Affinity Labeling of the Digitalis Receptor with p-Nitrophenyltriazene-Ouabain, a Highly Specific Alkylating Agent*

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitro- phenyltriazene (NPT) ethylenediamine to the perio-date-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, 11, and 111) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na’,K+)-ATPase) puri- fied from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NFT-ouabain I to the enzyme, and (iii) by the inhibition of r3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25°C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not signif-icantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain properties of interaction of NPT-ouabain with (Na',K')-ATP- ase using competition with purified

icantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as t h a t observed with t h e purified enzyme.
[3H]NPT-ouabain I w a s also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, 13H]NPTouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a M, = 93,000 is specifically labeled by [3H]NPT-ouabain I.
Digitalis preparations have proven to have great value clinically in the treatment of chronic congestive failure and other disorders associated with the cardiovascular system. In addition to its clinical importance, this family of molecules has attracted the interest of biochemists, physiologists, and biophysicists because of its rather unique action on the transmembranal transport of sodium and potassium. It is well known that the sodium pump in the plasma membrane of eukaryotic animal cells is inhibited by low concentrations of digitalis glycosides (1)(2)(3). Studies on the nature of the inter-* This work was supported by the Institut National de la Sante et de la Recherche Medicale (ATP 78-95), the Centre National de la recherche Scientifique, and the Commissariat a 1'Energie Atomique. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. actions of sodium-and potassium-activated ATPase with ouabain and related compounds indicate that pump inhibition is linked to enzyme inhibition (for reviews see Refs. 4

and 5).
Several covalent labels of the digitalis binding site have been synthesized in the last few years (6)(7)(8)(9)(10). The most effective of these have been photoaffinity derivatives of ouabain and strophanthidin (9)(10)(11). Recent reports have confinned the work of Ruoho and Kyte (6), who found that the major site of ouabain binding to (Na', K+)-ATPase' is the large polypeptide chain of M , = 100,OOO. This chain contains both the sugar and the steroid binding sites of the digitalis center of the enzyme (10,12).
Photoaffinity derivatives of drugs of pharmacological interest are being used with increasing frequency in attempts to specifically label their protein receptors (see for example Refs. 13 to 16). The great advantage of these derivatives is that, once irradiated, photoactivable groups produce radicals which will almost inevitably result in a covalent association with the receptor. There are several problems encountered in the use of these derivatives. First, the required UV irradiation can damage the receptor. Rogers and Lazdunski (10) encountered this difficulty when they used a repetitive cycle of binding, photolysis, and washing to increase the extent of covalent modification. Secondly, the high reactivity of the radicals formed results in covalent attachment of the affinity label to multiple sites on the receptor molecule and in many cases, to polypeptide chains which are in the vicinity of the receptor polypeptide chain (10,16). The interpretation of labeling patterns obtained with membrane preparations or homogenates in which the receptor represents only a small fraction of the total protein is frequently difficult. Photoactivable derivatives of ouabain and strophanthidin which gave easily interpretable labeling patterns with a pure (Na',K')-ATPase preparation yielded very complex patterns with membrane preparations of electric organ (10) or of cardiac cells.' Finally, good labeling results are usually obtained only if the photoaffinity label binds to its receptor with a high affinity.
This report describes the synthesis of a new affinity derivative of ouabain, one which is capable of alkylating the cardiac glycoside receptor. This affinity derivative is potentially much more useful than those which have been previously described. sariat a 1'Energie Atomique. ['HIOuabain (19 Ci/mmol) was obtained from New England Nuclear.
