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A new approach for quantitation of receptors and their ligands
.I. Mol.
Hid.
(1980) 143, 419-425
A New Approach
for Quantitation Ligands
A novel and sensitive method for quantitating described, using the calf uterine estrogen receptor When ligand conjugated to malate dehydrogenase
of Receptors
and their
receptors and their ligands is and estradiol as a model system. is incubated with the estrogen
receptor, the enzyme is inhibited proportionately to the concentration of receptor. However. receptor saturated with free ligand has almost no effect on the ligandconjugated enzyme. The assay can detect as little as three femtomoles of receptor and one femtomole
of estradiol
and can. in principle.
be applied
to any receptor-
ligand interaction. The formulation of assay procedures for physiologically relevant receptors or ligands has always been a formidable task. Radio-ligand assay or radioimmunoassay are currently the most frequently used systems (Hunter, 1978), although they suffer from several disadvantages, such as the tedious procedures for synthesizing a radiolabeled ligand, purification of the receptor, production of a high titre antibody. low shelf-life of radioisotopes, expensive equipment, radiation hazards, etc. (Scharpe et al., 1976). A desirable assay for receptors should be cheap. rapid, quantitative, specific and capable of measuring minute quantities even in crude extracts. The reversible nature of the estrogen-receptor interaction with rstradiol. as well as’its stereochemical specificity and high affinity (K,, = lop9 M at 4°C) led us to develop an assay system for this binding that depends exclusively on the specificity of the receptor-ligand interaction. The [ 3H]estradiol binding activity (Puca et al.. 1971) of the 4.5 S form of estrogen receptor isolated from calf uterus (Rica et al., 1973) was determined with [6,73H lest’radiol (50 Ci/mmol). Th e uterine cytosol (2.1 1; 15.1 mg protein/ml) bound 2.62 x 10’ disints/min of estradiol/ml (1.74 x 104 disints/min per mg protein). After fractionation wit,h (NH4)2S04, a portion was subjected to DEAE-cellulose chromatography (Puca et al., 1970) and the remainder was purified by affinity chromatograph,v (Sica et a/.. 1973). From the specific activity (2.4 x lo* disints/min per mg protein) of the affinity eluate (1.86 x lo6 disints/min per ml) it was possible to estimate the estradiol receptor per mg of protein in the cytosol and in the DEAE eluate (estradiol binding capacity 11.5 x lo6 disints/min per ml; 1.6 x 10’ disints/min per mg protein) to be 0.072 pg (1.09 pg receptor/ml) and 66.0 pg (48.0 pg receptor/ml), respectively. Proteins were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard. 17fi-Estradiol-17 hemisuccinate (400 pmol) was conjugat’ed to 1.35 mg of pigeon breast, muscle malic dehydrogenase (EC 1 .1,1.37: 5580 units/mg protein) according to the mixed anhydride technique (Erlanger et al.. 1959). Since about 50% of the enzyme activity was lost due to conjugation, active enz.vme (5010 units/mg protein) was separated by affinity chromatography on 419 002%2836/80/3204194)7
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$9 1980 Academic
Press
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Ltd.
420
I).
PAIN
AND
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SI:ROLIA
Cibacron-blue F3GA-Sepharose (Thompson et al.. 1975). One pmolar unit converts 1 .O prnol of oxaloacetate and /I-NADH to I,-malate and /I-NAD per minute at pH 7.5 at 25°C. The activity of the 178.estradiol-17 hemisuccinate conjugate of malate dehydrogenase in 0.1 M-phosphate buffer (pH 7.5) containing (Ply; bovine y-globulin was determined (Ochoa, 1955) to obtain an enzyme concentration curve at 25°C. All dilutions for the receptor and ligand were made with 10 mlvr-Tris.HCl (pH 7.5) containing 10 mM-KCl, 1 mM-EDTA and 0.1 “i;, bovine y-globulin. EstradiolMDHase+ (5 x 1 O- 3 units ; 10 ~1) was incubated for two hours at 4°C in a volume of 100 ~1 with varying concentrations of the receptor (DEAE-cellulose fraction) before assaying its activity. Similar experiments were carried out with crude tissue homogenate samples (100,OOOg supernatant), before and after addition of the partially purified receptor as an internal standard. To demonstrate that the calf ut’erine estrogen receptor specifically inhibits estradiol-MDHase, reversal of receptor-induced inhibition of estradiol-MDHa.se by the free estradiol was studied by incubating the receptor with an excess of free estradiol (10 to 50 pg in a total volume of 100 ~1) for two hours at 4°C and then with varying amounts of estradiolMDHase (in 10 ~1) for another two hours at 4°C. before assaying the enzyme activity. Varying amounts of estradiol were incubated with the amount of receptor that gave a 66% inhibition of estradiol-MDHase, for two hours at 4°C in a total volume of 100 ~1. Estradiol-MDHase (7.0 x 1O- 3 units in 10 ~1) was then added and. after two hours at 4”C, the extent of reversal of the enzyme inhibition was monitored. In the absence of receptor, the activity of estradiol-MDHase was proportional tc
Enzyme
unit
(x IO-‘)
FIG. 1. Effect of estradiol receptor on estradiol-MDHase activity. The enzyme activity of varying concentrations of estradiol-MDHase was determined at 25°C in the absence (0) and presence (A) of receptor (5 pg) in a total volume of 1 ml as described in t,he text. For reversal of receptor-induced inhibition ofestradiol-MDHase, the receptor was incubated with 50 pg of 17fi-estradiol for 2 h at 4°C in a total volume of 100 ~1, followed by addition of estradiol-MDHase. After 2 h at 4°C the enzyme activity was assayed at 25°C (m).
