scholarly journals Use of radioactive glucosamine in the perfused rat liver to prepare α1-acid glycoprotein (orosomucoid) with 3H- or 14C-labelled sialic acid and N-acetylglucosamine residues

1982 ◽  
Vol 203 (1) ◽  
pp. 141-148 ◽  
Author(s):  
N N Aronson
Keyword(s):  
Sialic Acid ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Deae Cellulose ◽  
Experimental System ◽  
Amino Sugar ◽  
Perfused Rat Liver ◽  
Rat Serum ◽  
Acid Glycoprotein ◽  
Sequential Digestion

1. A method was developed whereby [1-14C]glucosamine was used in a perfused rat liver system to prepare over 2 mg of alpha 1-acid glycoprotein with highly radioactive sialic acid and glucosamine residues. 2. The liver secreted radioactive alpha 1-acid glycoprotein over a 4-6 h period, and this glycoprotein was purified from the perfusate by chromatography on DEAE-cellulose at pH 3.6. 3. The sialic acid on the isolated glycoprotein had a specific radioactivity of 3.1 Ci/mol, whereas the glucosamine-specific radioactivity was 4.3 Ci/mole. The latter amino-sugar residues on the isolated protein were only 13-fold less radioactive than the initially added [1-14C]glucosamine. Orosomucoid with a specific radioactivity of 31.3 microCi/mg of protein was obtainable by using [6-3H]glucosamine. 4. The amino acid composition of the purified orosomucoid was comparable with that found by others for the same glycoprotein isolated from rat serum. A partial characterization of the carbohydrate structure was done by sequential digestion with neuraminidase, beta-D-galactosidase and beta-D-hexosaminidase. 5. Many other radioactive glycoproteins were found to be secreted into the perfusate by the liver. Thus this experimental system should prove useful for obtaining other serum glycoprotein with highly radioactive sugar moieties.

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 LABELING WITH 14C AMINO ACIDS OF ALBUMIN-LIKE PROTEIN BY RAT LIVER RIBONUCLEOPROTEIN PARTICLES

1963 ◽  
Vol 16 (3) ◽  
pp. 471-481 ◽  
Author(s):  
Alexandra von der Decken
Keyword(s):  
Amino Acids ◽  
Serum Albumin ◽  
Cellulose Acetate ◽  
Electrophoretic Mobility ◽  
Rat Liver ◽  
Deae Cellulose ◽  
Rat Liver Microsomes ◽  
Rat Serum ◽  
Ribonucleoprotein Particles ◽  
Rat Serum Albumin

Ribonucleoprotein particles were prepared by treatment of rat liver microsomes with detergents and high concentrations of KCl. They were active in incorporating 14C amino acids into protein when incubated with cell sap together with ATP, GTP, and a system to regenerate the triphosphates. The albumin of the incubation mixture, soluble at 105,000 g, and that of the fraction released by ultrasonication of the particles were studied by immunoelectrophoresis in agar gel. When the ribonucleoprotein particles were incubated with cell sap the immunological precipitation lines formed with antiserum to rat serum albumin were highly radioactive as tested by autoradiography. After zone electrophoresis on cellulose acetate, two immunologically reactive albumins were obtained which differed in their electrophoretic mobility from rat serum albumin. Labeled albumin, when purified on DEAE-cellulose columns, retained its radioactivity as tested by autoradiography following immunoelectrophoresis. On cellulose acetate this purified albumin showed an electrophoretic mobility higher than that of rat serum albumin.

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.

