CONTRACEPTION
Tim0
Zvpren,
130 Lundberf Department
of Biochemistry
and +Department University
Kim I:cttersson,
and Reijo
and Pharmacy,
of Obstetrics
Central
Xospital,
Iurnonen' Abe Akademi,
a?S Gynaecologg, S?-2OWO
Turku
,Ybo 50, Zinland
The binding of estroiyens to the estroceri binding protein in +~u:c::n mvometrisl cvtosol and the ecfect of Cu2+ ions on the bin,iing ckkacteristics have been investipated. The dissociation constants at 20 OC for the estrogen receptor comnlex were 0.081, ii.21 and 3.2L nY.1for estrediol, estriol respectively. and-estrore, 50th t?!e aifinity and the number of binding sites were afFected in tPie ;:resence of Cu2+ ions which indicated a mixed type inhibition of the binding of steroid to the receptor. ':'hesecond order association rate constants decreased in the Ix-esence of Cu2+ ions and the effect seemed to be reinforced by an increase in temperature. So chance in the sedimentation coefficient of the estrogen receptor con:‘lex occurred in the presence of ,Cu'+ ions alt?,ougk Tess steroid Cu>;'ion concentrations ahove IO )iY were required before remarkable alterations in t"e binding characteristics were established. !!s the copper level in human uterine fluid of women using a Cu-IiTD have been sbo~rn to be in the range of 20 pK, the-interaction of Cu2+ ions with the binding of estrogens to their specific receptors has to be considered as one factor responsible for the than{-es ir the uterus in tie presence of the device.
Accepted
AUGUST
for publication
1978 VOL. 18 NO. 2
JULY 6, 1978
181
CONTRACEPTION
Xany physio!oPical and hioc'.emical chances GCCU~ in the uterus in the rreserce of a copper intrauterine device which results in a contraceptive effect (1, 2). The cYhang:esdeoend on the continuous release of minute amounts of Cup+ ions&but the exact mechanism of action has not been elucidated. :;ost of the evidence is in f?vour of the interpretation t!:at a local Change in the intr?llterine enlrirOMPent prevents blastocgst implan tation (1, T). An alternative mechanism is based on the chemical reactions associated with tie dissolution of copier = (*' "ke ef-ect of steroid hormones in uterine tissue is mediated by specific receptors with 'high affinity and low capacity (5). Interest has uite recently been directed towards the influence of Cu 3+ ions on iormone binding (h-8). 130 t h estradiol and progesterone have shown a decrease in binding affinity to their specific receptors while the concentration of binding sites has not been altered. In a J:revious communication from our l-iborsltory (9) on the binding c?laracteristics of tk,e h>Jman myometrial estrogen binding protein, 501767results on the infilirnce of Cud+ ions was included tiat Tartly deviate from earlier o-o-ervations. In this presentation a detailed report is eiven en our observations on the effect of Cu2+ ions on the estrogen binding protein in human myomatrial qrtosol.