TAbbreviation
used: MDHase,
malate dehydrogenase.
LETTERS
TO
THE
EDITOR
&?I
the enzyme concentration (Fig. 1) However, about 92o/o loss of enzyme activity was observed with an excess of the receptor (5 pg), due to “conformational freezing” of the enzyme as a consequence of receptor binding (see below). That the decrease in enzyme activity was due only to the receptor-ligand interaction was established by the lack of inhibition of (1) native MDHase by the receptor, or (2) of estradioMDHase by 50 pg of riboflavin binding protein (Murthy et al., 1976) or y-globulin, or by heat-inactivated receptor (65°C for 5 min) (Puca et al., 1971). As expected. the enzyme activity in the conjugate decreased almost proportionately to the amount of receptor in the assay system (Fig. B(a)). Inhibition corresponding to 4.5 x lo- l4 and 9 x lo- l4 moles of receptor was observed when estradiol-MDHase (5 x 10m3 units in 10 ~1) was incubated with 100 ~1 each of 40 and 20 times diluted crude cytosol. Thus the amount of receptor per ml of crude cytosol calculated (1.12 pg/ml) was close to the value obtained from the radio-ligand assay (1.09 pg/ml). When known concentrations, as estimated by [3H]estradiol binding, of the receptor partially mol) were added purified on DEAE-cellulose (9 x 10-14, 15 x lo-l4 and 18 x lo-l4 to the above extract, inhibition corresponding to receptor concentrations of 13.5 x lo-14, 19.5x 10-14and22.5x lo-r4molesandof18x lo-14,24x 10-14and 27 x lo- l4 moles, respectively, was observed and a standard curve could be constructed. . Retention of estradiol-MDHase activity after incubation of the receptor wit#h varying amounts of 17p-e&radio1 is shown in Figure 2(b). Reversal of the inhibition of MDHase activity began at a certain concentration of 17g-estradiol, and then increased almost proportionately to the free 17fl-estradiol concentration, followed by nearly complete retention. The ability of free 178-e&radio1 to reverse the enzyme inhibition was compared to that of 6-carboxymethyloxime-17j3-estradiol, 17~ estradiol (a steroisomer of 17fl-estradiol), testosterone and hydrocortisone (steroid hormones without estrogenic activity) and the non-steroidal estrogen diethylstilboestrol. The amounts of 17p-estradiol, estriol, 6-carboxymethyloxime-178. estradiol and diethylstilboestrol required for 50% reversal were 4.3 x 10-14, 4.5 x lo-13, 2.5 x lop9 and 1.5 x lo- l4 moles, respectively. Testosterone, hydrocortisone and 17n-e&radio1 gave almost no reversal (<2oj), even at very high concentrations (5 x 1O-9 mol). The pattern of specificity is in good agreement with the reported potency of binding (Puca & Bresciani, 1969) and suggests that the inhibition and its reversal are due to the receptor-ligand interaction. It is worthwhile to compare the present approach with other systems. Selective adsorption by dextran-coated charcoal (McGuire, 1975), precipitation with protamine sulfate (Chamness et al., 1975) and gel filtration on Sephadex G-25 (Puca & Bresciani, 1968) are relatively simple and rapid but they are susceptible to influences from non-specific binding proteins and the protein concentration of the sample. With the present method. it is possible to assay estrogen receptor concentrations as low as 2.1 rig/ml, corresponding to a total protein concentration of 30 pg/ml. That is, it is at least 16 times more sensitive than the method developed recently using thin-layer gel filtration on Sephadex G-200 (Hazato et al., 1979), with the added advantage of rapidity. With the present method, e&radio1 receptor can be measured during various phases of the estrus cycle. Thus 0.25 x 1 O- “. 0.37 x 10-12. 0.53 x IO-l2 and 0.85 x lo-l2 moles of e&radio1 receptor per uterus
422
I).
