IMMUNOCHEMICAL CHARACTERIZATION OF PROTEINS IN CATTLE URINE AND THEIR COMPLEXES WITH SILICIC ACID

1978 ◽  
Vol 58 (3) ◽  
pp. 435-442
Author(s):  
C. B. BAILEY
Keyword(s):  
Urinary Tract ◽  
Sialic Acid ◽  
Silicic Acid ◽  
Serum Proteins ◽  
Gel Filtration ◽  
Urinary Calculi ◽  
The Other ◽  
Single Protein ◽  
Serum Components

Electrophoresis of total non-dialyzable solids (TNDS) from cattle urine showed the presence of components with mobilities covering the same range as noted for serum. Although most of the components had mobilities in the albumin to α-globulin region, they appeared to contain more glycoprotein than serum components with similar mobilities. Immunoelectrophoresis of TNDS against rabbit antibovine serum and rabbit anti-TNDS revealed the presence of four components giving reactions of identity with serum proteins and of one that originated from the urinary tract. Gel filtration of TNDS on Sephadex G-200 produced six poorly resolved peaks, four of which were shown by immunoelectrophoresis to contain the four serum components of TNDS. The fifth peak contained the urinary tract component. This component constituted about half the total weight of the proteinaceous constituents of TNDS and contained considerably more sialic acid than the other components. The substance represented by the sixth peak obtained by gel filtration was unidentified because it failed to elicit antibody formation in the rabbit. It was low in protein and sialic acid but had a much larger absorbance at 280 nm than the other constituents. Reaction of TNDS with polymerized silicic acid removed half the TNDS from solution by formation of an insoluble complex. One component was completely removed from solution and portions of others were partially removed by this procedure. It was concluded that in the process of formation of siliceous urinary calculi in cattle, precipitation of polymerized silicic acid is a generalized process that does not rely on the presence in urine of any single protein species.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

Systematic characterization of human prostatic fluid proteins with two-dimensional electrophoresis.

1984 ◽  
Vol 30 (12) ◽  
pp. 2026-2030 ◽  
Author(s):  
Y C Tsai ◽  
H H Harrison ◽  
C Lee ◽  
J A Daufeldt ◽  
L Oliver ◽  
...  
Keyword(s):  
Benign Prostatic Hyperplasia ◽  
Serum Proteins ◽  
Prostatic Hyperplasia ◽  
Affinity Column ◽  
Two Dimensional ◽  
Differential Analysis ◽  
Protein Patterns ◽  
Prostatic Fluid ◽  
Serum Components

Abstract We present a systematic analysis of human prostatic fluid with two-dimensional gel electrophoresis (the ISO-DALT system) and a characterization of normal and disease-related protein patterns. A reference map for prostatic fluid proteins was established by analysis of pooled prostatic fluids from 80 men (age less than or equal to 50 years) without prostatic lesions. Proteins in prostatic fluid that share immunogenicity with serum proteins were identified by use of antibody to whole human-serum protein in an affinity-column fractionation of a reference pool and differential analysis of the absorbed (serum components) and unabsorbed (non-serum components) fractions. Individual prostatic fluids from 30 patients (eight with prostatic cancer, 10 with prostatitis and benign prostatic hyperplasia, six with benign prostatic hyperplasia alone, and six with asymptomatic chronic prostatitis) were scored qualitatively with respect to the presence or absence of 57 major prostatic fluid proteins. Statistically significant, disease-correlated alterations were observed for at least eight of the proteins so scored.

Chemical characterization of urinary glycopeptides.

1987 ◽  
Vol 33 (12) ◽  
pp. 2214-2219
Author(s):  
H Matsue ◽  
K Takagaki ◽  
K Honda ◽  
Y Nakagawa ◽  
F Gejyo ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Carbohydrate Chain ◽  
Cetylpyridinium Chloride ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Small Peptide ◽  
Supernatant Liquid ◽  
Peptide Moiety ◽  
Peptide Linkage

Abstract We prepared human urinary glycopeptides from the supernatant liquid remaining after precipitation of the nondialyzable fraction with cetylpyridinium chloride. Using cation exchange and affinity chromatographies and gel filtration, we obtained 28 glycopeptide subfractions. By compositional analyses of sugar and amino acid, and by reducing-terminal analyses after reduction with NaBH4, we determined the size of the carbohydrate moiety and the types of carbohydrate-peptide linkage involved. We isolated several glycopeptides not previously described: six with sialic acid, two with fucose, two with glucose, and one with N-acetylgalactosamine. The sialic acid glycopeptides had a short carbohydrate chain of the O-glycoside type. The fucose-containing glycopeptides were fucosyllactosaminoglycans. The glucose glycopeptides were polymers linked to a small peptide moiety. The N-acetylgalactosamine-rich glycopeptide was found in an N-glycoside-type fraction, with N-acetylgalactosamine at the nonreducing terminal.

