Linked Life Data

2248950

Source:http://linkedlifedata.com/resource/pubmed/id/2248950

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
40
pubmed:dateCreated
1991-1-15
pubmed:abstractText
The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka approximately 10(8) M-1 at 4 degrees C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17 beta-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm.Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 degrees C show that, while between 0 and 1 M Gdm.Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5 alpha-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 degrees C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm.Cl above 1 M. The Gdm.Cl titration in the presence of steroids with equilibrium association constants less than 10(8) M-1 shows a plateau near 3 M Gdm.Cl at 11 degrees C; at this Gdm.Cl concentration, no DHE is bound. No plateau is observed at 21 degrees C. At higher Gdm.Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm.Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
9
pubmed:volume
29
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
9334-43
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Fluorescence investigation of the sex steroid binding protein of rabbit serum: steroid binding and subunit dissociation.
pubmed:affiliation
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
pubmed:publicationType
Journal Article, In Vitro, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.