0
PAIS
15 Receptor
AND
A. St’ROLIA
30
concentrotlon
(x IO+ mols)
(a)
45
I Log
2 [estrodiol]
3 (x b5
4 mols)
(b)
FIG. 2. Receptor induced inhibition of estradiol-MUease and the reversal of enzyme inhibition by free estradiol. Estradiol-MDHase (5.0 x 1 OF” units in 10 ~1) was incubated with varying concentrations of the receptor as described in the text and then assayed (a). Varying amounts of 17/Sestradiol were first incubated with the (fixed) amount of receptor that gave 66% inhibition and then with estradiol-MDHase (7 x 10e3 units in 10 ~1) and assayed (b). Details are given in the text.
were obtained during metestrus, diestrus, proestrus and estrus, respectively. Sensitivity of the radioimmunoassay for 17/Sestradiol is in the range of 20 pg/ml (Fishman & Fishman, 1974), whereas the present method can detect as little as 0.2 pg of 17/3-estradiol per ml. Moreover, a reduction in reaction volume by tenfold would lead to a tenfold higher sensitivity. The method can, in principle, be applied to any receptor-ligand interaction, eliminating the synthesis of a radiolabelled-ligand (radio-ligand assay) or production of an antibody (radioimmunoassay). Besides, with the present method it is easy to assay the receptor during various stages of purification and in various stages of cell or organ development. and to detect a putative receptor for a ligand by its effects on a ligand-conjugated enzyme. With the exception of equilibrium dialysis, which is lengthy and often difficult to quantitate (Baulieu & Raynaud, 1970), the present method may be the only procedure for assaying receptors and ligands where the separation ofthe free from the bound ligand is not required. Of course, the amount of receptor or the ligand that can be determined will depend on the affinity of the receptor for its specific ligand. Thus 0.1 femtomole of avidin can be detected using lectin using lactosylbiotin-MDHase (K,= 1015 Mm1 ) and 83 picomoles of Ricinw glucose-6-phosphate dehydrogenase (K, = lo8 M-') (our unpublished results). Mechanism
of InhibSon
of the Ligand-Malate Receptor
Dehydrogenase
Conjugate
by the
An experimental approach supporting the mechanism of inhibition of estradiolMDHase by estradiol receptor involving a “conformational freezing” of the enzyme due to binding of receptor to it is described here. No appreciable differences in the
LETTERS
TO THE
EDITOR
123
and p-NADH were observed for either estradioKm values for oxaloacetate MDHase or receptor-bound estradiol-MDHase from the native MDHase. This suggests that steric hindrance of the enzyme for either substrate by the bound receptor is not responsible for the enzyme inhibition. Assuming that all estradiols attached to a random set of enzyme sites are bound by the receptor. and that’ receptor binding to any estradiol attached to an “active” group of the enzyme causes 1OOqt enzyme inhibition. then the probability P, of inhibition of a givetl enzyme molecule is given by: p
m
= 1 _ (R-.4)!(R-m)! (R-d -m)!R!
(1)
’
where nr is the number of ligands bound to a set of R random active. When d = 1, equation (1) becomes :
site, A of which are
If rl is the average of randomly introduced ligands per enzyme, then the probability P, of finding an enzyme molecule bearing exactly m ligands is given by: P,=
n”‘(R-/z)~-“’
R !
m!(R-m)!RR
(3)
So, receptor bound to an “active” ligand inhibits the fraction P of enzyme molecules that is equal to the sum of the products of P, and P,, when m is ranging from zero to R; i.e. P=
; P,P, m=O
(4)
ff, however. because of steric constraints, only a limited number, C, of receptors can fit on the random set of ligands, the fraction of enzyme molecules inhibited becomes : C-l
P =
1 m=O
P,P,
+
;
P,P,C’/m.
(5)
m=C
When the difference between R and (7 is very small, the values of P obtained by equations (4) and (5) are more or less the same for practical purposes. Estradiol-MDHase with 21 estradiols (=R) conjugated to it, as estimated by conjugating [6,7-3H]17fl-estradiol, was mixed with an excess of estradiol receptor and subjected to gel filtration on Sepharose 6B. A major peak corresponding to a molecular weight of 1.2 x 106k20,000 was obtained, suggesting that 18 molecules of the receptor (M,=62,000) are bound per molecule of the conjugated MDHase. The values of R=21 and C= 18 were substituted in equation (5) and theoretical values of the enzyme inhibition corresponding to various occupancies of estradiolMDHase by the hormone receptor were computed considering the involvement, of either one or two active amino groups (A = 1,2) in the catalytic activity of the enzyme.
