Properties of triiodothyronine-binding proteins in liver cytosol of rat

1983 ◽  
Vol 61 (9) ◽  
pp. 1035-1041 ◽  
Author(s):  
D. Bellabarba ◽  
S. Bédard ◽  
J. -G. Lehoux
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Binding Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Gel Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Liver Cytosol

The electrophoretic mobility and the sedimentation coefficient were determined in partially purified preparations of both rat liver cytosol and serum triiodothyronine (T3)-binding proteins. Crude cytosol and serum, each labeled with [125I]T3, were filtered through a Sephadex G-100 column. The cytosol yielded a single T3-binding peak, whereas three binding components were recognized in the serum. Protamine sulfate precipitated the cytosol T3-binding protein, but had no effect on the serum T3-binders. The cytosol protein and the three binding proteins from serum were analyzed by polyacrylamide gel electrophoresis and sucrose density gradient centrifugation. The cytosol binder migrated as a single peak on gel electrophoresis with an Rf of 0.53, whereas the serum proteins had Rfs between 0.27–0.33. The sedimentation coefficient of the cytosol protein was 6.3 S, whereas it was 4.1 S for the major binding protein of the serum. These data indicate that: (i) preliminary purification by gel chromatography is a useful step for better characterization of the T3-binding proteins of the cytosol and serum; (ii) the cytosol binder is an acidic protein with completely different properties from those of the serum T3-binding proteins.

scholarly journals Affinity purification of cellular retinoic acid-binding protein on 14-carboxy-13-cis-retinamide-sepharose 4B

1989 ◽  
Vol 262 (3) ◽  
pp. 917-922 ◽  
Author(s):  
R K Singh ◽  
B P Sani ◽  
M I Dawson ◽  
Y F Shealy
Keyword(s):  
Retinoic Acid ◽  
Carboxy Group ◽  
Binding Proteins ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biologically Active ◽  
Amide Bond ◽  
Gel Chromatography ◽  
Fatty Acid Binding ◽  
Sepharose 4B

A biologically active bifunctional retinoid, ethyl 14-carboxyretinoate, has been synthesized and shown to bind cellular retinoic acid (RA)-binding protein (CRABP) via its free carboxy group. We describe herein the synthesis of 14-carboxy-13-cis-retinamide-Sepharose 4B, which is an affinity matrix bearing an all-trans-RA moiety, and thus was used to purify and characterize CRABP from chick-embryo skin. An amide bond was first formed between the free carboxy group of the retinoid and a primary amino group of aminohexyl-Sepharose 4B, by reaction with carbodi-imide, and the ester group of the resin-bound retinoid was then hydrolysed in an alkaline medium. Polyacrylamide-gel electrophoresis and f.p.l.c. Superose column-chromatographic analysis demonstrated that the affinity-purified CRABP (Mr 15,000) was close to electrophoretic homogeneity (greater than 90%) and specifically interacts with RA. By using affinity gel chromatography, conversion of holo-CRABP into apo-CRABP by treatment with p-hydroxymercuribenzoate and a possible involvement of a thiol group in RA binding to CRABP were established. This affinity procedure provides several advantages: (i) 14-carboxy-13-cis-retinamide-Sepharose exhibited high efficiency and selectivity for RA-binding protein (i.e. retinol- or fatty-acid-binding proteins did not bind); (ii) the presence of the amide linkage between the ligand and the matrix makes this affinity resin relatively stable to cytosolic enzymes; and (iii) other RA-binding proteins, e.g. nuclear receptor(s), may be purified.

Purification and Characterization of a 3,17 β-Hydroxysteroid Dehydrogenase from Streptomyces hydrogenans

1979 ◽  
Vol 34 (7-8) ◽  
pp. 533-540 ◽  
Author(s):  
Helmut Duchmann ◽  
Lothar Träger
Keyword(s):  
Gel Electrophoresis ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulfate ◽  
Sedimentation Coefficient ◽  
Hydroxysteroid Dehydrogenase ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization

3,17 β-Hydroxysteroid dehydrogenase has been enriched and purified from cytosol of Streptomyces hydrogenans. After ammonium sulfate precipitation and filtration on Sephadex G-100 the enzyme was finally purified by preparative gel electrophoresis and DEAE-Sephadex A-50 chro­matography. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate gave a single band of mobility corresponding to molecular weight of 70 200 ± 2 500. 3 β-. 17 β- as well as 20 β-hydroxy steroids were dehydrogenated by the enzyme in the presence of NAD+. The dehydrogenation proceeded faster than the reduction of the corresponding ketosteroids in the presence of NADH. The enzyme does not accent NADP+ or NADPH as co-substrates. The apparent Km values were calculated to be 11 μᴍ for 5 α-dihydrotestosterone, 20 μᴍ for testosterone ana 68 μᴍ for epiandrosterone in the NAD+-driven reaction, 1.8 x 10-4 m for NADH+ and 1.9 x 10-4 ᴍ for NADH. The catalytic activity was influenced by the ratio of NAD+/ATP. The inhibition by ATP appears to be of a competitive type with respect to NAD+ (Ki 1.15 x 10-3 ᴍ).After sucrose gradient centrifugation in a preparative ultracentrifuge the enzyme sediments with 4.1 ± 0.1 S as estimated in comparison to other proteins of known sedimentation coefficient. The isoelectric point was determined to be 3.9 with the LKB preparative isoelectric focusing col­umn (pH 2-11) and 4.1 with the analytical flat bed polyacrylamide isofocusing (pH 3 - 5). The number of SH groups was determined to be 2 mol/mol enzyme. In the presence of 6 M urea the fig­ure inceases to 3 mol SH/mol enzyme. In the presence of an excess of p-chloromercuribenzoate the enzyme activity decreases only partially.

