scholarly journals Actin affinity chromatography in the purification of human, avian and other mammalian plasma proteins binding vitamin D and its metabolites (Gc globulins)

1984 ◽  
Vol 218 (3) ◽  
pp. 805-810 ◽  
Author(s):  
J G Haddad ◽  
M A Kowalski ◽  
J W Sanger
Keyword(s):  
Vitamin D ◽  
Affinity Chromatography ◽  
Human Plasma ◽  
Plasma Protein ◽  
Binding Protein ◽  
Binding Activity ◽  
Single Step ◽  
Affinity Column ◽  
Vitamin D Metabolites ◽  
Molar Ratios

The human plasma protein binding vitamin D and its metabolites (Gc globulin; group-specific component) has been isolated from human plasma by column affinity chromatography on gels to which monomeric actin was covalently attached. Rabbit skeletal-muscle G-actin was covalently coupled to amino-agarose gels before the application of human plasma. At actin/protein molar ratios of 4-8:1, excellent recovery (approximately 58%) of purified binding protein was achieved. After 0.75 M-NaCl washes, the binding protein was eluted from the columns in 3 M-guanidinium chloride, dialysed and analysed. These eluates contained the binding protein as 34-100% of the total protein, reflecting a 130-fold average purification in this single step. In the presence of Ca2+, gelsolin (another plasma protein that binds actin) was apparently retained by the affinity column, but this was prevented by chelation of plasma Ca2+. The actin affinity step also was effective in the isolation of the binding protein from rat, rabbit and chicken plasma, as indicated by autoradiographs of purified fractions analysed by gel electrophoresis after incubation with 25-hydroxy[26,27-3H]cholecalciferol. Further isolation by hydroxyapatite chromatography yielded a purified binding protein which displayed characteristic binding activity toward vitamin D metabolites and G-actin, and retained its physicochemical features. This brief purification sequence is relatively simple and efficient, and should prove to be useful to investigators studying this interesting plasma protein.

Vitamin D-binding protein: multifunctional component of blood serum

2021 ◽  
Vol 76 (1) ◽  
pp. 103-110
Author(s):  
Alexandra A. Povaliaeva ◽  
Ekaterina A. Pigarova ◽  
Anastasia A. Romanova ◽  
Larisa K. Dzeranova ◽  
Artem Y. Zhukov ◽  
...  
Keyword(s):  
Vitamin D ◽  
Binding Sites ◽  
Binding Protein ◽  
Vitamin D Metabolites ◽  
Biological Functions ◽  
Vitamin D Binding Protein ◽  
Fatty Acid Binding ◽  
Physiological Properties ◽  
Steroid Binding ◽  
Tumor Pathogenesis

Vitamin D-binding protein (DBP) was discovered more than half a century ago as a polymorphic serum protein and is currently characterized by a variety of physiological properties. First of all, DBP carries the bulk of vitamin D metabolites circulating in the bloodstream, while albumin is the second most important transport protein, especially in patients with a low concentration of DBP in serum. Since it was discovered that only 12% of the total circulating DBP have occupied steroid binding sites, a vigorous study of other potential biological roles of DBP was initiated: actin utilization, regulation of inflammation and innate immunity mechanisms, fatty acid binding, effects on bone metabolism and participation in the tumor pathogenesis. This review focuses on the main known biological functions of DBP.

Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein

2017 ◽  
Vol 409 (10) ◽  
pp. 2547-2558 ◽  
Author(s):  
Pilar Canoa ◽  
Marcos L. Rivadulla ◽  
Jonathan Popplewell ◽  
René van Oosten ◽  
Generosa Gómez ◽  
...  
Keyword(s):  
Vitamin D ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Binding Protein ◽  
Binding Study ◽  
Vitamin D Metabolites ◽  
Vitamin D Binding Protein

Purification of the c-fos enhancer-binding protein

1987 ◽  
Vol 7 (10) ◽  
pp. 3482-3489
Author(s):  
R Prywes ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Binding Protein ◽  
Binding Activity ◽  
Affinity Column ◽  
Equilibrium Dissociation Constant ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Low Salt ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.

