Binding of the chymotrypsin substrate, tryptophan methyl ester, by rat α-fetoprotein

611

Biochimica et Biophysica Acta, 6 3 2 ( 1 9 8 0 ) 6 1 1 - - 6 1 8 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 29405

BINDING OF THE CHYMOTRYPSIN SUBSTRATE, T R Y P T O P H A N METHYL ESTER, BY RAT c~-FETOPROTEIN

M I C H A E L E. B A K E R , C A T H E R I N E S. M O R R I S a n d D A R R E L L D. F A N E S T I L

Department of Medicine, M-023, Division of Nephrology, University of California, San Diego, School of Medicine, La Jolla, CA 92093 (U.S.A.) (Received May 6th, 1980)

Key words: Chymotrypsin substrate binding; Tryptophan methyl ester; c~-Fetoprotein; (Rat)

Summary We studied h o w t r y p t o p h a n methyl ester and related c o m p o u n d s inhibit binding of estrone to rat ~-fetoprotein and find that: (a) like chymotrypsin, ~-fetoprotein binds t r y p t o p h a n esters with higher affinity than t r y p t o p h a n or its amides; (b) the affinity of ~-fetoprotein for tryptophan methyl ester is 3.7 • 10 -4 M, which is close to the affinity of chymotrypsin (10 -4 M); (c) a-fetoprotein binding of t r y p t o p h a n methyl ester is stereoselective and pH dependent. All of these observations suggest that there is a specific interaction between a-fetoprotein and the chymotrypsin substrate, tryptophan methyl ester, and that rat ~-fetoprotein contains a site with some structural similarities to the catalytic site in chymotrypsin. Since we also find that tryptophan methyl ester is a competitive inhibitor of estrone binding to ~-fetoprotein, it is possible that the protease substrate binding site on ~-fetoprotein is spatially close to the estrone binding site.

We reported that c o m p o u n d s which bind serine proteases can also inhibit binding of steroid hormones to' intracellular receptors and serum proteins [1,2]. In those studies we observed that rat a-fetoprotein b o u n d the chymotrypsin substrate, t r y p t o p h a n methyl ester (TrpOMe), with considerably higher affinity than did intracellular steroid hormone receptors. In this report we describe in more detail the nature of the TrpOMe binding site on a-fetoprotein. Our studies suggest that this site m a y have some structural properties similar to the TrpOMe binding site on chymotrypsin. 1

Abbreviations: Tes, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; Pipes, piperazine-N,N bis[2-ethane-sulfonie acid]; Bicine, N,N-bis[ 2-hydroxyethyl]glycine.

612 Materials and Methods The source of rat a-fetoprotein, serum from Buffalo rats with Morris hepat o m a 7777, was a gift from Dr. Stewart Sell. We have previously shown that TrpOMe inhibited 17/3-[3H]estradiol binding to rat a-fetoprotein [2]. In those studies we used Sephadex G-25 columns to separate steroid bound to a-fetoprotein from u n b o u n d steroid. However, this technique proved cumbersome in doing a Scatchard analysis of estrogen binding to rat a-fetoprotein in the presence of TrpOMe, where m a n y steroid concentrations are required for each experiment. Therefore, we adapted the Dextran-coated charcoal technique described by Baxter and Tomkins [3] for measuring the binding of [3H]estrone to a-fetoprotein. To optimize conditions for this assay, we employed two maneuvers to decrease the rate of dissociation of estrogen from a-fetoprotein. First, we used [3H]estrone, because it has a higher affinity (presumably due to a slower rate of dissociation) than does 17~[3H]estradiol [4,5]. Second, 15% glycerol was included in the incubation buffer because it has been shown to decrease the rate of dissociation of steroids from their receptors [6,7]. Initial studies using 20 mM Tes (pH 7.8)+ 2 mM EDTA indicated that Dextran-coated charcoal interacted with a-fetoprotein causing a loss of [3H]estrone binding which was not due to the on-off reaction of [3H]estrone with a-fetoprotein. We found that addition of 50 mM NaC1 to the incubation buffer and 10 mg/ml of ovalbumin to the Dextran-coated charcoal eliminated this stripping of [3H]estrone from rat a-fetoprotein. Employing these modifications, we obtained similar values for specific binding of [3H]estrone to a-fetoprotein with Sephadex G-25 columns and the Dextran-coated charcoal assay. While we were completing this study, Payne and Katzenellenbogen [8] reported results which also show that the dissociation of estrogen from rat a-fetoprotein is rapid, and that special care must be exercised in using non-equilibrium techniques for measuring steroid that is bound to a-fetoprorein. In most of our assays we diluted Morris hepatoma 7777 serum 1 : 10 000 into a buffer (20 mM Tes, Pipes, or Bicine) containing 2 mM EDTA, 50 mM NaC1, 15% glycerol and 0.2 mg/ml ovalbumin. This solution was incubated in glass tubes on ice with [3H]estrone (85--115 Ci/mmol New England Nuclear) usually for 2--3 h. Bound steroid was separated from u n b o u n d steroid using our modified Dextran-coated charcoal technique. We added 0.2 ml of 100 mg/ ml charcoal, 10 mg/ml Dextran and 10 mg/ml ovalbumin in buffer to 2.0 ml of the a-fetoprotein/steroid h o r m o n e solution. After vortexing and incubating for 60 s, the tubes were centrifuged at 6000 × g for 15 min. The radioactivity in 1.0 ml aliquots was determined in a liquid scintillation cocktail (phase two, Western Chemical Products). Specifically bound steroid was determined, by subtracting the a m o u n t of [3H]steroid bound in the presence of a 200-fold excess concentration of the same nonradioactive steroid, from the a m o u n t of [3H]steroid bound in the absence of the non-radioactive steroid. The rate of dissociation of [3H]estrone from rat a-fetoprotein was determined by incubating rat a-fetoprotein with 3 • 10 -9 M [3H]estrone for 2 h and then transferring aliquots to glass tubes on ice with either 1.5 • 10 -6 M [3H]estrone or 3 • 10 -6 M [3H]estrone. At different intervals aliquots were removed for analysis of specific [3H]estrone binding.