Synthesis of p-Nitrophenyltriazene Ethylenediamine-p-Nitrophenyltriazene ethylenediamine (l-nitrophenyl-3-(2-aminoethyl)triazene) was prepared using a modification of the procedure described by Sinnott and Smith (19). Typically, 14.65 mg of ethylenediamine (0.244 mmol) were dissolved in 3 ml of ice-cold water and the pH adjusted to 7.0 with HCI. p-Nitrophenyldiazonium tetrafluoroborate (28.9 mg, 0.122 nmol) was suspended in 2 ml of ice-cold water and then added to the ethylenediamine solution. The suspension was maintained at 0°C and stirred constantly. Throughout the reaction the pH was at pH 7.0 (Radiometer Titrator I1 equipped with a SBR2c recorder). After 5 min, the solid phase of the reaction medium was separated by filtration and washed with 2 ml of ice-cold water. The filtrate was extracted with 5 ml ice-cold diethyl ether which had been previously saturated with water. The aqueous phase was then extracted 4 times with 5 ml of ice-cold I-butanol. The combined butanol extracts were evaporated under vacuum at room temperature (15.8 mg, 62% yield). After quantitative determinations of primary amines (la), we used a molecular absorption coefficient of E:IYY = 22,000 M" X cm", a value in accordance with that reported by Sinnott and Smith (19) for p-nitrophenylpropyltriazene. The product was either immediately used or stored at -60°C and repurified before use on a Sephadex LH-20 column (elution with methanol).
Synthesis ofp-Nitrophenyltriazene Ethylenediamine Ouabain Derivatives (NPT-0uabain)"The synthesis of NPT-ouabain is outlined in Fig. 1. Schiff base formation between p-nitrophenyltriazene and / @ on the aldehyde groups of oxidized ouabain was followed by a reduction with NaCNBH,+ The synthesis yielded three NPT derivatives of ouabain.
Ouabain oxidation was performed according to Rogers and Lazdunski (10) except that iodate was removed on a QAE-Sephadex column (Pharmacia) instead of precipitation with lead (11) acetate.
Oxidized ouabain (10 mM) was incubated with 20 mM NPT ethylenediamine in absolute methanol a t room temperature. The reaction mixture was adjusted to an apparent pH between 5 and 6 (20) by adding acetic acid or triethylamine. NaCNBH., was then added to a final concentration of 20 mM. The reaction was followed by analytical thin layer chromatography. Three spots appeared with R p values of 0.40 (NPT-ouabain I), 0.49 (NPT-ouabain II), and 0.55 (NPT-ouabain 111). After 4 h, the reaction products were separated by preparative thin layer chromatography (2 mm). The three products, each representing approximately 5% of the starting material, were eluted with methanol. The three compounds had identical UV spectra, each consisting of two peaks (Fig. 2). The fist peak (A,,,., = 362 nm) represents the triazenyl group. A shift of h.,.. occurs during the coupling of NPT ethylenediamine (An,ax = 352 nm) to ouabain. The second peak represents the lactone ring of ouabain (cL0 = 14,500 M I X cm") and to some extent the secondary peak of p-nitrophenyltriazene ethylenediamine (€220 = 8,500 M" X cm". inset of Fig. 2).
Radiolabeled p-nitrophenyltriazene ouabain was synthesized by two methods, the usual procedure being similar to that for the synthesis of unlabeled NPT-ouabain. (i) ["HINPT ethylenediamine (25.8 nmol; specific radioactivity, 30 Ci/mmol) and 200 nmol of oxidized ouabain (10) were incubated in 100 pI of absolute methanol at pH 6.0 to 7.0. After 15 min, NaCNBH.i was added to a final concentration of 10 mM; reaction time was 5 h. Analysis by UV absorption indicates a total product yield relative to ouabain of 23% (approximately 8% for each of the three derivatives). Specific radioactivity of the product is about 40% relative to the specific radioactivity of ['H]ethylenediamine. (ii) Oxidized ['Hlouabain (2 pmol; specific activity, 6 Ci/mmol) and NPT ethylenediamine (4 pmol) were incubated in 620 pl of absolute ethanol at pH 6.0 to 7.0. After 15 min, NaCNBH.I (final concentration, 10 mM) was added and the reaction continued for 4 h. By this method, total product yield was again approximately 20%. The final specific radioactivity of oxidized ouabain was approximately 60% of that of the initial sample of ["HIouabain.