424
Receptor
molecules
/estradlol
-MDHase
moiecule
Es;. 3. Receptor-induced inhibition of estradiol-MDHase. The curve ( -) shows the theoretical 1 “active” group model where binding to the active group is assumed to cause lOOo/0 inhibition (R=21, C= 18). Even when 2 “active” groups are implicated, an almost identical pattern is observed (- - - --). Data points (a) indicate the experimentally observed values. In a separate set of experiments, a constant amount of estradiol-MDHase was mixed with 2.5,6, 10,15,20 and 25 molar excess ofthe receptor and, after incubation for one hour, they were subjected to gel filtration on Sepharose 6B. Peaks corresponding to molecular weight of 2 x 10’ +8000. 3.9 x 10’ ) 12,000, 57x 10s~15,000, 8.9~ 105+20,000, 1.05~ 106~20,000 and 1.20x 106f30,000 were obtained, indicating that 2. 5, 8, 13, 16 and 18 molecules of receptor, respectively, were bound per enzyme molecule. These peak fractions were then assayed for the enzyme activity (Fig. 3). An agreement between the experimental enzyme inhibition data with those obtained theoretically, assuming 1 (or 2) essential amino groups, substantiates our interpretation regarding the inhibition of the ligand-conjugated enzyme by the specific receptor. The striking effect of the estradiol receptor on the activity of estradiol-MDHase probably arises either from a conformational alteration imposed by the receptor binding to an active group, or by prevention of conformational changes essential for enzyme activity. In summary, inhibition of the ligand-conjugated MDHase is due to the specific interaction of the receptor with its ligand and arises mainly due to a “conformational freezing” of the enzyme. The authors thank Professor B. K. Bachhawat for his encouragement. supported by the Department of Science and Technology, India. Indian Institute of Experimental Medicine Jadavpur, Calcutta-700032, India Received 21 March
This work was
DEBKUMAK. AVADHESHA
1980, and in revised form 1 July 1980 REFERENCES
Baulieu, E. E. & Raynaud, J. P. (1970). Eur. J. Biochem. 13, 293-299. Chamness, G. C., Huff, K. & McGuire, W. L. (1975). Stwoids. 25, 627-635.
PAIX SLTROLIA
LETTERS
TO
THE
EDITOR
425
Erlanger. B. F., Borek, F., Beiser, S. M. & Lieberman, R. (1959). J. Riol. Chem. 234, 1090~1094. Metab. 39, 603-606. Fishman, J. & Fishman, J. H. (1974). J. Clin. Endocrinol. Hazato, T.. Murayama, A., Matsuzawa, A. & Yamamoto, T. (1979). Anal. Biochem. 94,29--35. Hunter, W. M. (1978). In Handbook of Experimental Immunology (Weir, D. M., ed.), 3rd edit., pp. 14.1-14.40, Blackwell Scientific Publications, Oxford. Lowry, 0. H., Rosebrough, N., Farr. A. 8: Randall, R. (1951). J. Hiol. Chem. 193, 26.5275. McGuire? W. L. (1975). In Methods in Enzymology (O’Malley. B. W. & Hardman. J. G.. rds). vol. 36. pp. 248-254, Academic Press, New York. Murthy, U. S., Podder, S. K. & Adiga, P. R. (1976). Biochim. Biophys. Acta, 434, 69-81. Ochoa, S. (1955). In Methods in Enzymology (Colowick, S. P. & Kaplan. N;. 0.. eds), vol. 1. pp. 735-739. Academic Press, London, New York. l’uca, G. A. 8: Bresciani, F. (1968). ‘Vatwe (London,). 218, 967-969. Puca, G. A. & Bresciani, F. (1969). ,Vature (London), 223, 74Fi-747. l’uca. (:. A.. De Sombre, E. R. &Jensen, E. V. (1970). In Research on Steroids (Finkelstein, RI., Klopper. A., Conti, (1. N. & Cassano. C., eds), vol. 4, pp. 274-289. Pergamon Press. New York.
Puca. (>. A.. Nola. E., Sica, V. & Bresciani, F. (1971). Riochrmistry. 10, 3769-3780. Scharpe, S. L., (looreman, W. M., Blomme, W. ?J. & Laekeman, (:. lfII. (1976). Clin. (‘hem. 22. 733-738. Sica. V., Parikh. 1.. Nola, E., Puca. (:. A. & Cuatrecasas, P. (1973). J. Riol. Ch,rm. 248. 6543-6558. Thompson. S. T., Cass, K. H. 8: Stellwagen. E. (1975). Proc. ‘Vat. ilcad. Ski., U.S.A. 72. 669-672.