613 Results

First we proceeded to define the conditions for attainment of equilibrium binding of [3H]estrone and D-TrpOMe to rat a-fetoprotein at 0°C, pH 7.8. Fig. 1 shows the time course of specific binding of [3H]estrone to rat a-fetoprotein in the presence and absence of 0.5 mM D-TrpOMe. It is clear that at 0°C, in the presence of 15% glycerol, equilibrium is achieved in 60--90 min. In the course of our efforts to develop an accurate Dextran-coated charcoal assay for the purpose of measuring [3H]estrone b o u n d to a-fetoprotein we determined the off-rate of [3H]estrone from rat a-fetoprotein. Fig. 2 shows that in the presence of 15% glycerol the half-time for dissociation is a b o u t 18 min, in good agreement with the results of Payne and Katzenellenbogen [8].

Specificity v f substrate binding to rat a-fetoprotein Our working hypothesis [1,2] is that a-fetoprotein and other steroid binding proteins have a site with a specific structure which recognizes protease inhibitors and substrates [9--11 ]. However, it is possible that the binding of protease substrates and inhibitors is nonspecific in nature, since the h y d r o p h o b i c properties of the site which binds steroids might also bind a h y d r o p h o b i c c o m p o u n d like tryptophan. To explore this question further, we compared the effects of the D and L stereoisomers of TrpOMe and t r y p t o p h a n , and of tryptophanamide, tryptamine and tryptophol (Fig. 3) on the binding of [3H]estrone to a-fetoprotein at 0°C,