Vitamin D metabolism in osteoporotic women during treatment with estrogen, an anabolic steroid, or calcitonin

1990 ◽  
Vol 122 (6) ◽  
pp. 715-721 ◽  
Author(s):  
Dorthe Hartwell ◽  
Christian Hassager ◽  
Kirsten Overgaard ◽  
Bente Juel Riis ◽  
Jan Pødenphant ◽  
...  
Keyword(s):  
Vitamin D ◽  
Serum Concentration ◽  
Binding Protein ◽  
Total Serum ◽  
Vitamin D Metabolites ◽  
Nandrolone Decanoate ◽  
Vitamin D Binding Protein ◽  
Double Blind ◽  
Vitamin D Metabolism ◽  
Norethisterone Acetate

Abstract. We assessed the effects of a continuous oral combination of estradiol and norethisterone acetate, nandrolone decanoate, or salmon calcitonin on the vitamin D endocrine system. One hundred and nineteen postmenopausal women, aged 55-75 years, with at least one osteoporotic fracture, were randomly allocated to one year of treatment with estradiol and norethisterone acetate, nandrolone decanoate, or calcitonin, all drugs with a beneficial effect on bone. All three trials were double-blind and placebo-controlled; 104 women (87%) completed the study. We measured the total serum concentration of 1,25-dihydroxyvitamin D (1,25(OH)2D) and vitamin D-binding protein, and estimated the free 1,25(OH)2D index and the "24-hydroxylase activity" initially, and at 6 and 12 months. Furthermore, the 24-h urinary excretions of calcium, phosphate, and adenosine 3'-5'-cyclic monophosphate were assessed initially and at 12 months. The serum concentration of vitamin D-binding protein and 1,25(OH)2D increased transiently during estradiol and norethisterone acetate treatment and vitamin D-binding protein decreased transiently during nandrolone decanoate treatment. None of the other parameters were significantly affected by any of the three treatments. The risk of type II errors was below 10 per cent for all vitamin D measurements. We conclude that the vitamin D metabolites are unlikely to be of major importance for the mechanism by which these drugs exert their positive skeletal effects.

scholarly journals Purification of the c-fos enhancer-binding protein.

1987 ◽  
Vol 7 (10) ◽  
pp. 3482-3489 ◽  
Author(s):  
R Prywes ◽  
R G Roeder
Keyword(s):  
Dna Sequences ◽  
Binding Protein ◽  
Binding Activity ◽  
Affinity Column ◽  
Equilibrium Dissociation Constant ◽  
Nuclear Extracts ◽  
Enhancer Binding Protein ◽  
Low Salt ◽  
Equilibrium Dissociation ◽  
Dyad Symmetry

We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.

Purification by affinity chromatography of a membrane dicarboxylate binding protein from Bacillus subtilis

1981 ◽  
Vol 27 (8) ◽  
pp. 795-800 ◽  
Author(s):  
William W. Kay
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Affinity Chromatography ◽  
Binding Protein ◽  
Membrane Vesicles ◽  
Binding Activity ◽  
High Affinity ◽  
Glutamate Binding ◽  
Tricarboxylate Cycle ◽  
Nonionic Detergents

Membrane vesicles of Bacillus subtilis W23 actively transport the C4 and C5 dicarboxylates of the tricarboxylate cycle by system(s) of relatively high affinity for their requisite substrates (Km 4–53 μM). Glutamate and succinate binding activities were readily solubilized from membrane vesicles by nonionic detergents, particularly by Lubrol WX. From this extract, glutamate binding activity was highly enriched by affinity chromatography on phloroglucinol-expanded Sepharose-6B to which L-aspartate was coupled via divinylsulfone. Another protein (41 000 molecular weight), which bound both L-glutamate and L-malate, was purified from affinity columns to which either L-glutamate or L-malate had been coupled via bisdiglycidyl ether. This protein bound labelled L-malate as well as L-glutamate with affinities similar to those seen with membrane vesicles (Kd's 8 μML-malate and 52 μML-glutamate).

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