Interaction of purified human transcortin with spin labelled steroids

Basset, M.; Defaye, G.; Chambaz, E.M.

doi:10.1016/0014-5793(75)80750-2

Cited by 12 publications

(4 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Affinity of SL-Vfor P-450scc. Concentrations of free SL-V were measured at various total concentrations of the spin-labeled compound (Benson et al, 1977;Basset et al, 1975) in the presence of a P-450 preparation that had a specific content of 4.0 nmol of P-450/mg of protein. From the Woolf plot analysis, the dissociation constant, KO, of the P-450scc-SL-V complex was calculated to be 1.1 µ (Figure 5).…”

Section: Resultsmentioning

confidence: 99%

“…The spin-labeled compounds were incubated with the P-450scc preparation for 20 min at room temperature prior to the measurements. The concentrations of the spin-labeled substances in solution were determined from the amplitudes of the unbroadened N-hyperfine lines or by double integration (Benson et al, 1977;Basset et al, 1975). Concentrations of the spin-labeled compounds, which were "immobilized" to various degrees by binding to a macromolecule, i.e., P-450scc, or in large selfaggregates of the steroid spin-labeled compound, were calculated from the difference between total spin-label added and free spin-label in solution (Benson et al, 1977;Basset et al, 1975).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

Seeley¹,

Schleyer²,

Kashiwagi³

et al. 1987

Biochemistry

View full text Add to dashboard Cite

The intramolecular site of P-450scc for conversion of cholesterol to pregnenolone involves a substrate site, an active site, and a site for transmission of electrons. The substrate site was studied with a high-affinity, high-potency nitroxide spin-labeled inhibitor of cholesterol side-chain cleavage. This substance, 17 alpha-hydroxy-11-deoxycorticosterone nitroxide (SL-V), has an affinity comparable to that of the most active substrate inhibitors ever reported and 2-50 times greater than that of the natural substrate cholesterol. Competition experiments with cholesterol and its analogues confirmed that SL-V binds reversibly to the substrate site. Titration experiments showed a single binding site on the P-450 molecule. The substrate site is on the apoprotein and has little or no direct interaction with the heme. Spin-spin interactions between the Fe3+ and side-chain or A-ring spin-labeled groups could not be demonstrated, which is consistent with carbons 22 and 20 being closest to the heme iron. We postulate that substrate disrupts a histidine nitrogen coordination with the heme iron and induces conformational changes in the apoprotein. These changes lead to increased affinity for iron-sulfur protein.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

Seeley¹,

Schleyer²,

Kashiwagi³

et al. 1987

Biochemistry

View full text Add to dashboard Cite

show abstract

“…Steroid hormones appear to enter the corticosteroid binding globulin with the A ring pointed to the interior (Basset et al, 1975). An inverted A ring structure has been proposed for high affinity binding of progesterone analogues to the receptor in rabbit uterus (Duax et al, 1978a).…”

Section: Discussionmentioning

confidence: 99%

Identification of histidine and methionine residues in the active site of the human uterine progesterone receptor with the affinity labels 11.alpha.- and 16.alpha.-(bromoacetoxy)progesterone

Holmes¹,

Smith²

1983

Biochemistry

View full text Add to dashboard Cite

The affinity labels 11 alpha- and 16 alpha-(bromo[2'-3H]acetoxy)progesterone (BAP) react covalently with amino acids present in the progesterone binding site of the human uterine progesterone receptor. Hydrolysis of the affinity labeled receptor followed by separation and analysis of the amino acid products demonstrated the sites of affinity labeling. The 11 alpha-BAP alkylates the 1-position of a histidine residue. The 16 alpha-BAP alkylates the 3-position of histidine, and a methionine residue. Affinity labeling did not occur in the presence of excess progesterone, and under the optimum conditions for affinity labeling of the receptor, heat-denatured receptor, bovine serum albumin, and 20 beta-hydroxysteroid dehydrogenase were not affinity labeled. This is the first report of the identification of specific amino acid residues in the binding site of a steroid hormone receptor.

show abstract

“…If then the immunoglobulin binding is directed towards the A-ring of the cortisol moiety, it is unlikely that transcortin-bound cortisol serves for antigenic stimulation of anti-cortisol antibodies. Electron-spinresonance studies indicate that the lateral chain at position-17 of transcortin-bound cortisol is orientated outwards (Basset et al, 1975). This moiety is significantly altered by the hemisuccinate group on the affinity gel and would also be sterically hindered by its proximity to the agarose matrix.…”

Section: Immunoglobulinsmentioning

confidence: 99%

Purification and characterization of human transcortin

Mueller¹,

Potter²

1981

Biochemical Journal

View full text Add to dashboard Cite

Human transcortin was purified to apparent homogeneity from plasma by a two-step procedure involving affinity and hydroxyapatite chromatography. The affinity gel incorporated denatured bovine serum albumin as the spacer and cortisol hemisuccinate as the ligand. Although isolated transcortin showed a propensity for spontaneous polymerization according to a geometric progression (1, 3, 9) only one band was observed on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Cortisol-binding activity of the isolated protein gave an apparent association constant of 2.5 X 10(8) M-1 at 4 degree C in equilibrium dialysis. Isoelectric focusing of purified native transcortin showed six discrete bands, five between pH 3.75 and 4.15 and another, possibly desialylated, at pH 6.15. Desialylated transcortin also gave six bands on isoelectric focusing, with pI values ranging from 4.90 to 6.30.

show abstract

Interaction of purified human transcortin with spin labelled steroids

Cited by 12 publications

References 18 publications

Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

Studies of the active site of cytochrome P-450 scc with a high-affinity spin-labeled inhibitor

Identification of histidine and methionine residues in the active site of the human uterine progesterone receptor with the affinity labels 11.alpha.- and 16.alpha.-(bromoacetoxy)progesterone

Purification and characterization of human transcortin

Contact Info

Product

Resources

About