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Fig. 1. T i m e c o u r s e o f [ 3 H ] e s t r o n e b i n d i n g t o ~ - f e t o p r o t e i n in t h e p r e s e n c e a n d a b s e n c e o f 0 . 5 m M D - t r y p t o p h a n m e t h y l e s t e r . ~ - F e t o p r o t e i n w a s i n c u b a t e d w i t h 1.5 • 10 -9 M [ 3 H ] e s t r o n e a l o n e , w i t h 3 " 10 -7 M e s t r o n e or w i t h 0 . 5 m M D - t r y p t o p h a n m e t h y l e s t e r a t 0 ° C , p H 7.8. A t d i f f e r e n t t i m e intervals, aliquots were removed and [3H]estrone specifically bound to ~-fetoprotein was determined u s i n g t h e D e x t r a n - c o a t e d c h a r c o a l t e c h n i q u e . E a c h p o i n t is t h e a v e r a g e f r o m t w o s a m p l e s . V a r i a t i o n b e t w e e n s a m p l e s w e r e less t h a n 1 0 % . Fig. 2. D i s s o c i a t i o n r a t e c o n s t a n t d e t e r m i n a t i o n f o r [ 3 H ] e s t x o n e f r o m ~ - f e t o p r o t e i n . ( ~ - f e t o p r o t e i n w a s i n c u b a t e d w i t h 1 . 5 • 10 -9 M [ 3 H ] e s t r o n e a t 0 ° C , p H 7.8 f o r 2 h. T h e n a l i q u o t s w e r e t r a n s f e r r e d to glass t u b e s o n ice c o n t a i n i n g 3.0 • 1 0 -6 M e s t e r o n e . A t d i f f e r e n t i n t e r v a l s a l i q u o t s w e r e r e m o v e d f o r analysis o f s p e c i f i c [ 3 H ] e s t r o n e b o u n d t o ~ - f e t o p r o t e i n . E a c h p o i n t is t h e a v e r a g e o f t w o s a m p l e s , v a r i a t i o n b e t w e e n s a m p l e s w a s less t h a n 1 0 % . T h e d a t a axe r e p r e s e n t e d b y a s e m i l o g a r i t h m i c p l o t w i t h a s l o p e o f 0 . 0 3 8 3 m i n -1 a n d c o r r e l a t i o n c o e f f i c i e n t o f - - 0 . 9 9 . T h e h a l f - t i m e o f d i s s o c i a t i o n o f c s t r o n e f r o m ~ - f e t o p r o t e i n w a s d e t e r m i n e d b y d i v i d i n g In 2 b y t h e s l o p e , o o, c o n t r o l , • • , 3 . 0 - 10 -6 M [ 3 H ] estrone.

614 H N

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Fig. 3. S t r u c t u r e of t r y p t o p h a n , t r y p t o p h a n m e t h y l e s t e r , t r y p t o p h a n a m i d e , t r y p t o p h o l , a n d t r y p t a m i n e . Fig. 4. I n h i b i t i o n o f [ 3 H ] e s t r o n e b i n d i n g to ~ - f e t o p r o t e i n b y L a n d D i s o m e r s o f t r y p t o p h a n m e t h y l e s t e r . ~ - F e t o p r o t e i n w a s i n c u b a t e d f o r 3 h at 0 " C , p H 7.8 w i t h 1.5 " 10 -9 M [ 3 H ] e s t r o n e a l o n e , w i t h 3 • 10 -7 e s t r o n e o r w i t h d i f f e r e n t c o n c e n t r a t i o n s o f L or D t r y p t o p h a n m e t h y l e s t e r . T h e n [ 3 H ] e s t r o n e s p e c i f i c a l l y b o u n d to ~ - f e t o p r o t e i n w a s d e t e r m i n e d u s i n g t h e D e x t r a n - c o a t e d c h a r c o a l a s s a y . E a c h p o i n t is t h e a v e r a g e f r o m t w o s a m p l e s , v a r i a t i o n b e t w e e n s a m p l e s w a s less t h a n 10%. o o L-TrpOMe, • •, D-TrpOMe.

pH 7.8. Table I and Fig. 4 show that a-fetoprotein recognizes TrpOMe and tryptophol, and that the L isomer of TrpOMe has a higher affinity for a-fetoprotein than the D isomer. As seen in Figs. 5 and 6, the binding of TrpOMe and tryptophan ethyl ester TrpOEt, but not tryptophol, is dependent on pH. The apparent pK of the binding of TrpOMe and TrpOEt to a-fetoprotein is about 7. TABLE I INHIBITION OF [3H]ESTRONE

BINDING TO c~-FETOPROTEIN

a - F e t o p r o t e i n w a s i n c u b a t e d w i t h 1.5 • 10 -9 [ 3 H ] e s t r o n e a l o n e , w i t h 3.0 • 10 -7 e s t r o n e , or w i t h 0 . 5 m M o f t h e b e l o w c o m p o u n d s , a t 0 ° C , p H 7.8 f o r 3 h. B o u n d [ 3 H ] e s t r o n e w a s d e t e r m i n e d u s i n g t h e D e x t r a n c o a t e d c h a r c o a l a s s a y . T h e r e s u l t s f r o m t w o e x p e r i m e n t s are s h o w n . I n e a c h e x p e r i m e n t t h e c o m p o u n d s w e r e t e s t e d in d u p l i c a t e , v a r i a t i o n b e t w e e n s a m p l e s w a s less t h a n 10%. S p e c i f i c a l l y b o u n d e s t r o n e in t h e c o n t r o l s a m p l e w a s 31 0 0 0 e p m / m l . Compound

Concentration (raM)

% Inhibition of specifically bound [ 3 H ] estrone

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0.5 0.5 0.5 0.5 0.5 0.5 0.5

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615

TABLE II EFFECT OF D-TRYPTOPHAN PROTEIN

M E T H Y L E S T E R O N T H E B I N D I N G O F E S T R O N E T O R A T o~-FETO-

Condition

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These results sugtgest that there is specific interaction between the chymotrypsin substrate TrpOMe and rat a-fetoprotein.

