Blue‐Dextran — Sepharose Affinity Chromatography: Recognition of a Polynucleotide Binding Site of a Protein

Drocourt, Jean-Louis; Thang, D.C.; Thang, M.N.

doi:10.1111/j.1432-1033.1978.tb12030.x

Cited by 48 publications

(11 citation statements)

References 20 publications

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“…Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7). The methodology was also applied to the characterization and purification of both viral and immune interferons (8)(9)(10)(11).…”

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confidence: 99%

“…We have recently described an affinity chromatography with blue dextran or polynucleotide as ligand for proteins interacting with nucleic acids (7). Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7).…”

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confidence: 99%

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Human leukocyte interferon: Relationship between molecular structure and species specificity

Thang

Chelbi-Alix

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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Human leukocyte interferon can be separated into two classes of subspecies by polynucleotide-agarose affinity chromatography; 30-40% of the molecular species have the polynucleotide-binding property and 60-70% lack affinity for the polynucleotide ligand. When analyzed on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the former class of interferon has a slower mobility corresponding to the migration of a polypeptide of 21,000 daltons, while the latter class has a faster mobility corresponding to a polypeptide of 13,500-15,000 daltons. By analogy to the behavior of other interferons and a class of nucleotidyl transferases on the polynucleotide-agarose chromatography, we suggest that the human leukocyte interferon having the polynucleotide-binding site is in a possibly "native" conformation and the loss of affinity for polynucleotide results from a degradative alteration of the native molecules. Moreover, the alteration of interferon is accompanied by an increase in heterospecific activity on bovine cells. It is suggested that the polypeptide domain responsible for species specificity may be closely related to the polynucleotide binding area. The modified interferon molecule, however, still conserves its antiviral activity. The simplicity and the high capacity of polynucleotide-agarose chromatography make this a powerful technique for the purification of interferon. The easy separation of these two classes of human leukocyte interferon makes the purification procedures more rational and will facilitate the preparation of both subspecies to a high degree of molecular homogeneity. Human leukocyte interferon (Hu Le IF) contains two subpopulations of active molecules, which are differentiated by their molecular sizes, charge properties, and even biological activities (1-6). Such heterogeneity, resulting from the presence of molecular species having different physicochemical properties, complicates the purification of leukocyte interferon.In particular, the strategy for purification has been mostly orientated by the recovery of as much initial activity as possible, necessitating copurification of the two subpopulations.We have recently described an affinity chromatography with blue dextran or polynucleotide as ligand for proteins interacting with nucleic acids (7). Such chromatography not only allows the separation of proteins having a polynucleotide-binding site from other classes of proteins but also can be used to separate one native enzyme from a degraded enzyme that has lost the polynucleotide attachment site (7). The methodology was also applied to the characterization and purification of both viral and immune interferons (8-11). We postulate that the polynucleotide-binding property, observed with mouse types I and II and human interferons, could be a basic characteristic of this antiviral protein. This property also could reveal a structural homology between the mouse types I and II interferons (10). In this paper, we show that Hu Le IF can be separated into two classes of molecules according...

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Human leukocyte interferon: Relationship between molecular structure and species specificity

Thang

Chelbi-Alix

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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“…Cibacron blue is a non-competitive inhibitor with respect to dinucleotide for dihydrofolate reductase, but competitive with dihydrofolate: this agrees with a binding in the substrate binding site [ 3 ] . Furthermore it has been suggested that the dye binds to the polynucleotide binding site in polynucleotide phosphorylase rather than in the mononucleotide binding site [8].…”

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confidence: 99%

“…Strong binding of blue dextran to NAD(P)+-dependent dehydrogenases seems to be frequent [3], but many other enzymes also interact with blue dextran or Cibacron: enzymes acting on NAD' like NAD' glycohydrolase [4], NAD' kinase [5] as well as nucleotide-dependent enzymes such as nucleotide kinase [6], nucleotidetransferase [6], adenylate cyclase [7], polynucleotide phosphorylase [8], phosphorylase a [9], hexokinase [lo] and flavocytochrome h~, a flavin-dependent enzyme [l 11. Conformational changes of Cibacron blue bound to dehydrogenases were detected by circular dichroism [12].…”

