Studies on Z-Fraction. I. Isolation and Partial Characterization of Low Molecular Weight Ligand-Binding Protein from Rat Hepatic Cytosol

1975 ◽  
Vol 53 (3) ◽  
pp. 493-500 ◽  
Author(s):  
Margaret Warner ◽  
Allen H. Neims
Keyword(s):  
Molecular Weight ◽  
Ligand Binding ◽  
Dissociation Constants ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Partial Characterization ◽  
Fraction I ◽  
Z Protein

The Z-fraction has been defined operationally as a ligand-binding (bilirubin sulfobromophthalein) portion of rat hepatic cytosol that elutes in the molecular weight region of 104 daltons after gel filtration. Polyacrylamide gel electrophoreses under different conditions, as well as binding stoichiometry, confirm the anticipated heterogeneity of the Z-fraction. Three factors have contributed to the subsequent resolution of the Z-fraction and partial characterization of that protein within the fraction with ligand-binding properties (Z-protein): (1) the use of hexachlorophene as ligand; (2) the inclusion of glycerol, 20%, during isolation to prevent aggregation and loss of binding-activity; and (3) the development of a charcoal binding assay. Upon ion exchange chromatography, the Z-fraction resolves into a group of distinct protein components and an unidentified material with a high 260/280 nm absorbancy ratio. The one protein component with binding capacity exhibits homogeneity on polyacrylamide gel electrophoresis (11% gel, Ann. N.Y. Acad. Sci. 121, 404–427, 1964; and 15% gel with SDS). With use of the charcoal method, apparent dissociation constants for the interaction between Z-protein and hexachlorophene, bilirubin and L-thyroxine, were found to be 20, 50, and 350 μM, respectively. The Scatchard plot generated upon extrapolation an n value of 1.0 with assumption of a molecular weight for Z-protein of 104 daltons.

Prothrombin Metz : Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M Ribieto ◽  
J Elion ◽  
D Labie ◽  
F Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion. Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Val-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin l (Pl) and the appearance of abnormal intermediates migrating faster than Pl.

Prothrombin Metz: Purification and Characterization of a Variant of Human Prothrombin

1979 ◽  
Author(s):  
M.J. Rabiet ◽  
J. Elion ◽  
D. Labie ◽  
F. Josso
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Polyacrylamide Gel ◽  
Cleavage Pattern ◽  
Purification And Characterization ◽  
Double Immunodiffusion ◽  
The Family

For the purification of the abnormal prothrombin (Pt Metz), advantage has been taken of the existence in the family of three siblings who, being double heterozygotes for Pt Metz and a hypoprothrombinemia, have no normal Pt. Purification procedures included barium citrate adsorption and chromatography on DEAE Sephadex as for normal Pt. As opposed to some other variants (Pt Barcelona and Madrid), Pt Metz elutes as a single symetrical peak. By SDS Polyacrylamide gel electrophoresis, this material is homogeneous and appears to have the same molecular weight as normal Pt. Comigration of normal and abnormal Pt in the absence of SDS, shows a double band suggesting an abnormal charge for the variant. Pt Metz exhibits an identity reaction with the control by double immunodiffusion.Upon activation by factor Xa, Pt Metz can generate amydolytic activity on Bz-Phe-Va1-Arg-pNa (S2160), but only a very low clotting activity. Clear abnormalities are observed in the cleavage pattern of Pt Metz when monitored by SDS gel electrophoresis. The main feature are the accumulation of prethrombin 1 (P1) and the appearance of abnormal intermediates migra-ti ng faster than P1.

Purification and Characterization of Ginsenoside-Hydrolyzing β-Glucosidase from Wheat Bran

2013 ◽  
Vol 641-642 ◽  
pp. 906-909
Author(s):  
Chun Zhi Zhang ◽  
Ming Chen ◽  
Hai Chen Guo ◽  
Guo Ren Zu ◽  
Li Chen
Keyword(s):  
Molecular Weight ◽  
Wheat Bran ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Purification And Characterization ◽  
Enzyme Solution ◽  
Crude Enzyme Solution

The ginsenoside-hydrolyzing β-glucosidase that can converse the major ginsenosides into the minor ginsenosides was isolated from wheat bran, and the enzyme was purified and characterized. The crude enzyme solution extracted from wheat bran could hydrolyse the protopanaxadiol-type ginsenosides such as Rb1, Rc, Rd and Rg3, but could not hydrolyse the protopanaxatriol-type ginsenosides such as Re and Rg2. The enzyme fractionated on the DEAE-Cellulose DE-52 column was purified to one spot in SDS polyacrylamide gel electrophoresis, and the molecular weight of enzyme in the fraction 34, 47, and 61 was approximately 62 kDa, 62 kDa, and 68 kDa, respectively.

In Yivo Behavior of Human Fibrinogen and Fragments X, D and E.

1975 ◽  
Author(s):  
Nicole Ardaillou ◽  
Lise Dray ◽  
Marie-José Larrieu
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Human Fibrinogen ◽  
Fraction I ◽  
Stage 1 ◽  
Lower Slope

Fragments X, D and E were obtained from plasmin digestion of purified human fibrinogen (fraction I-O). Fragment × was isolated from stage 1 digest by filtration on Sephadex G-200 by Budzinski and Marder, and fragments D and E in our laboratory by Pevikon block electrophoresis. The purified fragments were identified by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. Human fibrinogen and fragments X, D and E were labelled with 125iodine by the lactoperoxidase method. Plasma disappearance curves were studied in rabbits. A two exponential curve was obtained with all of them, with second exponential (lower slope) being clearly predominant. The half-lifes of the second exponentials were different for each fragment: there appears to be a relationship between the molecular weight of the fragments and their half-lifes. Values observed (mean±s.e.m.) were: fibrinogen 49.34±5.4 hours, fragment X 6.14±0.44 hours, fragment D 2.25±0.20 hours and fragment E 1.53±0.13 hours. No fragment was found either to polymerize or to bind to plasma proteins in vivo following injection into rabbits as demonstrated by the elution peaks obtained after filtration through Sephadex G-200.

Purification and partial characterization of cysteine-glutamate transaminase from rat liver

1977 ◽  
Vol 55 (9) ◽  
pp. 958-964 ◽  
Author(s):  
M. P. C. Ip ◽  
R. J. Thibert ◽  
D. E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Labile Hydrogen ◽  
Apparent Homogeneity

Cysteine-glutamate transaminase (cysteine aminotransferase; EC 2.6.1.3) has been purified 149-fold to an apparent homogeneity giving a specific activity of 2.09 IU per milligram of protein with an overall yield of 15%. The isolation procedures involve the preliminary separation of a crude rat liver homogenate which was submitted sequentially to ammonium sulfate fractionation, TEAE-cellulose column chromatography, ultrafiltration, and isoelectrofocusing. The final product was homogenous when examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). A minimal molecular weight of 83 500 was determined by Sephadex gel chromatography. The molecular weight as estimated by polyacrylamide gel electrophoresis in the presence of SDS was 84 000. The purified enzyme exhibited a pH optimum at 8.2 with cysteine and α-ketoglutarate as substrates. The enzyme is inactivated slowly when kept frozen and is completely inactivated if left at room temperature for 1 h. The enzyme does not catalyze the transamination of α-methyl-DL-cysteine, which, when present to a final concentration of 10 mM, exhibits a 23.2% inhibition of transamination of 30 mM of cysteine. The mechanism apparently resembles that of aspartate-glutamate transaminase (EC 2.6.1.1) in which the presence of a labile hydrogen on the alpha-carbon in the substrate is one of the strict requirements.

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