Preparation of Isiyometrial Cytosol. Yormal mvometrial tissue samples were obtained immediatelv after hvsterectomv from patients that were considered rre-menoTausaj_. The ’ samples were cooled on ice and homopenized 10iithin 1 hour of hvsterectomy with an Ultra-Turraw ?:i l?/lG homoPenizer (4 x 10 seconds) in 4O ml.1?ris-ilN?l buffer, pi! 7.4 (?-buffer, 4 ml/e. tissue). T'qe suoernatant obtained after 1 hour centrifugation at 100,000 Y g was used as the myometrial CTrtCSOl. The exrerime.nts were carried out with c:ltosol containinp 7-9 mp rrotein/ml. l'r.otrinwas quanticated accocdinc to T,owry (IO), Determination of Estrogen hindinp;. h constant amount of tritiated estrogen (IO0 ~1) was added to tllbes containint; cW~oso1 (ICO ~1) and different amounts of Cu2+ ions ilOC pi). The final concentration of estrone (El), estradiol rE2) and es+ricl (ET) were 1.6, 1.7 and 2.3 nK, res-ectivell The tubes were'incubated for 80 minutes at 20 0'2. The 4' -H-lipand receptor com;,lex was measured by usinp a dextran-charcoal
182
AUGUST
1978VOL.lSNO.2
CONTRACEPTION
suspension (9). A IOO-fold excess of unlabeled estrogen was used to estimate non-scjecific binding. To test the influence of Cu2+ ions on the dissociation const,ant and binding capacity of tritiated estrogens o1 the receptor, various concentrations ~0.05-4.0 nM) were used. The incubation time was 24 hours at 0 oc, 80 minutes at 20 oC and 60 minutes at 33 oC. Because of the instability of the receptor, only eotradiol binding collld be tested at 30 'C (9). Scatchard analysis was used for evaluation of the data (11). Determination of Association Rate Constants. The influence of CuL+ ions on the rate of association was studied at 0 oC and 20 oC for estrone, estradiol and estriol and at 30 oC for estradiol only. Cytosol (2.0 ml) was transferred into a tube containing a solution i.l.0 ml) of tritiated ligand and Cu2+ ions in T-buffer. Aliquots (300 pl) were taken after diCferent intervals of time and transferred to tubes containing a IOO-fold excess of nonradioactive lig2nd in 0.1 ml T-buffer at 0 oC in order to dilute free radioactive ligand and to stop the formation of rJ3ioactive complex. The dextrancharcoal susuension was used to remove free estropen. Second ? oraer plots accordinp to Sanborn --* et al ’'I?) were used for calculation of association rate constants. Sucrose Density Gradient hnalgsis. Cytosol (0.5 ml) was added to 15 nK tritiated estropen solution (0.5 ml) in T-buffer and incubated for 24 hours at 0 oC. Parallel samnles ContaininK a 100-vo?d excess of nonradioactive ehtropen were l:sed to asses nonspecific binding. Tinbound es'roF;en was removed with a nextran-cvarcoal s._us:3ension.Samples (300 pl) were l,e.veredonto 2.s-1-Z sucrose gradients (4.6 ml) in T-buffer 1n 'A ~I centrifuged at 40,GOD rpm i175,00C x g) ror 16 hours. 14
Tim0
Zvpren,
130 Lundberf Department
of Biochemistry
and +Department University
Kim I:cttersson,
and Reijo
and Pharmacy,
of Obstetrics
Central
Xospital,
Iurnonen' Abe Akademi,
a?S Gynaecologg, S?-2OWO
Turku
,Ybo 50, Zinland
The binding of estroiyens to the estroceri binding protein in +~u:c::n mvometrisl cvtosol and the ecfect of Cu2+ ions on the bin,iing ckkacteristics have been investipated. The dissociation constants at 20 OC for the estrogen receptor comnlex were 0.081, ii.21 and 3.2L nY.1for estrediol, estriol respectively. and-estrore, 50th t?!e aifinity and the number of binding sites were afFected in tPie ;:resence of Cu2+ ions which indicated a mixed type inhibition of the binding of steroid to the receptor. ':'hesecond order association rate constants decreased in the Ix-esence of Cu2+ ions and the effect seemed to be reinforced by an increase in temperature. So chance in the sedimentation coefficient of the estrogen receptor con:‘lex occurred in the presence of ,Cu'+ ions alt?,ougk Tess steroid Cu>;'ion concentrations ahove IO )iY were required before remarkable alterations in t"e binding characteristics were established. !!s the copper level in human uterine fluid of women using a Cu-IiTD have been sbo~rn to be in the range of 20 pK, the-interaction of Cu2+ ions with the binding of estrogens to their specific receptors has to be considered as one factor responsible for the than{-es ir the uterus in tie presence of the device.
