Bovine transferrin glycopeptide: the relevance of its structure to interaction with the mammalian hepatic lectin that binds asialoglycoproteins

1978 ◽  
Vol 56 (5) ◽  
pp. 339-344 ◽  
Author(s):  
M. W. C. Hatton ◽  
E. Regoeczi ◽  
H. Kaur
Keyword(s):  
High Voltage ◽  
Serum Proteins ◽  
Analytical Data ◽  
Gel Filtration ◽  
Commercial Sample ◽  
Sialic Acid Content ◽  
Human Transferrin ◽  
Bovine Transferrin ◽  
High Voltage Electrophoresis

After proteolytic digestion of bovine transferrin (phenotype AA), a glycopeptide fraction was isolated by gel filtration and high-voltage electrophoresis. Two glycopeptide bands were recovered, each of which contained one residue of aspartic acid, two of serine, two of galactose, three of mannose, and four of N-acetyl glucosamine together with some fucose. However, the bands differed with respect to sialic acid content (two and three residues respectively). Using the same isolation procedures, a commercial sample of human transferrin yielded four glycopeptide bands (two major, two minor) on high-voltage electrophoresis. On analysis, the two major bands differed in amino-acid residues but their carbohydrate compositions were very similar. The analyses largely resembled previously published analytical data for two human transferrin glycopeptides (Jamieson, G. A., Jett, M. &DeBernardo, S. L. (1971) J. Biol. Chem. 246, 3686–3693; Spik, G., Bayard, B., Fournet, B., Strecker. G., Bouquelet, S. &Montreuil, J. (1975) FEBS Lett. 50, 296–299) and the carbohydrate analyses showed many similarities to the bovine transferrin glycopeptide. From these data and earlier observations made on bovine and human transferrins (Hatton, M. W. C, Regoeczi, E. &Wong, K.-L. (1974) Can. J. Biochem. 52, 845–853), we have concluded that, in contrast with human transferrin, bovine transferrin contains only one heterosaccharide chain per molecule.Following tritiation, bovine transferrin asialoglycopeptide was compared with asialoglycopeptides from other serum proteins for their affinity to the rat liver in vivo. Considerable differences were observed and the following order of binding was established for the asialoglycopeptide preparations: bovine transferrin < human transferrin [Formula: see text] bovine fetuin [Formula: see text] human α1-acid glycoprotein.

Studies of the Metabolism of Asialotransferrins: Evidence that Transferrin Does not Undergo Desialylation in Vivo

1974 ◽  
Vol 46 (6) ◽  
pp. 763-774
Author(s):  
K.-L. Wong ◽  
P. A. Charlwood ◽  
M. W. C. Hatton ◽  
E. Regoeczi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biological Significance ◽  
Polyacrylamide Gel ◽  
Fractional Catabolic Rate ◽  
Sialic Acid Content ◽  
Catabolic Rate ◽  
Bovine Transferrin

1. Experiments are reported which aimed at determining whether transferrin loses sialyl residues from the carbohydrate side-chains during the biological lifetime of the molecule. To explore this possibility, transferrin fractions of relatively high sialic acid content (referred to as sialotransferrin) were prepared from purified rabbit and bovine transferrin by preparative polyacrylamide-gel electrophoresis. After labelling with 125I, the preparations were injected into a group of three rabbits each. From the plasma samples obtained between 1 h and 6–8 days after injection, transferrin was partially purified, mixed with 131I-labelled asialotransferrin of the corresponding species and run in preparative polyacrylamide-gel electrophoresis. In each specimen examined, the 125I radioactivity migrated ahead of the marker asialotransferrin, and no portion of the dose was detected with the electrophoretic mobility of asialotransferrin. 2. Evidence is presented that bovine transferrin desialylated in vitro remains detectable in the plasma of rabbits for intervals which are comparable with those found in previous studies with rabbit asialotransferrin. 3. A mathematical model is described for the computation of asialo- to sialotransferrin radioactivity ratios in the plasma, continuous desialylation of pulse-injected sialotransferrin being assumed. Calculations were made at various hypothetical rates of desialylation. 4. On the basis of the experimental data and the model it is concluded that transferrin (both rabbit and bovine) is not subjected to systematic desialylation in rabbits. Random desialylation of some transferrin could take place at rates less than 5% of the fractional catabolic rate of transferrin, which would be without any biological significance.