Studies on Acute Phase Proteins of Rat Serum. III. Site of Synthesis of Albumin and α1-Acid Glycoprotein and the Contents of These Proteins in Liver Microsome Fractions from Rats Suffering from Induced Inflammation

1973 ◽  
Vol 51 (7) ◽  
pp. 1034-1045 ◽  
Author(s):  
J. C. Jamieson ◽  
F. E. Ashton
Keyword(s):  
Liver Microsome ◽  
Acute Phase Proteins ◽  
Specific Radioactivity ◽  
Rat Serum ◽  
Experimental Animals ◽  
Acid Glycoprotein ◽  
Inflammatory Agent ◽  
Microsome Fraction ◽  
Rapid Stimulation ◽  
Short Times

Following injection of L-ieucine-3H or D-glucosamine-14C to normal rats or rats suffering from inflammation for 12 h there was a delay of 10–15 min before label appeared in albumin and α1-acid glycoprotein in serum; thereafter, there were rapid increases in specific radioactivities. The specific radioactivity of L-leucine-3H in albumin in serum from normal and experimental animals was not significantly different; however, there was a more rapid increase in specific radioactivities of both labelled compounds in α1-acid glycoprotein isolated from serum from experimental animals. Immunological techniques coupled with radioautography indicated that the microsome fraction of liver was the subcellular site of synthesis of albumin and of carbohydrate and polypeptide moieties of α1-acid glycoprotein in normal and experimental animals. The contents of albumin and α1-acid glycoprotein in liver microsome fractions from normal and experimental animals were determined by application of the quantitative precipitin technique to Lubrol-W extracts of microsome material. Little change in content of albumin associated with microsome material was found as a result of inflammation; however, there was a significant increase in the content of α1-acid glycoprotein in microsome material from experimental animals, reaching a maximum at 8–12 h after inflammation. On the basis of these latter results, it is suggested (1) that there is a fairly rapid stimulation of synthesis of α1-acid glycoprotein by liver in response to inflammation and (2) that the increased content of α1-acid glycoprotein associated with microsome material at short times of exposure to inflammatory agent is responsible for the increased content of this protein found in serum at longer times of exposure to inflammatory agent.

scholarly journals The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

1968 ◽  
Vol 107 (5) ◽  
pp. 637-644 ◽  
Author(s):  
Frank Maley ◽  
Anthony L. Tarentino ◽  
John F. McGarrahan ◽  
Rudolph DelGiacco
Keyword(s):  
Sialic Acid ◽  
Rat Liver ◽  
Soluble Fraction ◽  
Liver Extract ◽  
Perfused Rat Liver ◽  
Galactose Metabolism ◽  
Glycogen Synthetase ◽  
Dowex 1

d-[1−14C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1−14C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1−14C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.

scholarly journals Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver

1975 ◽  
Vol 150 (3) ◽  
pp. 495-509 ◽  
Author(s):  
A S Tavill ◽  
D Nadkarni ◽  
J Metcalfe ◽  
E Black ◽  
R Hoffenberg ◽  
...  
Keyword(s):  
Mathematical Model ◽  
Rat Liver ◽  
Specific Radioactivity ◽  
Compartmental Analysis ◽  
Constant Infusion ◽  
Isolated Perfused Rat Liver ◽  
Urea Synthesis ◽  
Perfused Rat Liver ◽  
Albumin Synthesis ◽  
Product Analysis

A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also provided an explanation for discrepancies observed between albumin synthesis measured radioisotopically and immunologically. This was based on a relative overestimation of [14C]urea specific radioactivity resulting from the rapid dynamics of [14C]carbonate and the [14C]urea subsystem relative to the labelled albumin subsystem. The effects of arginine compartmentalization could be minimized in the model by minor slowing of the rate of [14C]carbonate turnover or by constant infusion of [14C]carbonate, both of which permitted valid determination of albumin-synthesis rates.

scholarly journals Utilization by the isolated perfused rat liver of N-acetyl-d-[1-14C]galactosamine and N-[3H]acetyl-d-galactosamine for the biosynthesis of glycoproteins

1978 ◽  
Vol 174 (2) ◽  
pp. 421-426 ◽  
Author(s):  
A D MacNicoll ◽  
F S Wusteman ◽  
G M Powell ◽  
C G Curtis
Keyword(s):  
Sialic Acid ◽  
Rat Liver ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.

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.

Sign in / Sign up

Export Citation Format

Share Document