Competitive inhibition o f [3H]estrone binding to rat a-fetoprotein by D-TrpOMe Two other questions which are important for characterizing the binding of TrpOMe to a-fetoprotein are: is inhibition of [3H]estrone binding to a-fetoprorein by TrpOMe competitive or noncompetitive, and what is the affinity of D-TrpOMe for a-fetoprotein? To answer these questions, we used the m e t h o d of Scatchard [12] to measure both the equilibrium dissociation constant of [3H]estrone for a-fetoprotein and the apparent equilibrium dissociation constant of [3H]estrone for a-fetoprotein in the presence of D-TrpOMe. In Fig. 7 we show the results from one such experiment. The equilibrium dissociation constant of 2.8 • 10 -9 M f o r [3H]estrone is in good agreement with previously reported values [4,5,8]. In the presence of TrpOMe the apparent affinity of [3H]estrone for a-fetoprotein is lowered while the maximal number of binding sites remains unchanged. Table II summarizes results from several experiments similar to that shown in Fig. 7. These results are consistent with competitive inhibition of [3H]estrone binding to a-fetoprotein by TrpOMe. The equilibrium dissociation constant of D-TrpOMe for a-fetoprotein (gd TrpOMe) was calculated from the formula KTDrP O M e --

KdC

Kapp -- Kd where K d is the equilibrium dissociation constant of estrone, Kap p is the apparent dissociation constant of estrone in the presence of D-TrpOMe, and C is the concentration of D-TrpOMe [13]. The Kd of TrpOMe for a-fetoprotein is 3.7 • 10 -4 M.

TrpOMe is a reversible inhibitor o f estrogen binding to rat a-fetoprotein The observation that the maximal number of [3H]estrone binding sites is unaltered by TrpOMe indicates that TrpOMe reversibly binds to a-fetoprotein, because irreversible reaction with TrpOMe would produce a decrease in the maximal number of binding sites. Reversible binding of TrpOMe to a-fetoprotein has also been demonstrated by separating 1 mM D-TrpOMe from a-fetoprotein (by filtration on Sephadex

616

G-25 columns) and then demonstrating that the a-fetoprotein rebinds estrogen to the same extent as did a-fetoprotein which had not been previously incubated with D-TrpOMe. Discussion

Three aspects of our results (Table II, Figs. 4--7) support the hypothesis that a site on e-fetoprotein which regulates binding of [3H]estrone has properties in c o m m o n with the substrate binding site on chymotrypsin. 1. The K d of chymotrypsin for N-acetyl-tryptophan methyl ester (Ac-TrpOMe) is 1 ' 10 -4 M [14], a value which is close to the 3.7 • 10 -4 M Kd of a-fetoprotein for D-TrpOMe (Table II). (In unpublished experiments, we found that the Kd of a-fetoprotein for Ac-TrpOMe is 3.3 • 10 -4 M.) 2. D-TrpOMe and D-TrpOEt inhibit [3H]estrone binding to a-fetoprotein while tryptophan, t r y p t o p h a n a m i d e and tryptamine do not. Thus, both a-fetoprotein and chymotrypsin bind tryptophan esters with a higher affinity than t r y p t o p h a n or its amides [15,16]. 3. Tryptophol, a competitive inhibitor of chymotrypsin [17], also binds to a-fetoprotein, as evidenced b y its ability to inhibit binding of [3H]estrone by -fetoprotein. Furthermore, TrpOMe and TrpOEt binding to a-fetoprotein is stereoselective and pH dependent. Thus, these results reinforce the hypothesis [1,2] that there is a specific interaction between a-fetoprotein and these chymotrypsin substrates. We have previously suggested [1 ] that the site on steroid hormone receptors that binds protease inhibitors (e.g. diisopropyl fluorophosphate, phenylmethyl 100f loo