Sleep-promoting material from human urine and its relation to factor S from brain

1980 ◽  
Vol 238 (2) ◽  
pp. E116-E123
Author(s):  
J. M. Krueger ◽  
J. Bacsik ◽  
J. Garcia-Arraras
Keyword(s):  
Ion Exchange ◽  
Slow Wave ◽  
High Voltage ◽  
Human Urine ◽  
Time Course ◽  
Slow Wave Sleep ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Intraventricular Infusion ◽  
High Voltage Electrophoresis

A sleep-promoting factor was extracted from human urine. Intraventricular infusion of the purified material induced excess slow-wave sleep in rats and rabbits for 5--10 h after the infusion. Chemical properties of the urinary factor were similar to those of factor S derived from whole brains of sleep-deprived goats, sheep, and rabbits. The behavior of the urinary factor in two ion exchange chromatographic steps, high voltage electrophoresis, gel-filtration, and ultrafiltration was similar to that of factor S. Effects of the purified urinary factor on slow-wave sleep of rats and rabbits were similar in time-course and duration to those of factor S from brain. However, the factor obtained from human urine did not increase the amplitude of cortical slow waves to the same extent as did factor S from brains of sleep-deprived animals.

Pharmacokinetics, Serum Stability and Immunogenicity of A Polypeptide Inhibitor of B2GPI/Antibody Complexes Tested in Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2205-2205
Author(s):  
Andrew Porter ◽  
Natalia Beglova
Keyword(s):  
Human Serum ◽  
Serum Proteins ◽  
Conflicts Of Interest ◽  
Intraperitoneal Administration ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Mouse Serum ◽  
Carrier Protein ◽  
Serum Stability

Abstract Abstract 2205 Background: Antiphospholipid syndrome (APS) is an autoimmune disease with clinical features of thrombosis and pregnancy loss. Beta2-glycoprotein I (B2GPI) is the major antigen for APS-related antibodies. We engineered a polypeptide consisting of two ligand-binding A1 modules from the ApoE receptor 2. Previously, we demonstrated that this polypeptide, A1-A1, preferentially binds B2GPI/antibody complexes compared to B2GPI alone and efficiently inhibits the binding of B2GPI/antibody complexes to negatively charged phospholipids. Therefore, A1-A1 effectively interferes with two pathological mechanisms of B2GPI/antibody complexes: the binding to anionic phospholipids and ApoER2. In order to use A1-A1 to study pathological mechanisms of B2GPI/antibody complexes in vivo, we tested its pharmacokinetic, serum stability and immunogenicity in mice. To visualize A1-A1, we labeled it with a fluorescent probe Atto-488 attached to the N-terminus. Results: We monitored clearance of A1-A1 from the circulation after intraperitoneal and intravenous administration. After intraperitoneal administration, the concentration of A1-A1 in the blood reached its maximum at 30 min after injection and cleared from the blood in 6–8 hours. When A1-A1 was injected intravenously, 14% of A1-A1 remained in the blood 1 hour after administration and decreased to 4% in 3 hours. We assessed the binding of A1-A1 to serum proteins in both mouse and human serum by gel-filtration chromatography. Chromatograms of A1-A1 in both mouse and human serum collected just after mixing of A1-A1 with serum were almost identical to those collected after 2 hours of incubation at 37° C. About 90% of A1-A1 stays free from serum proteins. Previously, we demonstrated that A1-A1 has a favorable stability in human serum. More than 35% of A1-A1 remained in human serum after 15 days of incubation at 37° C. Here, we determined whether A1-A1 is cleaved by proteases in mouse serum. Degradation of A1-A1 was monitored by the reversed-phase HPLC by comparing the peak corresponding to intact A1-A1 and A1-A1 incubated with serum for 2 hours at 37° C. After incubation with mouse serum, A1-A1 eluted at the same time as intact A1-A1 and the intensity of the elution peak did not decrease, indicating that A1-A1 remains intact in mouse serum. To evaluate immunogenecity of A1-A1, we immunized mice with A1-A1, A1-A1 in the presence of adjuvant and A1-A1 conjugated to a carrier protein. A1-A1 did not induce detectable anti-A1-A1 IgG production even in the presence of adjuvant or carrier protein. Conclusions: A1-A1 has favorable properties for use in vivo. It stays in the circulation for more than one hour following intravenous injection. After intraperitoneal administration, A1-A1 is rapidly absorbed into blood and cleared in about 6 hours. A1-A1 is resistant to both human and mouse proteases, has low immunogenicity and its amount in the blood is not depleted by binding to serum proteins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Occupancy of sites of phosphorylation in inactive rat heart pyruvate dehydrogenase phosphate in vivo