Hid.
(1980) 143, 419-425
A New Approach
for Quantitation Ligands
A novel and sensitive method for quantitating described, using the calf uterine estrogen receptor When ligand conjugated to malate dehydrogenase
of Receptors
and their
receptors and their ligands is and estradiol as a model system. is incubated with the estrogen
receptor, the enzyme is inhibited proportionately to the concentration of receptor. However. receptor saturated with free ligand has almost no effect on the ligandconjugated enzyme. The assay can detect as little as three femtomoles of receptor and one femtomole
of estradiol
and can. in principle.
be applied
to any receptor-
ligand interaction. The formulation of assay procedures for physiologically relevant receptors or ligands has always been a formidable task. Radio-ligand assay or radioimmunoassay are currently the most frequently used systems (Hunter, 1978), although they suffer from several disadvantages, such as the tedious procedures for synthesizing a radiolabeled ligand, purification of the receptor, production of a high titre antibody. low shelf-life of radioisotopes, expensive equipment, radiation hazards, etc. (Scharpe et al., 1976). A desirable assay for receptors should be cheap. rapid, quantitative, specific and capable of measuring minute quantities even in crude extracts. The reversible nature of the estrogen-receptor interaction with rstradiol. as well as’its stereochemical specificity and high affinity (K,, = lop9 M at 4°C) led us to develop an assay system for this binding that depends exclusively on the specificity of the receptor-ligand interaction. The [ 3H]estradiol binding activity (Puca et al.. 1971) of the 4.5 S form of estrogen receptor isolated from calf uterus (Rica et al., 1973) was determined with [6,73H lest’radiol (50 Ci/mmol). Th e uterine cytosol (2.1 1; 15.1 mg protein/ml) bound 2.62 x 10’ disints/min of estradiol/ml (1.74 x 104 disints/min per mg protein). After fractionation wit,h (NH4)2S04, a portion was subjected to DEAE-cellulose chromatography (Puca et al., 1970) and the remainder was purified by affinity chromatograph,v (Sica et a/.. 1973). From the specific activity (2.4 x lo* disints/min per mg protein) of the affinity eluate (1.86 x lo6 disints/min per ml) it was possible to estimate the estradiol receptor per mg of protein in the cytosol and in the DEAE eluate (estradiol binding capacity 11.5 x lo6 disints/min per ml; 1.6 x 10’ disints/min per mg protein) to be 0.072 pg (1.09 pg receptor/ml) and 66.0 pg (48.0 pg receptor/ml), respectively. Proteins were estimated by the method of Lowry et al. (1951) using bovine serum albumin as the standard. 17fi-Estradiol-17 hemisuccinate (400 pmol) was conjugat’ed to 1.35 mg of pigeon breast, muscle malic dehydrogenase (EC 1 .1,1.37: 5580 units/mg protein) according to the mixed anhydride technique (Erlanger et al.. 1959). Since about 50% of the enzyme activity was lost due to conjugation, active enz.vme (5010 units/mg protein) was separated by affinity chromatography on 419 002%2836/80/3204194)7
$02.00/O
$9 1980 Academic
Press
Inc.
(London)
Ltd.
420
I).
PAIN
AND
A.
SI:ROLIA
Cibacron-blue F3GA-Sepharose (Thompson et al.. 1975). One pmolar unit converts 1 .O prnol of oxaloacetate and /I-NADH to I,-malate and /I-NAD per minute at pH 7.5 at 25°C. The activity of the 178.estradiol-17 hemisuccinate conjugate of malate dehydrogenase in 0.1 M-phosphate buffer (pH 7.5) containing (Ply; bovine y-globulin was determined (Ochoa, 1955) to obtain an enzyme concentration curve at 25°C. All dilutions for the receptor and ligand were made with 10 mlvr-Tris.HCl (pH 7.5) containing 10 mM-KCl, 1 mM-EDTA and 0.1 “i;, bovine y-globulin. EstradiolMDHase+ (5 x 1 O- 3 units ; 10 ~1) was incubated for two hours at 4°C in a volume of 100 ~1 with varying concentrations of the receptor (DEAE-cellulose fraction) before assaying its activity. Similar experiments were carried out with crude tissue homogenate samples (100,OOOg supernatant), before and after addition of the partially purified receptor as an internal standard. To demonstrate that the calf ut’erine estrogen receptor specifically inhibits estradiol-MDHase, reversal of receptor-induced inhibition of estradiol-MDHa.se by the free estradiol was studied by incubating the receptor with an excess of free estradiol (10 to 50 pg in a total volume of 100 ~1) for two hours at 4°C and then with varying amounts of estradiolMDHase (in 10 ~1) for another two hours at 4°C. before assaying the enzyme activity. Varying amounts of estradiol were incubated with the amount of receptor that gave a 66% inhibition of estradiol-MDHase, for two hours at 4°C in a total volume of 100 ~1. Estradiol-MDHase (7.0 x 1O- 3 units in 10 ~1) was then added and. after two hours at 4”C, the extent of reversal of the enzyme inhibition was monitored. In the absence of receptor, the activity of estradiol-MDHase was proportional tc
Enzyme
unit
(x IO-‘)
FIG. 1. Effect of estradiol receptor on estradiol-MDHase activity. The enzyme activity of varying concentrations of estradiol-MDHase was determined at 25°C in the absence (0) and presence (A) of receptor (5 pg) in a total volume of 1 ml as described in t,he text. For reversal of receptor-induced inhibition ofestradiol-MDHase, the receptor was incubated with 50 pg of 17fi-estradiol for 2 h at 4°C in a total volume of 100 ~1, followed by addition of estradiol-MDHase. After 2 h at 4°C the enzyme activity was assayed at 25°C (m).