scholarly journals A Polymeric form of Haemoglobin-Binding Protein in Sheep Following Metabolic and Hormonal Disturbance

1972 ◽  
Vol 25 (5) ◽  
pp. 941 ◽  
Author(s):  
IG Jarrett
Keyword(s):  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Serum Proteins ◽  
Binding Protein ◽  
Polymeric Form ◽  
Poly Acrylamide

The serum proteins of sheep have been examined by concave gradient poly-acrylamide gel electrophoresis. It would appear that sheep are usually lacking in haemoglobin-binding proteins (haptoglobins).

scholarly journals Purification of the constituent enzyme fractions of mycobacillin synthetase

1986 ◽  
Vol 235 (1) ◽  
pp. 81-85 ◽  
Author(s):  
S K Ghosh ◽  
N K Mukhopadhyay ◽  
S Majumder ◽  
S K Bose
Keyword(s):  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Sucrose Density Gradient Centrifugation ◽  
Final Purification

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.

scholarly journals Biochemical characterization of a leukemia-associated inhibitor (LAI) suppressing normal granulopoiesis in vitro

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 983-991 ◽  
Author(s):  
T Olofsson ◽  
I Olsson
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Charge Heterogeneity ◽  
Sucrose Density Gradient Centrifugation ◽  
Peripheral Cell

Low-density (less than 1.077 g/ml) marrow or blood cells from patients with acute or chronic leukemia release a high molecular weight substance called “leukemia-associated inhibitor” (LAI) that reduces the fraction of normal marrow CFU-c in S-phase as measured with the 3H-TdR suicide technique. LAI from conditioned media or 3M KCl extracts of subcellular fractions behaved homogeneously on gel chromatography, showing an apparent molecular weight greater than 500,000. However, ion- exchange chromatography and isoelectric focusing indicated considerable charge heterogeneity for LAI molecules. Results from SDS-polyacrylamide gel electrophoresis indicated that the biologic activity resides in a subunit of 150,000–170,000 daltons. The findings of marked affinity for Con-A-Sepharose, marked susceptibility to mild periodate treatment, partial susceptibility to protease digestion, and relative resistance to heating suggest that LAI is a glycoprotein. Data from radiolabeling of cell surface components and sucrose density gradient centrifugation are consistent with LAI being a peripheral cell membrane glycoprotein, which may suppress normal granulopoiesis in leukemia.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

scholarly journals On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin.

1981 ◽  
Vol 91 (1) ◽  
pp. 83-94 ◽  
Author(s):  
R Füssle ◽  
S Bhakdi ◽  
A Sziegoleit ◽  
J Tranum-Jensen ◽  
T Kranz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gradient Centrifugation ◽  
Membrane Damage ◽  
Sedimentation Coefficient ◽  
Water Soluble ◽  
Effective Diameter ◽  
Gel Chromatography ◽  
Alpha Toxin ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.

scholarly journals Biochemical characterization of a leukemia-associated inhibitor (LAI) suppressing normal granulopoiesis in vitro

Blood ◽  
1980 ◽  
Vol 55 (6) ◽  
pp. 983-991 ◽  
Author(s):  
T Olofsson ◽  
I Olsson
Keyword(s):  
Molecular Weight ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Gel Chromatography ◽  
Charge Heterogeneity ◽  
Sucrose Density Gradient Centrifugation ◽  
Peripheral Cell

Abstract Low-density (less than 1.077 g/ml) marrow or blood cells from patients with acute or chronic leukemia release a high molecular weight substance called “leukemia-associated inhibitor” (LAI) that reduces the fraction of normal marrow CFU-c in S-phase as measured with the 3H-TdR suicide technique. LAI from conditioned media or 3M KCl extracts of subcellular fractions behaved homogeneously on gel chromatography, showing an apparent molecular weight greater than 500,000. However, ion- exchange chromatography and isoelectric focusing indicated considerable charge heterogeneity for LAI molecules. Results from SDS-polyacrylamide gel electrophoresis indicated that the biologic activity resides in a subunit of 150,000–170,000 daltons. The findings of marked affinity for Con-A-Sepharose, marked susceptibility to mild periodate treatment, partial susceptibility to protease digestion, and relative resistance to heating suggest that LAI is a glycoprotein. Data from radiolabeling of cell surface components and sucrose density gradient centrifugation are consistent with LAI being a peripheral cell membrane glycoprotein, which may suppress normal granulopoiesis in leukemia.

scholarly journals Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage

1979 ◽  
Vol 183 (3) ◽  
pp. 539-545 ◽  
Author(s):  
M Paulsson ◽  
D Heinegård
Keyword(s):  
High Speed ◽  
Matrix Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Exchange Chromatography ◽  
Cartilage Matrix ◽  
Gel Chromatography ◽  
Sucrose Density Gradient Centrifugation

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.

scholarly journals Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles.

1987 ◽  
Vol 105 (2) ◽  
pp. 887-895 ◽  
Author(s):  
Y Y Toyoshima
Keyword(s):  
Electron Microscopy ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Time Courses ◽  
The One ◽  
Dynein Heavy Chains

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.

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