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X-Ray Studies of the Binding of Cibacron Blue F3GA to Liver Alcohol Dehydrogenase

et al. 1979

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“…CB, a sulfonated polyaromatic dye, has been shown to bind to several proteins that interact with nucleotide substrates, nucleotide coenzyme ligands, and polynucleotides (13)(14)(15)(16)(17)(18). In the case of enzymes that have polynucleotide binding sites in addition to nucleotide substrates, the dye interacts with the polynucleotide binding site preferentially (15,17,18). In associated with the cellulose pellet was measured in a Beckman scintillation counter (LS-250) using Bifluor scintillation fluid.…”

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Effect of Cibacron blue F3GA on oligonucleotide binding site of estradiol--receptor complexes of mouse uterine cytosol.

Kumar¹,

Beach²,

Dickerman³

1979

Proc. Natl. Acad. Sci. U.S.A.

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It is believed that steroid hormones induce differential gene expression in target cells by combining with specific cytoplasmic receptor proteins that subsequently translocate to the nuclear compartment where they interact with a limited number of acceptor sites (1). Although the nature of these nuclear acceptor sites is still unclear, it is well established that almost all steroid receptors described are able to combine with DNA (2-4). Andre and Rochefort (5) have demonstrated that the affinity of the estradiol receptor protein for DNA is increased by estrogen binding, analogous to the ligand-induced DNA binding property of cyclic AMP receptor protein, a prokaryotic gene regulatory protein (6). Several lines of evidence suggest that steroid receptor proteins, particularly estradiol and glucocorticoid receptor proteins, contain two distinct sites, one for the steroid hormone and the other for DNA. Limited proteolysis of the steroid receptor protein complexes destroys the DNA binding site without affecting the steroid binding site (7-9). In addition, compounds such as pyridoxal phosphate (10) and aurintricarboxylic acid (ATA) (11) have been shown to inhibit DNA binding of glucocorticoid and estradiol receptor proteins, respectively.Studies reported from this laboratory have shown that estradiol-receptor complexes (E2R) of mouse uterus and kidney bind to oligo(dT)-cellulose. The validity of this interaction as a model for the DNA binding property of estradiol-receptors has been well established (12). Using oligo(dT)-cellulose binding as an assay for DNA interaction, we studied the inhibitory effect of the dye Cibacron blue F3GA (CB) in order to characterize the DNA recognition site. CB, a sulfonated polyaromatic dye, has been shown to bind to several proteins that interact with nucleotide substrates, nucleotide coenzyme ligands, and polynucleotides (13)(14)(15)(16)(17)(18). In the case of enzymes that have polynucleotide binding sites in addition to nucleotide substrates, the dye interacts with the polynucleotide binding site preferentially (15,17,18). In the present study we have shown that CB interacts specifically with the DNA binding domain of the mouse uterine E2R. MATERIALS AND METHODS[3H]Estradiol (145 Ci/mol; 1 Ci = 3.7 X 1010 becquerels) was obtained from Amersham. Unlabeled estradiol was a product of Steraloids, Inc. (Wilton, NH). Olido(dT)-, oligo(dC)-, and oligo(dA)-celluloses were products of P-L Biochemicals. Calf thymus DNA and heparin were obtained from Sigma. Poly-L-glutamic acid was a product of Miles-Yeda (Rehovoth, Israel), and CB was a gift from CIBA (Basel, Switzerland). (vol/vol) glycerol with a Polytron P20ST homogenizer. The homogenate was centrifuged in a Beckman J-21 centrifuge with JA-20 rotor at 12,000 X g for 10 min, and the supernatant was recentrifuged at 300,000 X g for 60 min in a Beckman preparative ultracentrifuge (L-75) with a 75 Ti rotor. The supernatant fraction is referred to as the "cytosol." Testosterone (1.2 ,uM)

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Blue‐Dextran — Sepharose Affinity Chromatography: Recognition of a Polynucleotide Binding Site of a Protein

Cited by 48 publications

References 20 publications

Human leukocyte interferon: Relationship between molecular structure and species specificity

Human leukocyte interferon: Relationship between molecular structure and species specificity

X-Ray Studies of the Binding of Cibacron Blue F3GA to Liver Alcohol Dehydrogenase

Effect of Cibacron blue F3GA on oligonucleotide binding site of estradiol--receptor complexes of mouse uterine cytosol.

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