Proc. Nati. Acad. Sci. USAVol. 77, No. 8, pp. 4657-4661, August 1980Biochemistry

Juvenile hormone-binding protein from the cytosol ofDrosophila Kc cells

(insect hormones/insect cells/cytosolic receptor)

ERNEST S. CHANG*, THOMAS A. COUDRONt, MARILYN J. BRUCE*, BECKY A. SAGEt, JOHN D. O'CONNORt,AND JOHN H. LAWt*Department of Animal Science, University of California, Davis, and Bodega Marine Laboratory, P.O. Box 247, Bodega Bay, California 94923; tDepartmentof Biochemistry, The University of Chicago, 920 East 58th Street, Chicago, Illinois 60637; and *Department of Biology, University of California, Los Angeles,California 90024

Communicated by Herbert E. Carter, May 19, 1980

ABSTRACT Insect cells of an established line, DrosophilaKc cells, take up and metabolize juvenile hormone OH). Thecytoplasm of these cells contains a protein that binds JH withspecificity, saturability, and high affinity (Kd = 1.56 X 10-8 M).The kinetics for the specific binding and dissociation of JH Iwere independently measured, and the rate constants werefound to be ka = 1.3 X 106 M-' min-1, kd = 1.3 X 10-2 min-1,respectively. All three juvenile hormones bind to the proteinwith comparable affinities; the corresponding acid or diol me-tabolites of JH I are not bound. About 2500 hormone-bindingprotein molecules are present per cell. The protein has a mo-lecular weight of 80,000 as estimated by gel permeation chro-matography and by sucrose gradient sedimentation. The prop-erties of this protein suggest that it functions as a cytoplasmicreceptor for juvenile hormone.

Insect hormones thus far identified include peptides, steroids,and the unique sesquiterpene derivatives (1) known as juvenilehormones (H). We now have available some information aboutthe action of both peptide and steroid hormones at target tissues(1, 2). Recently, two papers have described the identificationof a receptor protein for ecdysteroids, the steroidal moltinghormones, from Drosophila melanogaster imaginal discs (3)and Kc cells (4).

Virtually nothing is presently known, however, about thedetails of action of the JH at their target cells. Insect JH is pro-duced in the corpora allata and transported through the he-molymph, often associated with specific carrier proteins, totarget tissues where the hormones exert a physiological effect(1). Schmialek and his colleagues (5, 6) attempted to identifyJH receptors in epidermal tissue of the mealworm, Tenebriomolitor, by incubating tissue with a labeled hormone analog,followed by disrupting membrane structures with the detergentTriton X-100. Unfortunately this procedure is complicated bythe fact that JH and the JH metabolites form mixed micelleswith Triton X-100 that behave as macromolecular complexes[see Akamatsu et al. (7) for discussion]. More recently, Riddifordand Mitsui (8) have provided evidence for a high-affinity (Kd= 2 X 10-8 M) JH receptor in nuclei from epidermal tissue ofManduca sexta. This presumably is analogous to the nuclearreceptor for steroid hormones in mammalian tissue (9).-The Drosophila melanogaster Kc cells seemed to be an at-tractive system in which to characterize a JH receptor, basedupon the successful use of this established insect cell line todemonstrate the presence of a cellular ecdysteroid receptor (4).In this paper we report the existence of a macromolecule in thecytoplasm of the Drosophila cells that binds JH and has the

properties expected for a receptor protein-i.e., specificity,saturability, and high affinity.

MATERLALS AND METHODSThe Kc cell line of Drosophila melanogaster, established byEchalier and Ohanessian (10), was maintained as a suspensionculture in 3-liter spinner flasks (Bellco Glass) in D-20 medium(11). The organic medium ingredients were from Sigma withthe exception of Yeastolate, which was from Difco; the inor-ganic ingredients were from Mallinckrodt. The cultures weremaintained at 250C at a stirring speed of 60 rpm. Humanforeskin fibroblast cells (Jackson 60) were grown in monolayersand were obtained from Glyn Dawson (The University ofChicago).The radiolabeled JH I, [10-3H]methyl (2E,6E,1OZ)-10,11-

epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate (racemic),was obtained from New England Nuclear (specific activity, 13.5Ci/mmol; 1 Ci = 3.7 X 1010becquerels). The tritiated acid anddiol of JH I were prepared according to the methods of Sladeand Zibitt (12). The products were located and purified bythin-layer chromatography (TLC). Unlabeled JH I, juvenilehormone II [JH II; methyl (2E,6E,10Z)-10,11-epoxy-3,7,11-trimethyl-2,6-tridecadienoate], and juvenile hormone III [JHIII; methyl (2E,6E)-10,11-epoxy-3,7,1 1-trimethyl-2,6-dode-cadienoate] (all racemic) were from Calbiochem. PonasteroneA (23,3#,14a,20R,22R-pentahydroxy-513-cholest-7-en-6-one)was a gift from D. H. S. Horn (Commonwealth Scientific andIndustrial Research Organization, Australia). Molecular weightstandards were obtained from Pierce and Schwarz/Mann.

Cells were harvested (density approximately 3 X 106 cells perml) by centrifugation at 800 X g for 8 min. The cells wereresuspended in 10 mM Tris/5 mM MgCl2, pH 6.9, at 4°C (4)to a final volume four times that of the cells and then repelleted.The cells were disrupted with 20 strokes of a Dounce homog-enizer. The homogenate was then centrifuged at high-speed(122,000 X g, 1.5 hr) to obtain supernatant fraction designatedcell cytosol. When stated, dilutions of the cell cytosol were madewith a standard buffer, 10 mM Tris/5 mM MgCl2/150 mMKC1, pH 7.4, at 220C (4) and 0.15 mM diisopropyl phospho-rofluoridate (DFP) from Aldrich.When stated, bound hormone was separated from free (un-

bound) hormone by the use of dextran-coated charcoal (DCC;13), prepared according to Kramer et al. (14), with a finalconcentration of 0.16% (wt/vol) in standard buffer. For theDCC assay, the incubation was carried out in tubes coated with

Abbreviations: JH, juvenile hormone; DCC, dextran-coated charcoal;DFP, diisopropyl phosphorofluoridate; TLC, thin-layer chromatog-raphy.

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a 1% solution of polyethylene glycol 20,000 (Fisher). Specifiedamounts of [3H]JH I and unlabeled JH I were added from stocksolutions in standard buffer, followed by the addition of cytosol.The incubation mixtures were held at 230C for 15 min withfrequent gentle mixing. To terminate the incubation, the tubeswere placed on ice, 50,Ml of an ice-cold 0.3% solution of DCCwas added, and the tubes were held for 15 min at 00C. Thesolutions were then centrifuged at 14,000 X g for 15 min topellet the charcoal. The amount of DCC used was sufficient toremove all of the JH from the incubation mixture. Aliquots ofthe supernatant were removed for scintillation spectrometry.The rate of association (ka) was determined by incubating

tubes containing 10 nM [3H]IJH I in 50 ,l of cell cytosol con-taining DFP with and without an excess of unlabeled JH L. Aftervarious incubation times, bound hormone was separated fromfree hormone by the DCC assay. The rate of dissociation (kd)was determined by first incubating cell cytosol containing DFPwith 10 nM of [3H]JH I for 3 hr. Excess unlabeled JH I was thenadded to the incubation mixture. After various time periods,aliquots were removed and bound hormone was separated fromfree hormone by the DCC assay.

Equilibrium binding data were obtained by incubating cy-tosol (final concentration of 12.5%) with the standard bufferthat contained DFP and various (2-20 nM) concentrations of[3H]JH I for 3 hr at 230C. After incubation, the bound hormonewas separated from free hormone by using the DCC assay.Column chromatography was performed with Sephadex

G-25 (Pharmacia; 1.5- X 20-cm bed) and Sephadex G-100 su-perfine (2.0- X 65-cm bed) with the standard buffer as the el-uant. When stated, the effluent fractions were monitored at 280nm by using a Gilford model 2000 spectrophotometer.