Enzyme a n d Membrane Preparations-Purified (Na',K')-ATPase from the electric organ of Electrophorus electricus was purified according to Dixon and Hokin (21). A microsomal fraction of the electric organ was prepared as described by Agnew et a f . (22). Crab axonal membranes and cardiac plasma membranes from 14-day-old chick embryonic hearts were prepared according to Balerna et a f . (23) and Paris et al. (24), respectively. Enzyme Assays-(Na',K')-ATPase activity was measured spectrophotometrically a t 340 nm with a pyruvate kinase-lactate dehydrogenase-linked system using conditions previously described by Gache et al. (25). Kinetics of interaction between ouabain or NPTouabain and (Na',K')-ATPase were followed by measuring the rate of inhibition of the enzyme activity with ATP. f3H]Ouabain and [3H]NPT-Ouabain Binding to (Na+,K+)-ATPuse-Specific activities of both compounds were 0.2 Ci/mmol. Unless indicated otherwise, purified (Na',K')-ATPase (75 pg of protein) was incubated a t 25°C for 30 min in 1.0 ml of triethanolamine buffer (50 mM, pH 7.4) containing 2 m~ ATP, 2 mM MgCL, 100 mM Na', 1 mM dithiothreitol, and various concentrations of radiolabeled ligands. (These conditions will be referred to elsewhere as standard incubation conditions.) Following incubation, the reaction mixture was passed through GSWP Millipore filters (0.22 pm) to separate [,'H]ouabain. or ["HINPT-ouabain. receptor complexes from excess ligands. Filters were rinsed three times with 5 ml of 50 mM triethanolamine (pH 7.4). Filters were then dissolved in Picofluor 30 (Packard) for radioactivity determinations. Nonspecific binding was estimated from parallel experiments in which enzyme preparations were preincubated with an excess (1 mM) of unlabeled ouabain. In all experiments, nonspecific binding represented less than 0.05%: of the total binding.
The properties of interaction of NPT-ouabain with (Na',K')-ATPase were also studied using competition experiments with [,'H]ouabain. Various concentrations of unlabeled NPT-ouabain were initially allowed to interact with the purified enzyme under the standard conditions which have been previously described. After  Polyacrylamide Gel Electrophoresis-Separation of membrane polypeptides was carried out using discontinuous sodium dodecyl sulfate gel electrophoresis (26). Gels were stained with Coomassie blue according to Fairbanks et al. (27) and then prepared for fluorography in 20% w/w 2,5-diphenyloxazole in dimethyl sulfoxide according to Laskey and Mills (28). Gel autoradiography was carried out on Kodak R P Royal X -0 mat films. Autoradiograms were recorded on a Vernon scanner densitometer.

Characterization of NPT-Ouabain Derivatives-Evidence
for the synthesis of p-nitrophenyltriazene ethylenediamine appears in Table I. All the results indicate a 1:l ratio of the two moieties in the final structure. Mass spectral analysis performed on a AEI MS 902 instrument was in agreement with the theoretical molecular weight of this product. The same type of strategy was used to estimate the molar ratio of ouabain on the one hand andp-nitrophenyltriazene ethylenediamine on the other hand in NPT-ouabain derivatives. Different methods of analysis gave a 1:1 stoichiometry of each of the two moieties in the final NPT-ouabain derivatives (Table  11).

