholipid/protein ratio of the smooth microsomes rose 1.5 times 3–4 h after injection of phenobarbital, while that of the rough microsomes fell slightly. The specific activity of NADPH cytochrome c reductase and NADPH diaphorase rose first in the rough microsomes, and subsequently in the smooth microsomes at a time coinciding with the return of the phospholipid/protein ratio to the control level. The rise in phospholipid/protein ratio was unaffected by actinomycin D or puromycin. These results indicate that the proliferating smooth membranes are the site of phospholipid synthesis, and that the phospholipid/protein ratio of these membranes may change independently.

Heterogeneity of phospholipid synthesis in rat liver endoplasmic reticulum during proliferation of smooth membranes

1976 ◽  
Vol 22 (1) ◽  
pp. 173-197
Author(s):  
J.A. Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Uniform Distribution ◽  
Specific Activity ◽  
Intact Cell ◽  
Smooth Endoplasmic Reticulum ◽  
Membrane Phospholipid ◽  
Phospholipid Synthesis ◽  
Protein Ratio ◽  
Cytochemical Localization ◽  
Heterogeneous Distribution

During proliferation of smooth endoplasmic reticulum (SER) induced by phenobarbital the specific activity of acyltransferases of the smooth microsomes increases, there is a transient rise in the phospholipid/protein ratio of these membranes, and an increased incorporation of [14C]glycerol into smooth-membrane phospholipid. Microsomes separated into subfractions on 2 gradients exhibited a heterogeneous distribution of these characteristics, indicating a non-uniform distribution of the site of phospholipid synthesis in the ER under these conditions. Cytochemical localization of acyltransferases on whole liver and smooth and rough microsomes confirmed this heterogeneity, and indicated that the distribution of this activity was not restricted to any morphologically distinct site in the ER of the intact cell. After 4 days of phenobarbital treatment the increased membrane is restricted to lighter subfractions and is similar in distribution to that of increased acyltransferase activity. These results indicate that the synthesis of membrane phospholipid and the growth of the SER in response to phenobarbital is not uniform but occurs at randomly dispersed sites in the SER while proteins may be added preferentially at these sites resulting in a final uniform distribution.

scholarly journals Re-examination of the subcellular localization of thyroxine 5′-deiodination in rat liver

1979 ◽  
Vol 180 (2) ◽  
pp. 273-279 ◽  
Author(s):  
D Auf Dem Brinke ◽  
R D Hesch ◽  
J Köhrle
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Rat Liver ◽  
Marker Enzyme ◽  
Cytochrome C Reductase ◽  
Substrate Saturation ◽  
Single Enzyme ◽  
Optimal Ph ◽  
Nadph Cytochrome C Reductase

We describe the existence of at least two thyroxine 5′-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5′-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5′-deiodination. However, this process is not mediated by only a single enzyme.

scholarly journals STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS

1972 ◽  
Vol 55 (2) ◽  
pp. 282-298 ◽  
Author(s):  
Joan A. Higgins ◽  
Russell J. Barrnett
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Normal Liver ◽  
Subsequent Treatment ◽  
Cell Fractionation ◽  
Acyltransferase Activity ◽  
Normal Rat

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.

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 Cytochrome c oxidase is preferentially synthesized in the rough endoplasmic reticulum-mitochondrion complex in rat liver

1982 ◽  
Vol 208 (2) ◽  
pp. 505-507 ◽  
Author(s):  
S Parimoo ◽  
N Rao ◽  
G Padmanaban
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Cytochrome C Oxidase ◽  
Rat Liver ◽  
Rough Endoplasmic Reticulum ◽  
Specific Activity ◽  
Mitochondrial Fraction

The specific activity and content of cytochrome oxidase in the rough endoplasmic reticulum-mitochondrion complex are higher than in the mitochondrial fraction. Radiolabelling studies with the use of hepatocytes and isolated microsomal and rough endoplasmic reticulum-mitochondrion fractions, followed by immunoprecipitation with anti-(cytochrome oxidase) antibody, reveal that the nuclear-coded cytoplasmic subunits of cytochrome oxidase are preferentially synthesized in the latter fraction. The results have a bearing on the mechanism of transport of these subunits into mitochondria.

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 Can mitochondria and synaptosomes of guinea-pig brain synthesize phospholipids?

1972 ◽  
Vol 126 (4) ◽  
pp. 805-821 ◽  
Author(s):  
E. K. Miller ◽  
R. M. C. Dawson
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Smooth Endoplasmic Reticulum ◽  
Cytochrome C Reductase ◽  
Nadph Cytochrome ◽  
Pig Brain ◽  
Guinea Pig Brain ◽  
Nadph Cytochrome C Reductase

1. The use of ‘marker’ enzymes for investigating the contamination by endoplasmic reticulum of mitochondrial and synaptosomal (nerve-ending) fractions isolated from guinea-pig brain was examined. NADPH–cytochrome c reductase appeared to be satisfactory. With the synaptosomal preparation there was a non-occluded enzymic activity believed to arise from contaminating microsomes and an occluded form released by detergent, which probably was derived from some type of intraterminal smooth endoplasmic reticulum. 2. Isolated brain mitochondria, both intact and osmotically shocked, could not synthesize more labelled phosphatidylcholine from CDP-[Me-14C]choline or phosphoryl[Me-14C]choline than could be accounted for by microsomal contamination. They could synthesize only phosphatidic acid and diphosphatidylglycerol from a [32P]Pi precursor and not nitrogen-containing phosphoglycerides or phosphatidylinositol. 3. The synaptosomal outer membrane and the intraterminal mitochondria could not synthesize phosphatidylcholine from CDP-[Me-14C]choline but the synaptic vesicles and probably the intraterminal ‘endoplasmic reticulum’ appeared to be capable of catalysing the incorporation of label from this substrate into their phospholipids. 4. Microsomal fractions and synaptosomes from guinea-pig brain could incorporate [Me-14C]choline into their phospholipids by a non-energy-requiring exchange process, which was catalysed by Ca2+. Fractionation of the synaptosomes after such an exchange had taken place revealed that the label was predominantly in the intraterminal mitochondria and not associated with membranes containing NADPH–cytochrome c reductase. 5. On the intraperitoneal injection of [32P]Pi into guinea pigs, incorporation of radioactivity into phosphatidylinositol and phosphatidic acid was much faster than into the nitrogen-containing phosphoglycerides. Mitochondria and microsomal fractions showed a roughly equivalent incorporation into individual phospholipids, and that into synaptosomes was appreciably less, whereas the phospholipids of myelin showed little 32P incorporation up to 10h.

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