1981 ◽  
Vol 193 (3) ◽  
pp. 935-946 ◽  
Author(s):  
G J Sale ◽  
P J Randle
Keyword(s):  
High Voltage ◽  
Pyruvate Dehydrogenase ◽  
Rat Heart ◽  
Alloxan Diabetes ◽  
Fold Increase ◽  
Diabetic Rats ◽  
Phosphorylation Sites ◽  
The Difference ◽  
High Voltage Electrophoresis

1. Inactive pyruvate dehydrogenase phosphate complexes were partially purified from hearts of fed, starved or alloxan-diabetic rats by using conditions that prevent phosphorylation or dephosphorylation. 2. Unoccupied sites of phosphorylation were assayed by incorporation of 32P from [gamma-32P]ATP into the complexes. Total sites of phosphorylation were assayed by the same method after complete reactivation, and thus dephosphorylation, of complexes by incubation with pyruvate dehydrogenase phosphate phosphatase. Occupancy is assumed from the difference (total sites–unoccupied sites). Percentage incorporation into individual sites was measured by high-voltage electrophoresis after tryptic digestion. 3. Values (means +/- S.E.M., in nmol of phosphate/unit of inactive complex) for total sites, occupied sites and percentage occupancies, with numbers of observations in parentheses were: fed, 2.1 +/- 0.04, 1.15 +/- 0.04, 54.8 +/- 1.6% (39); starved, 2.05 +/- 0.03, 1.85 +/- 0.03, 90.2 +/- 1.4% (28); alloxan-diabetic, 1.99 +/- 0.03, 1.72 +/- 0.03, 86.4 +/- 1.4% (68%). 4. Values (means +/- S.E.M. for percentage occupancy) for individual sites of phosphorylation in pyruvate dehydrogenase phosphate given in the order sites 1, 2 and 3 were : fed, 100 +/- 2.7, 27.8 +/- 1.6, 33.9 +/- .9; starved, 100 +/- 1.4, 76.2 +/- 2.0, 92.4 +/- 1.5; alloxan-diabetic, 100 +/- 1.2, 64.0 +/- 1.7, 94.6 +/- 1.4. 5. It is concluded that starvation or alloxan-diabetes leads to a 2–3-fold increase in the occupancy of phosphorylation sites 2 and 3 in pyruvate dehydrogenase phosphate in rat heart in vivo.

scholarly journals Molecular distinctions between heparan sulphate and heparin. Analysis of sulphation patterns indicates that heparan sulphate and heparin are separate families of N-sulphated polysaccharides

1985 ◽  
Vol 230 (3) ◽  
pp. 665-674 ◽  
Author(s):  
J T Gallagher ◽  
A Walker
Keyword(s):  
Uronic Acid ◽  
Heparan Sulphate ◽  
Analytical Data ◽  
Gel Filtration ◽  
Published Data ◽  
Sulphated Polysaccharides ◽  
Close Proximity ◽  
Sulphated Glycosaminoglycans ◽  
Very High ◽  
High Voltage Electrophoresis