TAbbreviation
used: MDHase,
malate dehydrogenase.
LETTERS
TO
THE
EDITOR
&?I
the enzyme concentration (Fig. 1) However, about 92o/o loss of enzyme activity was observed with an excess of the receptor (5 pg), due to “conformational freezing” of the enzyme as a consequence of receptor binding (see below). That the decrease in enzyme activity was due only to the receptor-ligand interaction was established by the lack of inhibition of (1) native MDHase by the receptor, or (2) of estradioMDHase by 50 pg of riboflavin binding protein (Murthy et al., 1976) or y-globulin, or by heat-inactivated receptor (65°C for 5 min) (Puca et al., 1971). As expected. the enzyme activity in the conjugate decreased almost proportionately to the amount of receptor in the assay system (Fig. B(a)). Inhibition corresponding to 4.5 x lo- l4 and 9 x lo- l4 moles of receptor was observed when estradiol-MDHase (5 x 10m3 units in 10 ~1) was incubated with 100 ~1 each of 40 and 20 times diluted crude cytosol. Thus the amount of receptor per ml of crude cytosol calculated (1.12 pg/ml) was close to the value obtained from the radio-ligand assay (1.09 pg/ml). When known concentrations, as estimated by [3H]estradiol binding, of the receptor partially mol) were added purified on DEAE-cellulose (9 x 10-14, 15 x lo-l4 and 18 x lo-l4 to the above extract, inhibition corresponding to receptor concentrations of 13.5 x lo-14, 19.5x 10-14and22.5x lo-r4molesandof18x lo-14,24x 10-14and 27 x lo- l4 moles, respectively, was observed and a standard curve could be constructed. . Retention of estradiol-MDHase activity after incubation of the receptor wit#h varying amounts of 17p-e&radio1 is shown in Figure 2(b). Reversal of the inhibition of MDHase activity began at a certain concentration of 17g-estradiol, and then increased almost proportionately to the free 17fl-estradiol concentration, followed by nearly complete retention. The ability of free 178-e&radio1 to reverse the enzyme inhibition was compared to that of 6-carboxymethyloxime-17j3-estradiol, 17~ estradiol (a steroisomer of 17fl-estradiol), testosterone and hydrocortisone (steroid hormones without estrogenic activity) and the non-steroidal estrogen diethylstilboestrol. The amounts of 17p-estradiol, estriol, 6-carboxymethyloxime-178. estradiol and diethylstilboestrol required for 50% reversal were 4.3 x 10-14, 4.5 x lo-13, 2.5 x lop9 and 1.5 x lo- l4 moles, respectively. Testosterone, hydrocortisone and 17n-e&radio1 gave almost no reversal (<2oj), even at very high concentrations (5 x 1O-9 mol). The pattern of specificity is in good agreement with the reported potency of binding (Puca & Bresciani, 1969) and suggests that the inhibition and its reversal are due to the receptor-ligand interaction. It is worthwhile to compare the present approach with other systems. Selective adsorption by dextran-coated charcoal (McGuire, 1975), precipitation with protamine sulfate (Chamness et al., 1975) and gel filtration on Sephadex G-25 (Puca & Bresciani, 1968) are relatively simple and rapid but they are susceptible to influences from non-specific binding proteins and the protein concentration of the sample. With the present method. it is possible to assay estrogen receptor concentrations as low as 2.1 rig/ml, corresponding to a total protein concentration of 30 pg/ml. That is, it is at least 16 times more sensitive than the method developed recently using thin-layer gel filtration on Sephadex G-200 (Hazato et al., 1979), with the added advantage of rapidity. With the present method, e&radio1 receptor can be measured during various phases of the estrus cycle. Thus 0.25 x 1 O- “. 0.37 x 10-12. 0.53 x IO-l2 and 0.85 x lo-l2 moles of e&radio1 receptor per uterus
422
I).
