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).

Partial purification and characterization of a membrane-associated steroid-binding protein from Pseudomonas testosteroni

1982 ◽  
Vol 60 (8) ◽  
pp. 798-803 ◽  
Author(s):  
Mike Francis ◽  
Mamoru Watanabe
Keyword(s):  
Binding Protein ◽  
Membrane Vesicles ◽  
Activity Analysis ◽  
Binding Activity ◽  
Regulatory Function ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Steroid Binding ◽  
Steroid Binding Protein

A steroid-binding protein obtained from the supernatant of the final wash from the preparation of membrane vesicles was purified severalfold to near homogeneity. The protein binds C18 and C19 steroids but has the highest affinity for androstenedione (Kd = 1.6 × 10−10 M). The molecular weight is 51 000 – 58 000. Binding activity is slightly inhibited by Cu2+, Ca2+, and Mg2+ and completely inhibited by Zn2+. The protein has no detectable steroid degradative activity. Analysis of androstenedione binding revealed negative cooperativity of binding for this ligand and may indicate a regulatory function for this protein. It is postulated that this protein binds the steroid after testosterone is converted to androstenedione.

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.

A high-affinity folate binding protein in normal human leukocytes: Ligand binding characteristics, ionic charge and molecular size

1985 ◽  
Vol 5 (8) ◽  
pp. 683-688 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
J⊘rgen Lyngbye
Keyword(s):  
Binding Protein ◽  
Molecular Size ◽  
Binding Activity ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Human Leukocytes ◽  
Binding Characteristics ◽  
High Affinity ◽  
Chromatographic Profile ◽  
Triton X

High-affinity binding of [3H]folate to supernatant from homogenized human leukocytes containing large amounts of binding protein displayed apparent positive cooperativity. The DEAE-Sepharose® CL-6B chromatographic profile of the supernatant at pH 6.3 contained a major peak of folate binding (Mr approx. 25 000) in the front effluent and a smaller more acidic peak (Mr approx. 25 000) that emerged after a rise in NaCl from 30 mmol/l to 1 mol/l. Triton X-100 solubilized ceil sediment from the leukocyte homogenate contained some high-affinity folate binding activity (Mr approx 25 000), typically 5–10% of the total binding activity.

High-affinity folate binding in human mammary gland

1993 ◽  
Vol 13 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen ◽  
Mimi Høier-Madsen
Keyword(s):  
Mammary Gland ◽  
Binding Protein ◽  
Cross Reactivity ◽  
Binding Activity ◽  
Gel Chromatography ◽  
Folate Binding Protein ◽  
High Affinity ◽  
Mammary Gland Tissue ◽  
Human Mammary Gland ◽  
Triton X

High-affinity 3H-folate binding in Triton X-100 solubilized human mammary gland tissue displayed characteristics, e.g. apparent positive cooperativity and increasing affinity with decreasing concentration of folate binding protein, shown to be typical of specific folate binding. Radioligand dissociation was slow at pH 7.4. A major fraction of the bound radioligand dissociated rapidly at pH 3.5, while a residual binding of 20% persisted even after prolonged dialysis at pH 3.5. Gel chromatography revealed two major folate binding proteins (Mr ≈ 100 kDa and 25 kDa). However, only one single band was detectable on SDS-PAGE immunoblotting. The highest folate binding activity per g protein was associated with the upper triglyceride-containing layer of the 1000 g supernatant of the homogenate. The folate binding protein extracted from this layer had a low cross-reactivity (<5%) with rabbit antibodies against 25 kDa human milk folate binding protein. The folate binding protein in the 1000 g pellet and the aqueous phase of the 1000 g supernatant was present at a low concentration and had a cross-reactivity of 100%.

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