scholarly journals Localization and Biosynthesis of NADH-Cytochrome b(5) reductase, an integral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover of the enzyme in various subcellular compartments

1980 ◽  
Vol 86 (1) ◽  
pp. 38-45 ◽  
Author(s):  
N Borgese ◽  
G Pietrini ◽  
J Meldolesi
Keyword(s):  
Cytochrome B ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Integral Membrane Protein ◽  
Rat Serum ◽  
Double Labeling ◽  
Nadh Cytochrome ◽  
Cell Fractions

The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.

scholarly journals The separation of intracellular serum albumin from rat liver

1971 ◽  
Vol 123 (4) ◽  
pp. 643-648 ◽  
Author(s):  
J. D. Judah ◽  
Marion R. Nicholls
Keyword(s):  
Serum Albumin ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Exchange Chromatography ◽  
Rat Serum ◽  
Radioactive Impurity ◽  
Simple Method ◽  
High Specific Radioactivity ◽  
Chromatographic Procedure

1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-14C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10–25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70–90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.

scholarly journals Retinol and retinyl esters in parenchymal and nonparenchymal rat liver cell fractions after long-term administration of ethanol.

1985 ◽  
Vol 26 (9) ◽  
pp. 1112-1119
Author(s):  
M Rasmussen ◽  
R Blomhoff ◽  
P Helgerud ◽  
L A Solberg ◽  
T Berg ◽  
...  
Keyword(s):  
Rat Liver ◽  
Liver Cell ◽  
Term Administration ◽  
Retinyl Esters ◽  
Cell Fractions

scholarly journals The availability of inorganic sulphate in blood for sulphate conjugation of drugs in rat liver in vivo. (35S)Sulphate incorporation into harmol sulphate

1978 ◽  
Vol 172 (2) ◽  
pp. 247-251 ◽  
Author(s):  
G J Mulder ◽  
E Scholtens
Keyword(s):  
Plasma Concentration ◽  
Rat Liver ◽  
Biliary Excretion ◽  
Time Course ◽  
Specific Radioactivity ◽  
Pool Size ◽  
Inorganic Sulphate ◽  
Initial Decrease ◽  
Sulphate Conjugate

1. When Na235SO4 is injected intravenously in rats, it is immediately available for sulphate conjugation of the phenolic drug harmol (7-hydroxyl-1-methyl-9H-pyrido[3,4-b]indole) in the liver. This was established by following the time course of the biliary excretion of the sulphate conjugate of harmol, and the incorporation of [35S]sulphate into harmol sulphate. 2. During the 10min immediately after injection of Na235SO4 re-distribution of [35S]sulphate took place, which resulted in a rapid initial decrease in the plasma concentration of [35S]sulphate; a concomitant decrease in the amount of [35S]sulphate incorporated into harmol sulphate was observed, indicating that the co-substrate of sulphation, adenosine 3′-phosphate 5′-sulphatophosphate, equilibrates rapidly with [35S]sulphate in plasma. 3. The results suggest that the pool size of adenosine 3′-phosphate 5′-sulphatophosphate is very small; therefore the specific radioactivity of [35S]sulphate in plasma determines the specific radioactivity incorporated into sulphate esters at any time.

scholarly journals CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

1971 ◽  
Vol 50 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Abraham Amsterdam ◽  
Michael Schramm ◽  
Itzhak Ohad ◽  
Yoram Salomon ◽  
Zvi Selinger
Keyword(s):  
Amino Acids ◽  
Secretory Granule ◽  
De Novo ◽  
Secretory Granules ◽  
Specific Radioactivity ◽  
Staining Intensity ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1974 ◽  
Vol 61 (3) ◽  
pp. 789-807 ◽  
Author(s):  
Gert Kreibich ◽  
David D. Sabatini
Keyword(s):  
Serum Proteins ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Rat Serum ◽  
Coomassie Blue ◽  
Specific Subset ◽  
Low Levels ◽  
Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

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.

scholarly journals Measurement of protein turnover in rat liver. Analysis of the complex curve for decay of label in a mixture of proteins

1976 ◽  
Vol 156 (3) ◽  
pp. 657-663 ◽  
Author(s):  
P J Garlick ◽  
J C Waterlow ◽  
R W Swick
Keyword(s):  
Rat Liver ◽  
Turnover Rate ◽  
Decay Curve ◽  
Specific Radioactivity ◽  
Liver Protein ◽  
Turnover Rates ◽  
A Value ◽  
Maximum Value ◽  
Single Exponential

The curve for decay of 14C in rat liver protein labelled by injection of NaH14CO3 was analysed to obtain the average turnover rate of mixed liver protein. Three different methods of analysis were used. (1) Unlike decay curves from homogeneous proteins, the curve did not fit a single exponential, but a good fit was obtained with three exponentials. By assuming that the mixture contained three major components with different turnover rates, the calculated value for the average turnover rate (k) was close to 40% per day. (2) k was also calculated from the area under the decay curve, a method which makes no assumptions about the number of proteins in the mixture. This method also gave a value close to 40% per day. (3) It was shown empirically, both by simulation of decay of label in model mixtures of protein and with the decay curve measured in vivo, that k can be calculated from the time taken for the specific radioactivity to fall to 10% of its maximum value. This is an advantage, since the other two methods require the decay curve to be measured over a much longer period of time.

scholarly journals Biocompatibility studies of fluorescent diamond particles-(NV)∼800nm (part V): in vitro kinetics and in vivo localization in rat liver following long-term exposure

2019 ◽  
Vol Volume 14 ◽  
pp. 6451-6464 ◽  
Author(s):  
Jonathan A Gerstenhaber ◽  
Cezary Marcinkiewicz ◽  
Frank C Barone ◽  
Mark Sternberg ◽  
Michael R D’Andrea ◽  
...  
Keyword(s):  
Rat Liver ◽  
Diamond Particles ◽  
In Vitro Kinetics
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