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7% 7'5 8'0 815 910 6L5 7LO 715 810 8'5 pH pH Fig. 5. Effect of pH on inhibition of [3H]estrone binding to c~-fetoprotein by tryptophan methyl ester. ~-Fetoprotein was incubated for 3 h at 0°C at, pH 6.5 (20 mM Pipes buffer)~ pH 7.2 (20 mM Tes buffer), pH 7.8 (20 mM Tes buffer), pH 8.5 (20 mM Bicine buffer) with 1.5 . 10 -9 M [3H]estrone alone, with 3 - 10 -7 M estrone with 0.5 mM tryptophol, or with either 0.5 mM L- or D-tryptophan methyl ester. Then [3H]estrone specifically bound to a-fetoprotein was determined using the Dextran-coated charcoal t e c h n i q u e . E a c h p o i n t is t h e a v e r a g e f r o m t w o s a m p l e s , v a r i a t i o n b e t w e e n s a m p l e s w a s l e s s t h a n 1 0 % . Percent inhibition was determined relative to the control at each pH. However, the binding of [3H]estrone by ~-fetoprotein between, pH 6.5 and 8.5 appears to be independent of pH with the buffers we used.~,0.5 mM tryptophol;0.05mM L-TrpOMe',O 0.5raM D-TrpOMe;pK a ~ 7.0. F i g . 6. E f f e c t o f p H o n i n h i b i t i o n o f [3 H ] e s t r o n e b i n d i n g t o ~ - f e t o p r o t e i n e x p e r i m e n t w a s p e r f o r m e d a s d e s c r i b e d i n t h e l e g e n d f o r F i g . 5. o 0.5 mM D-TrpOEt; pK a ~ 7.0.

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F i g . 7. S e a t c h a r d a n a l y s i s o f t h e e f f e c t o f 0 . 5 m M t r y p t o p h a n m e t h y l e s t e r o n t h e b i n d i n g o f [3 H ] e s t r o n e t o ~ - f e t o p r o t e i n . a - F e t o p r o t e i n w a s i n c u b a t e d w i t h [ 3 H ] e s t r o n e , ( 7 . 5 • 1 0 - 1 0 M t o 1 . 5 • 1 0 -8 M ) -+ u n l a b e l l e d e s t r o n e or 0.5 m M T r p O M e for 3 h at 0 ° C , p H 7.8. B o u n d s t e r o i d was s e p a r a t e d f r o m u n b o u n d steroid using Dextran-coated charcoal. Ordinate: specifically bound [3H]estrone (cpm/ml) divided by free estrone (cpm/ml). Abscissa: specifically bound [3H]estrone (mol/ml). o c, [ 3 H ] e s t r o n e , K d = 2 . 8 1 0 -9 M , r = - - O . 9 3 ; • • + 0 . 5 r a m T r p O M e , K d = 6.7 • 10 -9 M , r = - - 0 . 9 6 .

sulfonyl fluoride, tosylamide-phenylethyl-chloromethyl ketone, tosyl-lysine chloromethyl ketone) and substrates (e.g. TrpOMe and TosArgOMe) has some structural similarities to the substrate binding site in chymotrypsin and other serine proteases [9--11]. This hypothesis is strengthened by our findings reported here that a-fetoprotein binding of the chymotrypsin substrate, TrpOMe, has specificity and resembles in some ways the interaction of chymotrypsin with this substrate, and recent studies, still in progress, show, that a histidine selective reagent, diethypyrocarbonate [18], inhibits estrogen binding to a-fetoprotein. From our results we cannot determine whether protease inhibitors and substrates inhibit steroid hormone binding to a-fetoprotein by binding at the steroid hormone binding site or by binding t o an allosteric site. Our previous finding that the M r 20 000 mero-receptor fragment of the progesterone receptor in chick oviduct could bind protease inhibitors and substrates [2] suggested that the steroid hormone binding and protease inhibitor binding sites are n o t widely separated on the intact receptor and that the sites could possibly overlap. More compelling, but not conclusive, evidence for some spatial overlap of the steroid hormone and protease substrate binding sites comes from our finding that D-TrpOMe is a competitive inhibitor of [3H]estrone binding to a-fetoprotein (Fig. 7) and [3H]dexamethasone binding to HTC cell cytosol [19]. Acknowledgements This work was supported by NIH grants GM24190 and AM14915 and grant SG868 from the American Cancer Society, California Division.

618

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10 11 12 13 14 15 16 17 1S 19

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