Proc. Nati. Acad. Sci. USAVol. 76, No. 5, pp. 2263-2267, May 1979Biochemistry

Antibodies raised against purified fl-adrenergic receptorsspecifically bind f3-adrenergic ligands

(agarose-alprenolol/affinity chromatography/catecholamines/immunogen/model receptor binding site)

MARC G. CARON*t4, YOGAMBAL SRINIVASANt§, RALPH SNYDERMANt11 , AND ROBERT J. LEFKOWITZtt§§Howard Hughes Medical Institute Laboratory and Departments of tMedicine, tBiochemistry, IMicrobiology, and tIlmmunology, Duke University MedicalCenter, Durham, North Carolina 27710

Communicated by James B. Wyngaarden, March 8, 1979

ABSTRACT Antibodies raised against purified jl-adrenergicreceptors themselves specifically bind 1&-adrenergic ligands.Digitonin-solubilized frog (Rana pipiens) erythrocyte #-adren-ergic receptors, purified 100- to 200-fold by adsorption to analprenolol-agarose affinity support and specifically eluted fromthe affinity resin by 1-100 mM (±)isoproterenol, were used toimmunize six rabbits. All immune sera, in contrast to preim-mune sera, bound the P-adrenergic antagonist [3H]dihydroal-prenolol with high affinity (Kd = 1 nM). [3H]Dihydroalprenololbinding activity was due to immunoglobulins. By competitionstudies, antibody [3H]dihydroalprenolol binding was found todisplay a specificity and stereoselectivity resembling that of theB-adrenergic receptor, i.e., (-)isoproterenol > (-)epinephrine> (-)norepinephrine; alprenolol propranolol >> phentolamine= aloperidol; and (-) isomers of both agonists and antagonists10-100 times more potent than (+) isomers]. A portion of the13H]dihydroalprenolol binding antibodies could be specificallyadsorbed onto purified frog erythrocyte membranes, whereasXenopus and human erythrocyte membranes, both of which arealmost devoid of f3-adrenergic receptors, were ineffective inadsorbing [3H]dihydroalprenolol binding antibodies. We suggestthat the likely immunogen was a fl-adrenergic receptor-iso-proterenol complex and that immunization with drugs nonco-valently bound to their receptors might be a means of raisingantibodies to biologically active otherwise nonimmunogenicsmall molecules. Such antibodies, whose specificity mimics thatof a receptor, should also provide useful models for the studyof the structure of the receptor binding sites.

The adenylate cyclase-coupled f3-adrenergic receptors of frogerythrocyte plasma membranes have been extensively char-acterized (1). In addition to being identified initially by directligand-binding techniques using the f3-adrenergic antagonist[3H]dihydroalprenolol ([3H]DHA) (2), these receptors have beensolubilized with detergents (3) and more recently purified byaffinity chromatography (4). In order to further characterizethis receptor it was of interest to raise an antibody to the par-tially purified receptors. Antibodies against hormone receptorshave previously been raised (5-7) or found in the circulationof patients with specific diseases (8-10). Circulating antibodiesagainst thyrotropin receptors, insulin receptors, and the cho-linergic receptors have been implicated in the mechanisms ofsuch diseases as hyperthyroidism, the syndrome of insulin re-sistance with acanthosis nigricans, and myasthenia gravis. Inthe course of immunizing rabbits with affinity chromatogra-phy-purified preparations of the frog erythrocyte 3-adrenergicreceptors, we obtained antibodies that unexpectedly were foundto bind adrenergic agonists and antagonists with a specificityresembling that of the f3-adrenergic receptor. Moreover, aportion of the antibodies capable of binding f3-adrenergic li-gands could be adsorbed specifically to frog erythrocytemembranes. The properties of these unique antibodies and their

implications for studies of hormone and drug receptor structureand function are described in this communication.

MATERIALS AND METHODSMaterials. (±)-Alprenolol was a gift from Hassle Pharma-

ceutical (Molndal, Sweden). Freund's adjuvant was from Difco.[3H]DHA was from New England Nuclear. All other drugs andchemicals were from sources described before (2-4) and wereof the highest grade available.

