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 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.

Studies on Acute Phase Proteins of Rat Serum. IV. Pathway of Secretion of Albumin and α1-Acid Glycoprotein from Liver

1973 ◽  
Vol 51 (9) ◽  
pp. 1281-1291 ◽  
Author(s):  
J. C. Jamieson ◽  
F. E. Ashton
Keyword(s):  
Endoplasmic Reticulum ◽  
Acute Phase ◽  
Rough Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Acute Phase Proteins ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Serum ◽  
Experimental Animals ◽  
Acid Glycoprotein ◽  
Ionic Detergent

Normal rats and those suffering from inflammation for 12 h were given pulse injections of L-leucine-3H and D-glucosamine-14C and were killed at 3–60 min thereafter. Rough and smooth microsome fractions and a Golgi-enriched fraction were prepared from liver and the fractions were extracted with the non-ionic detergent Lubrol-W. Albumin and acute phase α1-acid glycoprotein were isolated from the extracts by application of a quantitative precipitin technique employing antisera to albumin and α1-acid glycoprotein, and specific radioactivities were determined. The results indicated that the pathway of secretion of albumin and α1-acid glycoprotein was rough endoplasmic reticulum → smooth endoplasmic reticulum → Golgi complex → blood. Although there was little difference in the pathway or rate of secretion of the proteins under study in normal and experimental animals, there was an increase in specific radioactivities of labelled leucine and glucosamine associated with α1-acid glycoprotein when isolated from subcellular fractions from livers from experimental animals.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals Glycosyltransferase activities in Golgi complex and endoplasmic reticulum fractions isolated from African trypanosomes.

1984 ◽  
Vol 99 (2) ◽  
pp. 569-577 ◽  
Author(s):  
D J Grab ◽  
S Ito ◽  
U A Kara ◽  
L Rovis
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Smooth Endoplasmic Reticulum ◽  
Surface Glycoprotein ◽  
Relative Distribution ◽  
African Trypanosomes ◽  
Independent Form ◽  
Variable Surface ◽  
Dependent Form

Highly enriched Golgi complex and endoplasmic reticulum fractions were isolated from total microsomes obtained from Trypanosoma brucei, Trypanosoma congolense, and Trypanosoma vivax, and tested for glycosyltransferase activity. Purity of the fractions was assessed by electron microscopy as well as by biochemical analysis. The relative distribution of all the glycosyltransferases was remarkably similar for the three species of African trypanosomes studied. The Golgi complex fraction contained most of the galactosyltransferase activity followed by the smooth and rough endoplasmic reticulum fractions. The dolichol-dependent mannosyltransferase activities were highest for the rough endoplasmic reticulum, lower for the smooth endoplasmic reticulum, and lowest for the Golgi complex. Although the dolichol-independent form of N-acetylglucosaminyltransferase was essentially similar in all the fractions, the dolichol-dependent form of this enzyme was much higher in the endoplasmic reticulum fractions than in the Golgi complex fraction. Inhibition of this latter activity in the smooth endoplasmic reticulum fraction by tunicamycin A1 suggests that core glycosylation of the variable surface glycoprotein may occur in this organelle and not in the rough endoplasmic reticulum as previously assumed.

Fibrinogen Biosynthesis

1975 ◽  
Author(s):  
B. Blombach ◽  
C. Redman ◽  
B. Kudryk
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Fibrinogen Level ◽  
Smooth Endoplasmic Reticulum ◽  
Time Intervals ◽  
Plasma Fraction ◽  
Polypeptide Chains ◽  
Dog Liver

A mixture of 3H-labeled amino acids was injected into dogs. Blood was collected at specific time intervals and fibrinogen from each corresponding plasma fraction was isolated by affinity chromatography. After reduction and alkylation, the fibrinogen chains were separated either by CM-cellulose chromatography or preparative SDS-gel electrophoresis. In one dog whose fibrinogen level was approximately 8 mg/ml, it was found that both the γ and B β chains had lower radioactivity when compared to the Aα chain, at least in the early times of appearance in blood. When this experiment was repeated in another dog whose fibrinogen level was 2 mg/ml and non-labeled amino acids were given as “chaser”, the radioactivity in the γ and Bβ chain did not reach the same value as that in the Aα chain, even after a three hour period. The significance of these findings with respect to rate of synthesis, secretion and assembly of the polypeptide chains will be discussed.Experiments were also performed using dog liver slices incubated with radioactive amino acids. Labeled, fibrinogen-related, material from detergent-solubilized rough endoplasmic reticulum (HER) could be recovered either by affinity chromatography or immunoprecipitation. Fractions were also isolated from smooth endoplasmic reticulum and Golgi. Analysis of the material isolated from BEB suggests that it may not consist of all three chains but rather one or possibly two of them to be either in degraded (during isolation procedures) or precursor form.(Supported by N.I.H. grants HL 09011–11 and AM 13620.)

scholarly journals Proteolytic activation and solubilization of endoplasmic-reticulum cyclic AMP phosphodiesterase activity

1983 ◽  
Vol 213 (1) ◽  
pp. 99-105 ◽  
Author(s):  
S R Wilson ◽  
M D Houslay
Keyword(s):  
Endoplasmic Reticulum ◽  
Proteinase Inhibitors ◽  
Smooth Endoplasmic Reticulum ◽  
Limited Proteolysis ◽  
Proteolytic Activation ◽  
Freeze Thaw ◽  
Membrane Bound ◽  
Time Courses ◽  
Triton X ◽  
Thiol Proteinase

Dithiothreitol led to the activation and solubilization of the cyclic nucleotide phosphodiesterase activities associated with the smooth and various rough subfractions of rat liver endoplasmic reticulum. The activity in each of the subfractions exhibited somewhat different time courses, and sensitivities to dithiothreitol concentration, in respect of their solubilization and activation. Both activation and solubilization by dithiothreitol could be blocked by either thiol proteinase inhibitors or excess bovine serum albumin. Freeze-thaw solubilization was not blocked by the thiol proteinase inhibitor antipain and did not lead to the activation of the enzyme. After dithiothreitol-induced solubilization, all of the enzymes exhibited non-linear Lineweaver-Burk plots indicative of apparent negative co-operativity. In contrast, after freeze-thaw solubilization the enzyme in the smooth-endoplasmic-reticulum-plus-Golgi fraction still obeys Michaelis kinetics, as does the membrane-bound enzyme. It is possible to mimic the action of dithiothreitol in solubilizing and activating the enzyme by limited proteolysis with trypsin. Triton X-100 is highly efficient at solubilizing these enzymes, yet has little effect on their activities. Charged detergents exhibit highly selective effects on the enzymes as regards their solubilization and activity expressed.

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.

Sign in / Sign up

Export Citation Format

Share Document