Accepted
AUGUST
for publication
1978 VOL. 18 NO. 2
JULY 6, 1978
181
CONTRACEPTION
Xany physio!oPical and hioc'.emical chances GCCU~ in the uterus in the rreserce of a copper intrauterine device which results in a contraceptive effect (1, 2). The cYhang:esdeoend on the continuous release of minute amounts of Cup+ ions&but the exact mechanism of action has not been elucidated. :;ost of the evidence is in f?vour of the interpretation t!:at a local Change in the intr?llterine enlrirOMPent prevents blastocgst implan tation (1, T). An alternative mechanism is based on the chemical reactions associated with tie dissolution of copier = (*' "ke ef-ect of steroid hormones in uterine tissue is mediated by specific receptors with 'high affinity and low capacity (5). Interest has uite recently been directed towards the influence of Cu 3+ ions on iormone binding (h-8). 130 t h estradiol and progesterone have shown a decrease in binding affinity to their specific receptors while the concentration of binding sites has not been altered. In a J:revious communication from our l-iborsltory (9) on the binding c?laracteristics of tk,e h>Jman myometrial estrogen binding protein, 501767results on the infilirnce of Cud+ ions was included tiat Tartly deviate from earlier o-o-ervations. In this presentation a detailed report is eiven en our observations on the effect of Cu2+ ions on the estrogen binding protein in human myomatrial qrtosol.
Preparation of Isiyometrial Cytosol. Yormal mvometrial tissue samples were obtained immediatelv after hvsterectomv from patients that were considered rre-menoTausaj_. The ’ samples were cooled on ice and homopenized 10iithin 1 hour of hvsterectomy with an Ultra-Turraw ?:i l?/lG homoPenizer (4 x 10 seconds) in 4O ml.1?ris-ilN?l buffer, pi! 7.4 (?-buffer, 4 ml/e. tissue). T'qe suoernatant obtained after 1 hour centrifugation at 100,000 Y g was used as the myometrial CTrtCSOl. The exrerime.nts were carried out with c:ltosol containinp 7-9 mp rrotein/ml. l'r.otrinwas quanticated accocdinc to T,owry (IO), Determination of Estrogen hindinp;. h constant amount of tritiated estrogen (IO0 ~1) was added to tllbes containint; cW~oso1 (ICO ~1) and different amounts of Cu2+ ions ilOC pi). The final concentration of estrone (El), estradiol rE2) and es+ricl (ET) were 1.6, 1.7 and 2.3 nK, res-ectivell The tubes were'incubated for 80 minutes at 20 0'2. The 4' -H-lipand receptor com;,lex was measured by usinp a dextran-charcoal
182
AUGUST
1978VOL.lSNO.2
CONTRACEPTION
suspension (9). A IOO-fold excess of unlabeled estrogen was used to estimate non-scjecific binding. To test the influence of Cu2+ ions on the dissociation const,ant and binding capacity of tritiated estrogens o1 the receptor, various concentrations ~0.05-4.0 nM) were used. The incubation time was 24 hours at 0 oc, 80 minutes at 20 oC and 60 minutes at 33 oC. Because of the instability of the receptor, only eotradiol binding collld be tested at 30 'C (9). Scatchard analysis was used for evaluation of the data (11). Determination of Association Rate Constants. The influence of CuL+ ions on the rate of association was studied at 0 oC and 20 oC for estrone, estradiol and estriol and at 30 oC for estradiol only. Cytosol (2.0 ml) was transferred into a tube containing a solution i.l.0 ml) of tritiated ligand and Cu2+ ions in T-buffer. Aliquots (300 pl) were taken after diCferent intervals of time and transferred to tubes containing a IOO-fold excess of nonradioactive lig2nd in 0.1 ml T-buffer at 0 oC in order to dilute free radioactive ligand and to stop the formation of rJ3ioactive complex. The dextrancharcoal susuension was used to remove free estropen. Second ? oraer plots accordinp to Sanborn --* et al ’'I?) were used for calculation of association rate constants. Sucrose Density Gradient hnalgsis. Cytosol (0.5 ml) was added to 15 nK tritiated estropen solution (0.5 ml) in T-buffer and incubated for 24 hours at 0 oC. Parallel samnles ContaininK a 100-vo?d excess of nonradioactive ehtropen were l:sed to asses nonspecific binding. Tinbound es'roF;en was removed with a nextran-cvarcoal s._us:3ension.Samples (300 pl) were l,e.veredonto 2.s-1-Z sucrose gradients (4.6 ml) in T-buffer 1n 'A ~I centrifuged at 40,GOD rpm i175,00C x g) ror 16 hours. 14