Reversible Binding of NPT-Ouabain to Its Receptor Site-
The time course of the inhibition of activity of (Na',K')-ATPase purified from the electric organ of E. electricus is nearly the same for ouabain and NPT-ouabain I (data not shown). The inhibitory potencies of the different ouabain derivatives were compared to the inhibitory potency of ouabain under a variety of experimental conditions (Table 111). In the presence of ATP, Mg", and Na', the three NPT derivatives are nearly equipotent to ouabain itself. In the presence of Na' alone, none of the compounds has any inhibitory activity. Differences which are seen between N P T derivatives and ouabain in the presence of ATP, Mg", and K' probably reflect a lower rate of association of the NPT-ouabain derivatives with (Na',K')-ATPase, The data in Fig. 3 indicate that in the presence of ligands which favor inhibition by ouabain and NPT-ouabain deriva- Chemical methods used to identifv a n d quantify thep-nitrophenyltriazene ethylenediamine group associated with ouabain after the coupling step -___-  Direct evidence that NPT-ouabain I and ouabain interact with the same receptor site in the (Na',K')-ATPase molecule came from observations that unlabeled NPT-ouabain I displaces the ["Hlouabain bound to the enzyme (Fig. 4). The concentration of NPT-ouabain that is able to induce 50% displacement of labeled ouabain bound to its receptor is 0.65 p~. The following equation was used to calculate I C 2 , the true dissociation constant of the complex:

A/B molar ratio in NPT-ouabain
where K , , dissociation constant for the VHIouabain. receptor complex; K2, dissociation constant for the NPT-ouabain. re-  Fig. 3.

Covalent Labeling of the Receptor Sites for Cardiac Glycosides using [3H]NPT-Ouabain-NPT-ouabain derivatives
formed covalent linkages with their receptor site in the (Na+,K')-ATPase molecule when they were incubated with the enzyme for long periods of time (Fig. 5). NPT-ouabain I is the best of the three derivatives as an affinity label of the digitalis binding site. With this derivative, irreversible labeling of the enzyme reached 18% of the total possible binding after 6 h. The extent of covalent labeling obtained with NPTouabain I1 was similar to that obtained with NPT-ouabain I; however, the kinetics were slower. NPT-ouabain I11 was much less effective than the other two in covalently modifying the (Na',K')-ATPase. Repetitive additions of any of the triazene derivatives of ouabain failed to increase the extent of the covalent modification of the enzyme.
The identification of polypeptide chains which were covalently labeled by ["HINPT-ouabain I is presented in Fig. 6 using a purified preparation of (Na',K')-ATPase and a variety of other membrane preparations. Following covalent modification of the purified (Na',K')-ATPase by ["HINPT-ouabain I, radioactivity was recovered in two bands designated a and c. About 90% of the total radioactivity was incorporated in a band which can be identified as the larger polypeptide chain of the enzyme (Mr = 93,000), and 10% in band c, in a region where a small polypeptide of molecular weight less than 15,000 is stained by Coomassie blue (Fig. 6 A ) . Covalent labeling of the large chain was reduced approximately 90% by exposure of the enzyme to 1 mM unlabeled ouabain. When labeling studies were carried out with a crude membrane preparation from the electric organ, the results were essentially the same as those obtained with the purified (Na',K')-ATPase (Fig.  6 B ) .