0
PAIS
15 Receptor
AND
A. St’ROLIA
30
concentrotlon
(x IO+ mols)
(a)
45
I Log
2 [estrodiol]
3 (x b5
4 mols)
(b)
FIG. 2. Receptor induced inhibition of estradiol-MUease and the reversal of enzyme inhibition by free estradiol. Estradiol-MDHase (5.0 x 1 OF” units in 10 ~1) was incubated with varying concentrations of the receptor as described in the text and then assayed (a). Varying amounts of 17/Sestradiol were first incubated with the (fixed) amount of receptor that gave 66% inhibition and then with estradiol-MDHase (7 x 10e3 units in 10 ~1) and assayed (b). Details are given in the text.
were obtained during metestrus, diestrus, proestrus and estrus, respectively. Sensitivity of the radioimmunoassay for 17/Sestradiol is in the range of 20 pg/ml (Fishman & Fishman, 1974), whereas the present method can detect as little as 0.2 pg of 17/3-estradiol per ml. Moreover, a reduction in reaction volume by tenfold would lead to a tenfold higher sensitivity. The method can, in principle, be applied to any receptor-ligand interaction, eliminating the synthesis of a radiolabelled-ligand (radio-ligand assay) or production of an antibody (radioimmunoassay). Besides, with the present method it is easy to assay the receptor during various stages of purification and in various stages of cell or organ development. and to detect a putative receptor for a ligand by its effects on a ligand-conjugated enzyme. With the exception of equilibrium dialysis, which is lengthy and often difficult to quantitate (Baulieu & Raynaud, 1970), the present method may be the only procedure for assaying receptors and ligands where the separation ofthe free from the bound ligand is not required. Of course, the amount of receptor or the ligand that can be determined will depend on the affinity of the receptor for its specific ligand. Thus 0.1 femtomole of avidin can be detected using lectin using lactosylbiotin-MDHase (K,= 1015 Mm1 ) and 83 picomoles of Ricinw glucose-6-phosphate dehydrogenase (K, = lo8 M-') (our unpublished results). Mechanism
of InhibSon
of the Ligand-Malate Receptor
Dehydrogenase
Conjugate
by the
An experimental approach supporting the mechanism of inhibition of estradiolMDHase by estradiol receptor involving a “conformational freezing” of the enzyme due to binding of receptor to it is described here. No appreciable differences in the
LETTERS
TO THE
EDITOR
123
and p-NADH were observed for either estradioKm values for oxaloacetate MDHase or receptor-bound estradiol-MDHase from the native MDHase. This suggests that steric hindrance of the enzyme for either substrate by the bound receptor is not responsible for the enzyme inhibition. Assuming that all estradiols attached to a random set of enzyme sites are bound by the receptor. and that’ receptor binding to any estradiol attached to an “active” group of the enzyme causes 1OOqt enzyme inhibition. then the probability P, of inhibition of a givetl enzyme molecule is given by: p
m
= 1 _ (R-.4)!(R-m)! (R-d -m)!R!
(1)
’
where nr is the number of ligands bound to a set of R random active. When d = 1, equation (1) becomes :
site, A of which are
If rl is the average of randomly introduced ligands per enzyme, then the probability P, of finding an enzyme molecule bearing exactly m ligands is given by: P,=
n”‘(R-/z)~-“’
R !
m!(R-m)!RR
(3)
So, receptor bound to an “active” ligand inhibits the fraction P of enzyme molecules that is equal to the sum of the products of P, and P,, when m is ranging from zero to R; i.e. P=
; P,P, m=O
(4)
ff, however. because of steric constraints, only a limited number, C, of receptors can fit on the random set of ligands, the fraction of enzyme molecules inhibited becomes : C-l
P =
1 m=O
P,P,
+
;
P,P,C’/m.
(5)
m=C
When the difference between R and (7 is very small, the values of P obtained by equations (4) and (5) are more or less the same for practical purposes. Estradiol-MDHase with 21 estradiols (=R) conjugated to it, as estimated by conjugating [6,7-3H]17fl-estradiol, was mixed with an excess of estradiol receptor and subjected to gel filtration on Sepharose 6B. A major peak corresponding to a molecular weight of 1.2 x 106k20,000 was obtained, suggesting that 18 molecules of the receptor (M,=62,000) are bound per molecule of the conjugated MDHase. The values of R=21 and C= 18 were substituted in equation (5) and theoretical values of the enzyme inhibition corresponding to various occupancies of estradiolMDHase by the hormone receptor were computed considering the involvement, of either one or two active amino groups (A = 1,2) in the catalytic activity of the enzyme.
