scholarly journals Isolation and characterization of a mannose/N-acetylglucosamine/fucose-binding protein from rat liver

1981 ◽  
Vol 194 (1) ◽  
pp. 209-214 ◽  
Author(s):  
R Townsend ◽  
P Stahl
Keyword(s):  
Affinity Chromatography ◽  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Calcium Dependent ◽  
Isolation And Characterization

A rat liver mannan-binding protein was isolated by affinity chromatography on invertase–Sepharose by a modification of the method of Kawasaki, Etoh & Yamashina [(1978) Biochem. Biophys. Res. Commun. 81, 1018-1024] and by a new method involving chromatography on mannose-Sepharose. The binding protein appears as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with an apparent mol.wt. of approx. 30000. Binding of 125I-labelled mannan is saturable and inhibited by mannose, N-acetylglucosamine, or L-fucose but not by galactose or mannose 6-phosphate. Neoglycoproteins containing mannose, N-acetylglucosamine, or L-fucose, but not galactose, are inhibitory. The neoglycoproteins are 10000-fold more effective (based on moles of sugar) than are free monosaccharides as inhibitors. 125I-labelled mannan binding to the binding protein is calcium-dependent.

The isolation, purification and some properties of pheromaxein, the pheromonal steroid-binding protein, in porcine submaxillary glands and saliva

1988 ◽  
Vol 118 (1) ◽  
pp. 47-NP ◽  
Author(s):  
W. D. Booth ◽  
C. A. White
Keyword(s):  
Liquid Chromatography ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Submaxillary Gland ◽  
Fast Protein Liquid Chromatography ◽  
Steroid Binding ◽  
Steroid Binding Protein

ABSTRACT Pheromaxein, the 16-androstene steroid-binding protein with a relative molecular mass of 15 000 was isolated in sub-milligram quantities from the submaxillary gland and saliva of the Gottingen miniature boar, after a fourfold purification involving the following methods: ultrafiltration for submaxillary gland cytosols and ethanol precipitation for saliva, Concanavalin-A-Sepharose affinity chromatography, sodium dodecyl sulphate polyacrylamide gel electrophoresis, 'Extractigel-D' affinity chromatography (to remove sodium dodecyl sulphate) and fast protein-liquid chromatography. Yields of purified pheromaxein obtained after fast protein-liquid chromatography represented 10–20% of total protein present in an ultrafiltrate of a submaxillary gland cytosol. Fast protein-liquid chromatography separated the α- and β-charge isomers of pheromaxein which were shown to have isoelectric points of 4·78 and 5·35 respectively on flat-bed isoelectric focusing. Some data are provided for the variable occurrence of the isomeric forms of pheromaxein in relation to different breeds of pig. Five 16-unsaturated steroids showed the highest binding to pheromaxein. Other steroids of the 5α- and 5β-androstane series also showed some binding to pheromaxein, i.e. 17β-hydroxy-5α-androstan-3-one (19·2%), with 5α-androstan-3-one, which has a similar urinous odour to 5α-androst-16-en-3-one, showing the greatest binding (42·6%) relative to 5α-androst-16-en-3-one (100%). J. Endocr. (1988) 118, 47–57

Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization

1989 ◽  
Vol 67 (8) ◽  
pp. 411-421 ◽  
Author(s):  
Om P. Malhotra
Keyword(s):  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Dependent ◽  
Isolation And Characterization ◽  
Carboxyglutamic Acid ◽  
K Deficiency ◽  
Agar Gel Electrophoresis

Isolation and characterization of γ-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate Polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.

Glycosylation of chicken haptoglobin: Isolation and characterization of three molecular variants and studies of their distribution in hen plasma before and after turpentine-induced inflammation

1988 ◽  
Vol 66 (3) ◽  
pp. 208-217 ◽  
Author(s):  
Francisco Delers ◽  
Gérard Strecker ◽  
Robert Engler
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Isolation And Characterization ◽  
Molecular Variants ◽  
Hemoglobin Binding ◽  
Significant Difference ◽  
Before And After ◽  
Carbohydrate Unit

Chicken haptoglobin (Hp), a hemoglobin-binding protein isolated from chicken plasma, is composed of three molecular variants that react differently with concanavalin A (ConA). These glycosylation variants of chicken Hp have been isolated by affinity chromatography using Sepharose-bound ConA. They differ in their molecular weight, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of the glycopeptides obtained after pronase digestion of these variants yielded two types of structures: one, reactive with ConA, corresponded to a biantennary N-linked carbohydrate unit and one, unreactive with ConA, corresponded to a triantennary unit. The strongly ConA-reactive Hp variant bears only two biantennary units and the nonreactive Hp variant bears only two triantennary units; the weakly reactive Hp variant bears equal amounts of both units. The distribution of Hp glycosylation variant does not show any significant difference when obtained from the plasma of laying hens before and after turpentine-induced inflammation.

Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

1983 ◽  
Vol 29 (10) ◽  
pp. 1361-1368 ◽  
Author(s):  
Thomas P. Poirier ◽  
Stanley C. Holt
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Alkaline Phosphatases ◽  
Purification And Characterization ◽  
Molecular Weights ◽  
Sds Page ◽  
Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

scholarly journals Use of immuno-blot techniques to discriminate between the glutathione S-transferase Yf, Yk, Ya, Yn/Yb and Yc subunits and to study their distribution in extrahepatic tissues. Evidence for three immunochemically distinct groups of transferase in the rat

1986 ◽  
Vol 233 (3) ◽  
pp. 779-788 ◽  
Author(s):  
J D Hayes ◽  
T J Mantle
Keyword(s):  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Glutathione S Transferase ◽  
Specific Expression ◽  
Glutathione S Transferases ◽  
Extrahepatic Tissues ◽  
High Concentrations

The glutathione S-transferases are dimeric enzymes whose subunits can be defined by their mobility during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as Yf (Mr 24,500), Yk (Mr 25,000), Ya (Mr 25,500), Yn (Mr 26,500), Yb1 (Mr 27,000), Yb2 (Mr 27,000) and Yc (Mr 28,500) [Hayes (1986) Biochem. J. 233, 789-798]. Antisera were raised against each of these subunits and their specificities assessed by immuno-blotting. The transferases in extrahepatic tissues were purified by using, sequentially, S-hexylglutathione and glutathione affinity chromatography. Immune-blotting was employed to identify individual transferase polypeptides in the enzyme pools from various organs. The immuno-blots showed marked tissue-specific expression of transferase subunits. In contrast with other subunits, the Yk subunit showed poor affinity for S-hexylglutathione-Sepharose 6B in all tissues examined, and subsequent use of glutathione and glutathione affinity chromatography. Immuno-blotting was employed to identify a new cytosolic polypeptide, or polypeptides, immunochemically related to the Yk subunit but with an electrophoretic mobility similar to that of the Yc subunit; high concentrations of the new polypeptide(s) are present in colon, an organ that lacks Yc.

scholarly journals Purification and characterization of a new cytosolic glutathione S-transferase (glutathione S-transferase X) from rat liver

1983 ◽  
Vol 215 (3) ◽  
pp. 617-625 ◽  
Author(s):  
T Friedberg ◽  
U Milbert ◽  
P Bentley ◽  
T M Guenther ◽  
F Oesch
Keyword(s):  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Glutathione S Transferase ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases

A hitherto unknown cytosolic glutathione S-transferase from rat liver was discovered and a method developed for its purification to apparent homogeneity. This enzyme had several properties that distinguished it from other glutathione S-transferases, and it was named glutathione S-transferase X. The purification procedure involved DEAE-cellulose chromatography, (NH4)2SO4 precipitation, affinity chromatography on Sepharose 4B to which glutathione was coupled and CM-cellulose chromatography, and allowed the isolation of glutathione S-transferases X, A, B and C in relatively large quantities suitable for the investigation of the toxicological role of these enzymes. Like glutathione S-transferase M, but unlike glutathione S-transferases AA, A, B, C, D and E, glutathione S-transferase X was retained on DEAE-cellulose. The end product, which was purified from rat liver 20 000 g supernatant about 50-fold, as determined with 1-chloro-2,4-dinitrobenzene as substrate and about 90-fold with the 1,2-dichloro-4-nitrobenzene as substrate, was judged to be homogeneous by several criteria, including sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, isoelectric focusing and immunoelectrophoresis. Results from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration indicated that transferase X was a dimer with Mr about 45 000 composed of subunits with Mr 23 500. The isoelectric point of glutathione S-transferase X was 6.9, which is different from those of most of the other glutathione S-transferases (AA, A, B and C). The amino acid composition of transferase X was similar to that of transferase C. Immunoelectrophoresis of glutathione S-transferases A, C and X and precipitation of various combinations of these antigens by antisera raised against glutathione S-transferase X or C revealed that the glutathione S-transferases A, C and X have different electrophoretic mobilities, and indicated that transferase X is immunologically similar to transferase C, less similar to transferase A and not cross-reactive to transferases B and E. In contrast with transferases B and AA, glutathione S-transferase X did not bind cholic acid, which, together with the determination of the Mr, shows that it does not possess subunits Ya or Yc. Glutathione S-transferase X did not catalyse the reaction of menaphthyl sulphate with glutathione, and was in this respect dissimilar to glutathione S-transferase M; however, it conjugated 1,2-dichloro-4-nitrobenzene very rapidly, in contrast with transferases AA, B, D and E, which were nearly inactive towards that substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Accumulation of UDP-glucose pyrophosphorylase by axenically grown amoebae of Dictyostelium discoideum

1979 ◽  
Vol 177 (1) ◽  
pp. 21-28 ◽  
Author(s):  
B D Hames ◽  
B A Hodson
Keyword(s):  
Affinity Chromatography ◽  
Dictyostelium Discoideum ◽  
Sodium Dodecyl Sulphate ◽  
Vegetative Growth ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Klebsiella Aerogenes ◽  
Axenic Medium ◽  
Axenic Growth

Amoebae of the slime mould Dictyostelium discoideum AX2 possess only low UDP-glucose pyrophosphorylase activity when grown on autoclaved Klebsiella aerogenes (approx. 30 units/mg of protein), but accumulate the enzyme to approx. 150-200 units/mg of protein during vegetative growth in axenic medium. The vegetative accumulation of UDP-glucose pyrophosphorylase by axenically grown cells is prevented if autoclaved K. aerogenes are included in the axenic medium, suggesting the absence of a specific inducer. Affinity chromatography using anti-(UDP-glucose pyrophosphorylase) antibody and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicate that the enzyme accumulated during axenic growth and that normally accumulated during development are immunologically cross-reactive and that both are composed of two subunits with mol.wts. 55,600 and 57,500 present in approximately equal amounts in the active enzyme.

scholarly journals Purification and characterization of a plasminogen activator secreted by a pig kidney cell line

1984 ◽  
Vol 219 (3) ◽  
pp. 971-978 ◽  
Author(s):  
M Sudol ◽  
E Reich
Keyword(s):  
Plasminogen Activator ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Pig Kidney ◽  
Reducing Conditions ◽  
Pig Kidney Cells

The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.

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