Preparation of the Immunogen. Soluble f3-adrenergic re-ceptor preparations from frog (Rana pipiens) erythrocytemembranes were obtained by digitonin treatment and werepurified 100- to 200-fold in a single step on a Sepharose 6B-alprenolol affinity gel as described (4). Briefly, 40-50 ml ofsoluble receptor preparation was chromatographed on 5-8 mlof Sepharose 6B-alprenolol gel. About 80-90% of the receptoractivity was routinely bound, whereas as low as 3-5% of totalprotein was retained by the column. After the column waswashed, receptor activity was eluted specifically by additionof 1-100 mM (+)-isoproterenol to the equilibration buffer (0.2%digitonin/50 ,uM dithiothreitol/100 mM NaCl/10 mM Tris-HCI, pH 7.4). The eluate was then concentrated to 6-7 ml bylyophilizing and then chromatographing on Sephadex G-50.This chromatography removed most of the free isoproterenolso that it was possible to assay [3H]DHA binding without in-terference by using saturating concentrations of the radioligand.In separate experiments in which [3H]isoproterenol was usedwe documented that after Sephadex G-50 chromatography ofaffinity column eluates the residual isoproterenol concentrationwas about 0.1 uM or lower. Soluble receptor preparations ob-tained in this way were routinely purified 100- to 200-fold.

Immunization. Routinely, 5-10 pmol of receptor in 7 ml of0.2% digitonin/100 mM NaCl/10 mM Tris-HCl, pH 7.4, wasemulsified with 7 ml of complete Freund's adjuvant. Onemilliliter was injected intradermally in 10-12 sites on the backand 1 ml intramuscularly in the subscapular muscle region toeach of six New Zealand White rabbits. Injections were repeatedat intervals of 2-3 weeks.Immune Sera and Immunoglobulin Fractions. Twelve days

after the fourth booster injection and periodically thereafteranimals were bled from the ear artery. Blood was allowed tocoagulate for 2 hr at room temperature and serum was removedand stored in aliquots at -20°C. An immunoglobulin-richfraction was prepared from some sera by precipitation withammonium sulfate to 50% saturation. After resuspension of theprecipitate in 0.01 M potassium phosphate at pH 8.0, dialysis,and centrifugation, the material was stored frozen. The con-

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2263

Abbreviation: DHA, dihydroalprenolol.* To whom correspondence and reprint requests should be addressed

at: Box 3287, Duke University Medical Center, Durham, NC27710.

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centration of immunoglobulin was measured by absorbanceat 280 nm with bovine gamma globulin as the standard.

Detection of Antibody Activity. The ability of the immunesera to interact with adrenergic drugs was assessed by directligand binding, using the 3-adrenergic antagonist [3H]DHA.As with particulate f-adrenergic receptor preparations, it wasfound that [3H]DHA bound to immune sera could be separatedfrom free ligand by rapid vacuum filtration on glass fiber filters.We further documented that [3H]DHA binding to immune seracould also be assessed by filtration on Millipore filters, bySephadex G-50 chromatography (3), or by ammonium sulfateprecipitation. These methods appeared to be qualitativelyequivalent, and the glass fiber filtration assay was routinely usedfor the results reported here. [3H]DHA specifically bound wasassessed (2, 3) by defining the nonspecific binding as that whichwas not competed for by 10 ,tM unlabeled (±)-alprenolol or 100,M unlabeled (-)-isoproterenol. Most assays were performedwith whole antiserum at a final dilution of 1:500 unless other-wise stated. Under these conditions specific [3H]DHA bindingrepresented 95-98% of total binding.

RESULTSCharacteristics of binding of ,B-adrenergic agents toantiseraInitially, immune sera were tested for their ability to interactwith the 0-adrenergic receptor by examining their effects onthe binding of [3H]DHA to purified or solubilized frog eryth-rocyte membranes by using the rapid filtration or the SephadexG-50 chromatography technique. It was then realized thatimmune sera themselves were capable of binding the ligand[3H]DHA. Of the six animals immunized, all developed de-tectable [3H]DHA binding activity in their serum. The generalbinding properties of these six antisera were similar, thoughdifferences in detailed binding specificity were found. Thebinding data presented below were obtained with the antiserumfrom one animal (no. 349) unless otherwise noted.[3H]DHA binding to these immune serum sites was rapid and

rapidly reversible (Fig. 1). At 23°C the rates of association anddissociation were too fast to be determined with any accuracy;that is, within 1 min the whole process was complete. However,at -4oC the rate constant of association ki was found to be 9.5X 107 min-1 M-1, whereas the rate constant of dissociation k2