424
Receptor
molecules
/estradlol
-MDHase
moiecule
Es;. 3. Receptor-induced inhibition of estradiol-MDHase. The curve ( -) shows the theoretical 1 “active” group model where binding to the active group is assumed to cause lOOo/0 inhibition (R=21, C= 18). Even when 2 “active” groups are implicated, an almost identical pattern is observed (- - - --). Data points (a) indicate the experimentally observed values. In a separate set of experiments, a constant amount of estradiol-MDHase was mixed with 2.5,6, 10,15,20 and 25 molar excess ofthe receptor and, after incubation for one hour, they were subjected to gel filtration on Sepharose 6B. Peaks corresponding to molecular weight of 2 x 10’ +8000. 3.9 x 10’ ) 12,000, 57x 10s~15,000, 8.9~ 105+20,000, 1.05~ 106~20,000 and 1.20x 106f30,000 were obtained, indicating that 2. 5, 8, 13, 16 and 18 molecules of receptor, respectively, were bound per enzyme molecule. These peak fractions were then assayed for the enzyme activity (Fig. 3). An agreement between the experimental enzyme inhibition data with those obtained theoretically, assuming 1 (or 2) essential amino groups, substantiates our interpretation regarding the inhibition of the ligand-conjugated enzyme by the specific receptor. The striking effect of the estradiol receptor on the activity of estradiol-MDHase probably arises either from a conformational alteration imposed by the receptor binding to an active group, or by prevention of conformational changes essential for enzyme activity. In summary, inhibition of the ligand-conjugated MDHase is due to the specific interaction of the receptor with its ligand and arises mainly due to a “conformational freezing” of the enzyme. The authors thank Professor B. K. Bachhawat for his encouragement. supported by the Department of Science and Technology, India. Indian Institute of Experimental Medicine Jadavpur, Calcutta-700032, India Received 21 March
This work was
DEBKUMAK. AVADHESHA
1980, and in revised form 1 July 1980 REFERENCES
Baulieu, E. E. & Raynaud, J. P. (1970). Eur. J. Biochem. 13, 293-299. Chamness, G. C., Huff, K. & McGuire, W. L. (1975). Stwoids. 25, 627-635.
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Erlanger. B. F., Borek, F., Beiser, S. M. & Lieberman, R. (1959). J. Riol. Chem. 234, 1090~1094. Metab. 39, 603-606. Fishman, J. & Fishman, J. H. (1974). J. Clin. Endocrinol. Hazato, T.. Murayama, A., Matsuzawa, A. & Yamamoto, T. (1979). Anal. Biochem. 94,29--35. Hunter, W. M. (1978). In Handbook of Experimental Immunology (Weir, D. M., ed.), 3rd edit., pp. 14.1-14.40, Blackwell Scientific Publications, Oxford. Lowry, 0. H., Rosebrough, N., Farr. A. 8: Randall, R. (1951). J. Hiol. Chem. 193, 26.5275. McGuire? W. L. (1975). In Methods in Enzymology (O’Malley. B. W. & Hardman. J. G.. rds). vol. 36. pp. 248-254, Academic Press, New York. Murthy, U. S., Podder, S. K. & Adiga, P. R. (1976). Biochim. Biophys. Acta, 434, 69-81. Ochoa, S. (1955). In Methods in Enzymology (Colowick, S. P. & Kaplan. N;. 0.. eds), vol. 1. pp. 735-739. Academic Press, London, New York. l’uca, G. A. 8: Bresciani, F. (1968). ‘Vatwe (London,). 218, 967-969. Puca, G. A. & Bresciani, F. (1969). ,Vature (London), 223, 74Fi-747. l’uca. (:. A.. De Sombre, E. R. &Jensen, E. V. (1970). In Research on Steroids (Finkelstein, RI., Klopper. A., Conti, (1. N. & Cassano. C., eds), vol. 4, pp. 274-289. Pergamon Press. New York.
Puca. (>. A.. Nola. E., Sica, V. & Bresciani, F. (1971). Riochrmistry. 10, 3769-3780. Scharpe, S. L., (looreman, W. M., Blomme, W. ?J. & Laekeman, (:. lfII. (1976). Clin. (‘hem. 22. 733-738. Sica. V., Parikh. 1.. Nola, E., Puca. (:. A. & Cuatrecasas, P. (1973). J. Riol. Ch,rm. 248. 6543-6558. Thompson. S. T., Cass, K. H. 8: Stellwagen. E. (1975). Proc. ‘Vat. ilcad. Ski., U.S.A. 72. 669-672.