scholarly journals CHEMICAL CHARACTERIZATION OF GLYCOPEPTIDES FROM HUMAN γM-GLOBULINS

1968 ◽  
Vol 128 (4) ◽  
pp. 699-713 ◽  
Author(s):  
Joseph M. Davie ◽  
C. Kirk Osterland
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Gel Filtration ◽  
Chemical Characterization ◽  
Carbohydrate Composition ◽  
Total Percentage ◽  
Group I ◽  
Group Ii ◽  
Group 1

A survey of human pathological macroglobulins revealed that γM can be divided into at least two groups on the basis of carbohydrate composition. Differences between the two groups exist in the total percentage of carbohydrate (10.69 ± 1.49% for group 1, 7.71 ± 0.65% for group II) which is attributable to variation in hexose content. Glycopeptides from macroglobulins of each group were purified from pronase digests and characterized chemically. Macroglobulins from each group contain three types of oligosaccharides. Glycopeptide I for each group consisted of mannose, galactose, and NAG with a ratio of 3:2:1 for group I and a ratio of 2:1:2 for group II. Glycopeptide II consisted of mannose, galactose, and NAG (9:1:2) for group I, and mannose, fucose, galactose, and NAG (2:1:3:2) for group II. Glycopeptide III in both groups consisted of mannose, fucose, galactose, NAG, and sialic acid with a ratio of 6:2.5:2.5:5.5:2 for group I and a ratio of 5:1:1:6:1 for group II. Molecular weight estimations by gel filtration indicates that there are 10 glycopeptides I and II and 20 units of glycopeptide III per molecule of γM.

scholarly journals Isolation and fractionation of glycopeptides from porcine thyroglobulin

1971 ◽  
Vol 123 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Minoru Fukuda ◽  
Fujio Egami
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Chemical Composition ◽  
Column Chromatography ◽  
Gel Filtration ◽  
Paper Electrophoresis ◽  
Alkaline Treatment ◽  
The Other

1. Glycopeptides were isolated by gel filtration on Sephadex G-25 and Sephadex G-50 from a Pronase digest of porcine thyroglobulin. 2. Isolated glycopeptides were separated into five main fractions on a column of DEAE-Sephadex A-25. Of these fractions I to III were further purified by SE-Sephadex C-25 or DEAE-Sephadex A-25 column chromatography. Several of the purified glycopeptides were homogeneous on paper electrophoresis. 3. Based on the chemical composition and molecular weight of the fractionated glycopeptides, two distinct types of heterosaccharide chain were demonstrated. 4. One type of the heterosaccharide unit consisted of four to eight residues of mannose and two residues of glucosamine and had a molecular weight of 1000–1700. The other type of unit contained sialic acid, fucose and galactose in addition to mannose and glucosamine and had a molecular weight of about 3600. 5. Mild alkaline treatment of the glycopeptide did not result in the destruction of threonine and serine. 2-Acetamido-1-N-(4-l-aspartyl)-2-deoxy-β-d-glucopyranosylamine was isolated from partial acid hydrolysates.

SOLUBILIZATION AND PARTIAL CHARACTERIZATION OF THYROID MEMBRANE TSH BINDING PROTEINS

1979 ◽  
Vol 92 (3) ◽  
pp. 512-521 ◽  
Author(s):  
B. Czarnocka ◽  
J. Nauman ◽  
G. Adler ◽  
W. Kiełczyński
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Gel Filtration ◽  
Soluble Proteins ◽  
The Other ◽  
High Affinity ◽  
Triton X ◽  
Maximal Capacity

ABSTRACT Crude plasma membranes obtained from bovine thyroids were found to possess one class of high affinity, low capacity binding sites for TSH with average association constant (Ka) of 1.301 × 109 m−1 and maximal capacity 8.76 × 10−10 m/mg of protein. Treatment of crude membranes fraction with 0.1 % Triton X-100 and the subsequent sonication in ultrasonic disintegrator resulted in solubilization of membranes proteins with mean recovery of 40.0 ± 6.2 %. Soluble proteins retained the property to bind [125I]TSH, but the binding of the hormone was decreased. The removal of the detergent from the solubilizate by gel filtration on Sephadex LH-20 increased the binding of TSH well above that demonstrated for crude thyroid membranes. The chromatography of soluble proteins on Ultrogel AcA-44 revealed the presence of two TSH binding proteins, one with the molecular weight (m.w.) above 130 000 daltons and the other with the m.w. approximately 30 000 daltons. The electrofocusing of solubilizate on Ampholine resulted in two protein peaks, one at pH 4.0–4.1 and the other at pH 4.4–4.6. The latter peak was shown to bind [125I]TSH specifically. The present results have confirmed the heterogeneous character of solubilized TSH receptor preparation and have shown that the hormone binding sites belong to acid proteins.