150

X ~ ~~* No addition&7120 0

E4- x0

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rS60 | 10 ,gM (±)-alprenololI

3030

(o , L..L.., .., I0 2 4 1 0 30 35 40 45

Time, min

FIG. 1. Association of [3H]DHA with and dissociation of[;HJDHA from antiserum 349 as a function of time at 0C. Dissocia-tion of bound [3H]DHA was assessed after the addition of 10 ,1M(±)-alprenolol to incubation mixtures at equilibrium. The data areshown as fmol of specific binding per 50 jul of 1:500 dilution of theantiserum. The association and dissociation rate constants k1 and k2were calculated as described (4). Results shown are representativeof two or three experiments.

was 0.054 min-1. The ratio k2/kl provides an estimate of thevalue of the dissociation constant (Kd) of these sites for[3H]DHA, 0.6 nM.

Scatchard analysis of equilibrium binding data for [3H]DHAto these sites at 230C indicated a saturable binding process andyielded a monophasic curve (Fig. 2). From these data a valueof Kd for [3H]DHA binding of 2-4 nM was obtained. The Kdvalue obtained by kinetic analysis was in close agreement withthe value of 0.7 nM obtained for (-)-alprenolol by bindingcompetition (Table 1). The Kd value derived by equilibriumbinding (2-4 nM) was determined on an earlier bleed of theimmune serum and correlated well with the potency of (-)-alprenolol binding in that immune serum.The specificity of binding of [3H]DHA to these sites was

examined by measuring the ability of agonists and antagoniststo compete for [3H]DHA binding. As shown in Fig. 3, f-adrenergic agonists were potent competitors of binding, witha potency order (-)-isoproterenol > (-)-epinephrine > (-)-norepinephrine that is identical to the order of potency forcompetition of binding to the 3-adrenergic receptor. Bindingalso displayed the stereoselectivity that characterizes beta-adrenergic receptor interactions, because (-)-isorpoterenolcompeted 10-fold more effectively than (+)-isoproterenol.,B-Adrenergic antagonists such as (-)-alprenolol and (-)-pro-pranolol (not shown) were also very potent competitors and the(-) isomers of these drugs were also preferentially recognizedby these binding sites over their (+) isomers (Fig. 3, Table 1).The detailed specificity of the interaction of several adrenergicagents with immune serum 349 as assessed by competition for[3H]DHA binding is shown in Table 1. Table 1 also comparesthe Kd values obtained for the antiserum binding sites withthose previously obtained for the interaction of these drugs withthe membrane-bound 0-adrenergic receptors of frog erythro-cytes (12). It can be seen that striking similarities exist betweenthe pharmacological properties of both binding sites. Dopa-mine, the dopaminergic antagonist haloperidol, and the a-

0.12

0.10 0

0

' 0.08 \

Kd = 3.7 nMo r = 0.9630D 0.06

I0

_ 0.04

0.02

0 0.1 0.2 0.3 0.4 0.5(3HIDHA bound, nM

FIG. 2. Scatchard plot of [3HJDHA binding to antiserum 349.Antiserum at 1:500 final dilution was incubated with increasingconcentrations of [3H]DHA (0.3-50 nM) for 30 min at 23°C. Non-specific binding, which ranged from 5% to 15% of total binding, wasdetermined in parallel incubations in the presence of 10 IM(i)-alprenolol. Binding was assessed by filtration. Results are represen-tative of two experiments.