Three polypeptide chains were labeled by ["HINPT-ouabain in purified crab axonal membrane preparation (Fig. 6C). The polypeptide corresponding to a M , = 95,000 was identified FIG. 3 (left). Specific binding of [3H]ouabain (0) and [3H]-NPT-ouabain I (0) to (Na+,K+)-ATPase. The enzyme is a purified solubilized preparation from the electric organ of E. electricus (21). Measurements were carried out a t 25°C for 30 min in 50 mM triethanolamine-HC1 buffer at pH 7.4 in the presence of 100 mM Na+, 2 mM Mg2+, 2 m ATP, 1 mM dithiothreitol, and increasing concentrations of labeled ligands. Bound ligand was separated from unbound ligand by filtration. Unspecific binding was estimated from measurements in the presence of 1 m unlabeled ouabain. FIG. 4 (center). Competition between NPT-ouabain I and [3H]ouabain for the binding to (Na',K+)-ATPase. Conditions are the same as for Fig. 3   as the large subunit of (Na',K')-ATPase (23). Labeling of this chain by ["HINPT-ouabain was specifically inhibited by an excess of unlabeled ouabain. The other two chains correspond to M , = 63,000 and t15.000. The former is acetylcholinesterase (23). the major protein in the crab membrane preparation, while the latter is probably the polypeptide observed in studies with the purified (Na+,K')-ATPase. With membrane preparations from embryonic chick hearts, only two bands in the gel were labeled; these represented polypeptides of M , = 95,000 and ~15,000. This is noteworthy, since these membrane preparations contain at least 40 different polypeptide chains. Incorporation of tritium into the larger of the two chains was completely blocked by excess ouabain, whereas that in the small chain was reduced by 40%. DISCUSSION Previously described alkylating derivatives of cardiac glycosides were in fact found to be poorly efficient affinity reagents ( 7 , 29). NPT-ouabain derivatives have been prepared which interact at the digitalis site of purified (Na',K')-ATPase with properties very similar to those of ouabain. They can be prepared in practical quantities with specific radioactivities of at least 8 Ci/mmol. Two of the three NPT-ouabain derivatives readily alkylated their receptor site, probably by a mechanism involving nucleophile-catalyzed generation of a highly reactive carbonium ion. Such a mechanism was described by Sinnott and Smith for P-D-galactopyranosyl methyl-p-nitrophenyltriazene which covalently labelsp-galactosidase (19). The extent of the covalent linkage of NPTouabain I (or 11) to the purified (Na',K')-ATPase reaches a satisfactory level of 20% relative to noncovalent binding. The incomplete covalent labeling is probably due to the fact that the reactive intermediate formed from NPT-ouabain reacts preferentially with the solvent. Sinnott and Smith (19) have previously shown that the reactive intermediate formed from their triazene derivative could react with water up to an extent of 805.
There are at least two types of subunits in the (Na+,K+) recent indications that a small proteolipid ( M , = 12,000) may also be part of the sodium pump (9,11).
In the purified (Na',K')-ATPase from electric organ of E. electricus, NPT-ouabain I is covalently linked to the M , = 93,000 chain at the digitalis receptor site. The incorporation is said to be specific since it is protected by an excess of unlabeled ouabain. There is no labeling of the glycoprotein chain. There is a labeling in the region corresponding to peptides of about M , = 12,000, but this labeling is unprotected by an excess of unlabeled ouabain.
The pattern of labeling of a crude membrane preparation of the electric organ of E. electricus by NPT-ouabain I is the same as that found with purified (Na',K')-ATPase (Fig. GB).
When a radioactive photoactivable derivative of ouabain was used under the same experimental conditions, a more complex pattern was observed with additional labeling a t bands of M , = 45,000 and M , = 50,000 (10). The purified crab axonal membrane preparation used in this work contained about 300 pmol of (Na',K')-ATPase/mg of protein (23). This (Na',K')-ATPase has a low affinity for ouabain, inhibition constant for the ouabain-ATPase complex is 100 p~ (25). Because of these two properties (i.e. low (Na',K')-ATPase content and low affinity for ouabain), the number of ouabain binding sites could not be determined by titration with [.'H]ouabain. Nevertheless, [:1H]NPT-ouabain I was an excellent label for the membrane preparation. Radioactivity was specifically incorporated into only one of the membrane polypeptides, the large chain of (Na',K')-ATPase which is also labeled by ['"PIATP (25).
The plasma membrane of chick embryo cardiac cells also contains a (Na',K')-ATPase with a low affinity for ouabain (IC,, = 44 p~, Ref. 31). This membrane preparation, which contains a large number of polypeptide chains, was labeled by ["HINPT-ouabain I on only two chains. However, only the larger of these ( M , = 95,000) is completely protected against covalent labeling by ["HINPT-ouabain I in the presence of an exces ouabain. Previous labeling of cardiac membranes with photoaffinity derivatives of ouabain or strophanthidin gave much more complex patterns.
Covslent labeling of polypeptides in the low molecular