scholarly journals Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1971 ◽  
Vol 123 (5) ◽  
pp. 845-853 ◽  
Author(s):  
D. Snary ◽  
A. Allen
Keyword(s):  
Sialic Acid ◽  
Gastric Mucosa ◽  
Gel Filtration ◽  
Water Soluble ◽  
Group Activities ◽  
Isolation And Characterization ◽  
Two Peaks ◽  
Sepharose 4B

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with 14C-labelled carbohydrate or with 3H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-14C]glucose or [G-3H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s025,w values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B–C, which were chemically, biosynthetically and immunologically very similar.

scholarly journals Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein

1984 ◽  
Vol 221 (2) ◽  
pp. 379-392 ◽  
Author(s):  
S R Carlsson ◽  
T Stigbrand
Keyword(s):  
Gel Filtration ◽  
Glycosylation Site ◽  
The Other ◽  
Complex Type ◽  
Mouse Thymocyte ◽  
Lentil Lectin ◽  
High Mannose ◽  
Sepharose 4B ◽  
Oligosaccharide Structures

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary ‘complex-type’ with terminal fucose; IIA, biantennary ‘complex-type’ without fucose; IIB, biantennary ‘complex-type’ with fucose; III, a mixture of ‘high-mannose’ chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of ‘high-mannose’ type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with ‘complex-type’ chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three ‘complex-type’ chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.

Characterization of an immunogenic fraction of Pasteurella multocida culture filtrates

1977 ◽  
Vol 23 (2) ◽  
pp. 197-201 ◽  
Author(s):  
Kunwar K. Srivastava ◽  
John W. Foster
Keyword(s):  
Culture Filtrate ◽  
Pasteurella Multocida ◽  
Gel Filtration ◽  
The Other ◽  
Rabbit Skin ◽  
Culture Filtrates ◽  
Homologous Challenge

An immunogenic fraction (IF) of Pasteurella multocida strain P-1059 was separated from culture filtrate by Sephadex gel filtration. Additional fractionation of IF with aqueous ether resulted in the glycoprotein-like preparation (GLP) while extraction with aqueous phenol provided the lipopolysaccharide-like preparation (LPP). The unextracted IF contained carbohydrate, protein, and lipid; the GLP contained carbohydrate and protein; and the LPP contained carbohydrate and lipid. The GLP was maximally protective for mice against homologous challenge, and was medially toxic in rabbit skin when compared to the other culture-filtrate preparations; the LPP was maximally toxic in rabbit skin, and was least protective for mice; and the unextracted IF was medially protective for mice, and was least toxic in rabbit skin.

Purification and characterization of exo-β-1,3-glucanase from a hatching supernatant of Strongylocentrotus intermedius

1983 ◽  
Vol 61 (1) ◽  
pp. 54-62 ◽  
Author(s):  
Kiyoshi Takeuchi
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Protein Band ◽  
Divalent Ions ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Single Protein

Exo-β-1,3-glucanase from the sea urchin embryos was purified 114-fold from the initial hatching supernatant by the following procedures: (a) gel filtration on Sephadex G-100; (b) hydrophobic chromatography on 4-phenylbutylamine-Sepharose (PBA-Sepharose); (c) two ion-exchange chromatographic steps on DEAE-cellulose; (d) gel filtration on Ultrogel AcA 34; (e) gel filtration on Sephadex G-100. The purified enzyme contained 2.2% carbohydrate and gave a single protein band corresponding to a molecular weight of 136 000 following electrophoresis on sodium dodecyl sulfate (SDS) – urea – polyacrylamide gel. Gel filtration on Ultrogel AcA 34 with a nondenaturing solvent gave a molecular weight of 130 000 ± 6000. The enzyme displayed an optimum pH at 5.0–5.5 and hydrolysed laminarin and PS(curdlan)-beads at the nonreducing ends, releasing glucose. Although activity of the purified enzyme was not affected by SDS, urea, some divalent ions, and 2-mercaptoethanol, both dithiothreitol and Hg2+ were markedly inhibitory.

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