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Trable 1. Dissociation constants (Kd) of various adrenergic agentsobtained by binding competition of [3HJDHA

Kd,,UMFrog erythrocyte

Antiserum membraneCompounds 349 3-adrenergic receptors

Antagonists(-)-Alprenolol 0.0007 0.0034(+)-Alprenolol 0.084 0.150(-)-Propranolol 0.001 0.0046(+)-Propranolol 0.031 0.286(W)-Dichloriso- 0.002 0.57

proterenol(W)-Practolol 0.007 20.9(W)-Phentolamine -

Agonists(-)-Isoproterenol 0.04 0.40(+)-Isoproterenol 0.4 183(-)-Epinephrine 9.3 4.6(-)-Norepinephrine 280 49(-)-Soterenol 0.006 0.7(±)-Hydroxybenzyl 7.7 0.06

isoproterenolDopamineCarbachol

[3H]DHA (8-10 nM) binding assessed by the filtration techniquewas determined on 50-yl aliquots of 1:100-1:500 diluted antiserum349 in the presence and absence of five to seven concentrations of eachof the drugs shown. Kd values for each drug were calculated from theconcentration of the drug that competed for 50% of specific [3H]DHAbinding according to the method of Cheng and Prusoff (11), using adissociation constant of 3.7 nM obtained by Scatchard analysis for[3HIDHA binding to the antiserum. -, No effect of the drug at con-centrations of 100 ,M or effects too slight to permit calculation of aKd value. Results shown are from one to three experiments performedin duplicate for each drug. Results on the specificity of [3H]DHAbinding to erythrocyte membranes were taken from previously pub-lished work (12).

adrenergic antagonist phentolamine did not interact with theantibody sites at concentrations as high as 10-100 ,M; this issimilar to their lack of effect on binding to the fl-adrenergic

Dopamine, Carbachol

+1001±)Dp

0

80-

C

v6 60IC (-)-AL P (-)-IS t , -S )-N

O0 (-_)IEI20-

0.

0 -9 -8 -7 -6 -5 -4 -3

log ladrenergic agent] (M)

FIG. 3. Ability of agonists, antagonists, and other compounds tocompete for [3HJDHA binding to antiserum 349. Antiserum 349 (50,ul of a 1:100 dilution) was incubated with 8 nM [3H]DHA (final vol-ume 500 1l) in the presence and absence of various concentrations ofthe different drugs shown. The results, expressed as % of total binding(100% control binding was 0.52-0.57 pmol) are from an experimentin duplicate and are representative of three such experiments. ALP,alprenolol; ISO, isoproterenol; EPI, epinephrine; NE, norepinephrine;DHMA, dihydroxymandelic acid.

receptor. The catechol analogs dihydroxphenylalanine anddihydroxymandelic acid were also without effect at 100 AM.

Despite these similarities to the specificity of the 13-receptor,detailed studies revealed a number of striking differences. Thusthe 3-antagonists (+)-dichlorisoproterenol and (±)-practololwere, respectively, 300- and 3000-fold more potent in com-peting for the antibody than for the receptor binding sites, andthe agonist (+)-hydroxybenzylisoproterenol was only 1/100thas potent at the antibody sites. Moreover, whereas all antisera[3H]DHA binding activities clearly possessed a fl-adrenergicspecificity, each differed from the other on detailed examina-tion (data not shown).

Immunoglobulin nature of the [3H]DHA binding sitesin immune seraIn order to document that [3H]DHA binding activity in theimmune sera was in fact due to the presence of immunoglob-ulin, the following tests were performed. First, [3H]DHAbinding activity in the immune sera was quantitatively pre-cipitable by 50% saturation with ammonium sulfate. Second,[3HJDHA binding activity was stable to heating at 560C for 30min. Third, [3H]DHA binding activity could be fully precipi-tated by goat antiserum to rabbit IgG (Fig. 4). Fourth, the[3H]DHA binding activity was stable to a reduction of the pHto 3.5 for 10 min. Finally, the binding activity was found tocochromatograph with authentic IgG on Sephadex G-200.None of six preimmune sera contained any [3H]DHA binding

activity. Further, several hyperimmune sera obtained in re-sponse to other antigens failed to show any binding activity.Similarly, sera from rabbits immunized with isoproterenol-containing digitonin solutions as well as isoproterenol-con-taining soluble preparations depleted of f3-adrenergic receptorsby affinity chromatography failed to show any [3H]DHAbinding activity. These results indicate that the [3H]DHAbinding activity observed in these immune sera is due to animmunoglobulin fraction raised in response to the partiallypurified ,B-adrenergic receptor preparations used as the im-munogen.