Heparan sulphate and heparin are chemically related alpha β-linked glycosaminoglycans composed of alternating sequences of glucosamine and uronic acid. The amino sugars may be N-acetylated or N-sulphated, and the latter substituent is unique to these two polysaccharides. Although there is general agreement that heparan sulphate is usually less sulphated than heparin, reproducible differences in their molecular structure have been difficult to identify. We suggest that this is because most of the analytical data have been obtained with degraded materials that are not necessarily representative of complete polysaccharide chains. In the present study intact heparan sulphates, labelled biosynthetically with [3H]glucosamine and Na2(35)SO4, were isolated from the surface membranes of several types of cells in culture. The polysaccharide structure was analysed by complete HNO2 hydrolysis followed by fractionation of the products by gel filtration and high-voltage electrophoresis. Results showed that in all heparan sulphates there were approximately equal numbers of N-sulpho and N-acetyl substituents, arranged in a similar, predominantly segregated, manner along the polysaccharide chain. O-Sulphate groups were in close proximity to the N-sulphate groups but, unlike the latter, the number of O-sulphate groups could vary considerably in heparan sulphates of different cellular origins ranging from 20 to 75 O-sulphate groups per 100 disaccharide units. Inspection of the published data on heparin showed that the N-sulphate frequency was very high (greater than 80% of the glucosamine residues are N-sulphated) and the concentration of O-sulphate groups exceeded that of the N-sulphate groups. We conclude from these and other observations that heparan sulphate and heparin are separate families of N-sulphated glycosaminoglycans.

Chromatographie and Electrophoretic Properties of Synthetic Human Fibrinopeptides

1974 ◽  
Vol 32 (02/03) ◽  
pp. 651-658
Author(s):  
Robin McKenzie ◽  
D. S Pepper ◽  
A. B Kay
Keyword(s):  
Thin Layer ◽  
High Voltage ◽  
Low Voltage ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Molecular Weights ◽  
Electrophoretic Mobilities ◽  
Rf Values ◽  
Layer Chromatography ◽  
High Voltage Electrophoresis

SummarySome properties of synthetic human fibrinopeptides were studied by thin-layer chromatography, thin-layer electrophoresis and low voltage and high voltage paper electrophoresis. The Rf values and electrophoretic mobilities of the peptides in these systems were determined. In high voltage electrophoresis synthetic and natural (fibrinogen-derived) peptides migrated in an identical fashion.When gel filtration was performed in 0.05 M pyridine or 0.1 N ammonia, synthetic fibrinopeptides A and B appeared to be aggregated. In contrast, when filtration was performed in 1.3 M formic acid, the peptides eluted in positions corresponding to their monomeric molecular weights.In addition it was possible to quantitate synthetic fibrinopeptides by two colorimetric assays, the Sakaguchi reaction and the Folin-Ciocalteu method. Ultraviolet extinction coefficients for each peptide were also determined.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

In Vivo Studies on the Inhibition of Coagulation by Fractionated Heparin and by a Heparin Analogue I. Effects of Heparin Fractions

1981 ◽  
Vol 46 (03) ◽  
pp. 612-616 ◽  
Author(s):  
U Schmitz-Huebner ◽  
L Balleisen ◽  
F Asbeck ◽  
J van de Loo
Keyword(s):  
Molecular Weight ◽  
Day Treatment ◽  
Gel Filtration ◽  
Weight Fraction ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor ◽  
Heparin Fractions

SummaryHigh and low molecular weight heparin fractions obtained by gel filtration chromatography of sodium mucosal heparin were injected subcutaneously into six healthy volunteers and compared with the unfractionated substance in a cross-over trial. Equal doses of 5,000 U were administered twice daily over a period of three days and heparin activity was repeatedly controlled before and 2, 4, 8 hrs after injection by means of the APTT, the anti-Xa clotting test and a chromogenic substrate assay. In addition, the in vivo effect of subcutaneously administered fractionated heparin on platelet function was examined on three of the volunteers. The results show that s.c. injections of the low molecular weight fraction induced markedly higher anti-Xa activity than injections of the other preparations. At the same time, APTT results did not significantly differ. Unfractionated heparin and the high molecular weight fraction enhanced ADP-induced platelet aggregation and collagen-mediated MDA production, while the low molecular weight fraction hardly affected these assays, but potently inhibited thrombin-induced MDA production. All heparin preparations stimulated the release of platelet Factor 4 in plasma. During the three-day treatment periods, no side-effects and no significant changes in the response to heparin injections were detected.

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