100C

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. 0

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I

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FIG. 4. Immunoprecipitation of [3HJDHA binding activity inimmune serum 349 by goat antiserum to rabbit IgG. A 1:10 dilutionof the immune serum was incubated in 25mM Tris-HCl/2mM MgCl2,pH 7.4, at 25°C for 24 hr with various concentrations of the anti-rabbitIgG. The precipitate was removed by centrifugation and the super-natant was assayed for [3H]DHA binding (12 nM). Results shown arefrom an experiment performed in duplicate.

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Frog Xenopus Human

FI(.. 5. Interaction of antiserum 349 with various erythrocytemembrane preparations. Frog, Xenopus, and human erythrocytemembranes were all prepared as described (3,. 4) for the frog eryth-rocyte membranes. Membranes (1.1-1.4 mg of protein per ml) wereincubated at 250C for 11/2 hr in 25 mM Tris-HCl/5 mM MgCI2, pH 7.4,with a 1:50 final dilution of whole antiserum 349 (hatched bars) or

normal rabbit serum (empty bars) preheated at 560C for 30 min toinactivate complement. Membranes were washed twice with 5-mlsamples of the incubation buffer and [3H]DHA binding was measuredon the resuspended membranes by filtration on GF/C glass fiber fil-ters. Membrane preparations incubated with normal rabbit serumshowed the same [3H]DHA binding activity as untreated preparations.Results ± SEM shown are the means of 3 different experiments per-formed in duplicate. These experiments are representative of 8-10similar experiments.

Interaction of the antibodies with erythrocytemembranesThe evidence described thus far indicates that the antibodiesraised in response to partially purified preparations of frogerythrocyte f3-adrenergic receptor possess specificity towardcatecholamines and related f-adrenergic drugs. Studies werenext performed to elucidate whether these immune sera alsopossessed affinity for components of erythrocyte membranes.As shown in Fig. 5, a portion of the [3H]DHA binding activitypresent in immune sera was specifically adsorbed by frogerythrocyte membranes, whereas Xenopus erythrocytemembranes as well as human erythrocyte membranes, both ofwhich are essentially devoid of f3-adrenergic receptors, were

almost ineffective in adsorbing [3H]DHA binding activity fromthe antisera.

DISCUSSIONIn this communication we report that antibodies raised inrabbits against partially purified preparations of the frogerythrocyte f-adrenergic receptors appear to recognize fi-

adrenergic catecholamines as well as components of theerythrocyte membrane. As evidenced by the results obtainedwith the specific f-adrenergic radioligand [3H]DHA, it is clearthat these antisera binding sites display specificity that bearsstriking similarities to that of the physiological f3-adrenergicreceptors. In addition, the data indicate that the [3H]DHAbinding activity in these sera is due to immunoglobulins, be-cause binding was stable to exposure to 560C for 30 min andto acidic conditions (pH 3.5), was precipitated by 50% saturatedammonium sulfate, and was quantitatively immunoprecipi-tated by goat antiserum to rabbit IgG. None of these propertiesis shared by either membrane-bound or solubilized f-adren-ergic receptors.

Proc. Natl. Acad. Sci. USA 76 (1979)

The properties of the antibodies described in this commu-nication appear to be unique. Although antibodies have pre-viously been raised to several hormone receptors, such as thosefor insulin (7), prolactin (6), and acetylcholine (5), in none ofthese cases did the antibodies themselves mimic the bindingspecificity of the receptor. In one case an antibody that mim-icked the effects of insulin was obtained (13), but this antibodywas raised against an anti-insulin antibody. Attempts to raiseantibodies to catecholamines (14, 15) or to adrenergic antago-nists (16, 17) have to date met with very limited success. Thisis not surprising in view of the chemical nature of these agents,which are small molecules with molecular weights of only about300. Thus, in the few cases in which an antibody has beensuccessfully raised against an adrenergic drug or antagonist thishas required covalent linkage of the agent to a protein carrierin order to form the immunogen. In addition, the antibodiesobtained have possessed very restricted specificities. Antibodiesraised to catecholamine analogues have had very low affinitiesfor the native catecholamines (14, 15). In no case have any ofthese antibodies displayed a specificity even vaguely reminis-cent of an adrenergic receptor.

What, then, is the explanation for the unique antibodiesobtained in the present studies? Several explanations will beconsidered, though a firm conclusion may not be possible at thistime. One possibility is that two separate immunogens havebeen involved, the receptors and free isoproterenol in the re-ceptor preparations, not removed by the Sephadex G-50chromatographic step used prior to injection of partially puri-fied receptors. This seems unlikely on several grounds. First,as noted above, the free catecholamine would certainly not beexpected to be antigenic by itself. Second, if two totally distinctimmunogenic stimuli were involved, the antibodies that canbe specifically adsorbed on the erythrocyte membrane and thatare presumably directed against a component of the membranewould not be expected to bind [3HJDHA too; the data in Fig.5 did demonstrate such binding.

Another possibility is that the antibodies studied have beenraised in response to partially purified receptor alone. This alsoseems somewhat unlikely. There would be no reason to antic-ipate that such an antibody, even if directed at the activebinding site of the receptor, would itself display a bindingspecificity that so closely mimics that of the receptor. In fact,a somewhat different or complementary specificity might bepredicted.A third possiblity is that antibodies formed against the re-

ceptor have in turn served as antigens leading to the formationof "anti-idiotypic" antibodies, the binding specificity of whichnow resembles that of the receptor. In this case too, however,the antibodies that can be adsorbed by the erythrocyte mem-branes would not be expected to be the same as those that canbind DHA.An additional possibility is that the immune sera contain

soluble anti-receptor antibody-receptor complexes and thatbinding found in such sera is due to the presence of receptors.This is unlikely because, in contrast to the antibody [3H]DHAbinding sites, soluble fl-adrenergic receptor binding activityis completely destroyed by heating to 560C for 30 min, or bydecreasing the pH to 3.5. At low pH, putative antibody-re-ceptor complexes would be dissociated and the receptors theninactivated. Upon chromatography of antiserum 349 onSephadex G-200, the [3H]DHA binding activity was found tocochromatograph with authentic rabbit IgG. The presence ofantibody-receptor complexes would have produced a peak ofbinding activity of larger apparently molecular size. In addition,the specificity of the antiserum is readily distinguishable fromthat of the receptors as noted above.

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Proc. Natl. Acad. Sci. USA 76 (1979) 2267

Perhaps the most attractive hypothesis is that the antibodiesstudied have been formed in response to a complex of isopro-terenol bound to the purified receptors. Viewed in this light,the receptor-bound isoproterenol might be immunologicallyequivalent to a hapten conjugated to a carrier and hence beantigenic (18). That some of the antibodies to the hapten (iso-proterenol) also recognize determinants on the carrier (receptor)would thus not be surprising. That the specificity for bindingcatecholamines and related drugs so closely resembles that ofthe 3-adrenergic receptor in terms of rank order of potencies,stereospecificity, etc. might reflect the particular conformationin which isoproterenol was held by the receptors.

Regardless of the exact mechanism leading to the generationof these unusual antibodies, their further characterizationshould provide important new insights into f3-adrenergic re-ceptor structure and function. Among the potentially interestingavenues which could be explored are: (i) The possible biologicalactivity of the antibodies. (ii) The use of the antibodies as a toolfor purification of the receptors. (iii) The use of the antibodiesto probe the structure of the specific adrenergic drug bindingsite. Because the antibodies are available in much largerquantities than the P-receptors and bind drugs with specificitiesand affinities similar to those of the receptor, purification andcharacterization of the antibody binding sites might help shedlight on the nature of the structural features that contribute tothe specificity of the receptor. (iv) Development of anti-cate-cholamine antibodies for radioimmunoassay. Another pointraised by these studies that deserves mention is that immuni-zation with drugs noncovalently bound to their receptor maybe a means of raising antibodies to otherwise nonimmunogenicbiologically active small molecules.

The expert technical assistance of Miss Laurie Card is greatly ap-preciated. This work was supported by U.S. Public Health Service.Grants HL16037 and HL20339 and a grant-in-aid from the AmericanHeart Association.

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5. Patrick, J. & Lindstrom, J. (1973) Science 180, 871-872.6. Shiu, R. P. C. & Friesen, H. G. (1976) Science 192,259-261.7. Jacobs, S., Chang, K.-J. & Cuatrecasas, P. (1978) Science 200,

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