Velocity sedimentation centrifugation was conductedthrough a preformed continuous sucrose gradient in the stan-dard buffer. After centrifugation fractions were collected, theywere analyzed by scintillation spectrometry.The identity of the bound hormone was confirmed by the

parallel incubations of 500 ,l of cytosol with [3H]IJH I (ap-proximately 105 dpm) for 15 min at 230C. The tubes were thenchilled on ice, and DCC was added to one of the tubes. After15 min the tubes were centrifuged, and ethanol was added tothe supernatant fractions (final concentration of 75% vol/vol).The precipitated protein was pelleted, and the supernatantfractions, to which 20 ,g of unlabeled juvenile hormone wasadded, were extracted twice with hexane (1:1, vol:vol). Thecombined hexane extracts were reduced in volume under anitrogen stream, and the residues were analyzed by thin-layerchromatography. The radioactive fractions from the G-100column were also analyzed to determine the identity of thehormone. Aliquots were removed, 20,ug of JH I was added, andthe solutions were extracted three times with an equal amountof chloroform:methanol (2:1, vol:vol). The organic phases werecombined, the combined volume was reduced under a nitrogenstream, and the residue was analyzed by thin-layer chroma-tography.

Metabolism of the hormone by intact cells was determinedby incubating approximately 109 cells with 10 nM [3H]JH I at23°C for 20 min. The cells were separated from the labeledmedium by centrifugation, and then the pellet and supernatantfraction were separately extracted with 75% (vol/vol) ethanolafter the addition of unlabeled JH I (20 ,g) to each fraction.After centrifugation, the supernatant phases were reduced involume under a nitrogen stream, and the residues were ana-lyzed by thin-layer chromatography.

Analytical thin-layer chromatography was performed onsilica gel G plates [Analtech, Newark, DE]. The plates weredeveloped in ethyl acetate:hexane (3:7, vol:vol). Radiolabeled

JH I, JH I acid, and JH I diol were used as standards and hadRF values of 0.75, 0.50, and 0.25, respectively, in this proce-dure.

All scintillation counting was performed in a toluene-basedfluor and a Nuclear Chicago Isocap (300) or a Beckman (LS-lOOC) spectrometer. Protein concentrations were determinedby the biuret method (15).

RESULTSPreliminary experiments showed that radiolabeled hormoneadded to the cell medium at 10 nM was taken up quickly by Kccells; no intact hormone remained in the medium after 35 min,but acid and acid diol metabolites (12) appeared. The amountof radioactive compounds associated with the cells after 35 minwas at least six times that remaining in the medium; of the totalradioactive compounds in the cells, 38% was intact hormoneand the rest represented metabolites. These results indicatedthat the cells were able to concentrate the hormone from themedium near physiological hormone levels for most animals.Presumably this process could require binding to a macro-molecule. Furthermore, cellular enzymes were capable ofmetabolizing the hormone, and the metabolites were returnedto the medium, because no metabolism of the hormone wasobserved in fresh or spent medium lacking cells.JH I binding was subsequently examined in the supernatant

fraction of Kc-cell homogenates obtained at high speed. Theelution profile (Fig. 1) shows the results of gel permeationchromatography on Sephadex G-25 of cytosol preincubatedwith [3H]JH I. These data indicate that radiolabeled hormonedid associate with a macromolecule that eluted in the voidvolume (fractions 15-18) and that the binding of labeled JH Icould be prevented by competition with a 2000-fold excess ofunlabeled hormone. The bound radioactive compound in thecytosol, after the removal of free hormone using dextran-coatedcharcoal, was shown to be intact juvenile hormone by thin-layerchromatography. Most of the unbound hormone in the incu-bation mixture was authentic JH I as well, although smallamounts of acid, diol, and acid diol were present.

Gel permeation chromatography of cytosol that had beenincubated with [3H]JH I was also conducted with SephadexG-100 to separate the macromolecules more efficiently (Fig.

3.0-

'-4

x 2.0-

1.0 i~

20 40 60Fraction

FIG. 1. Sephadex G-25 (fine, 2 cm X 1.2-cm inside diameter)chromatography of Kc-cell cytosol (150 ul) incubated with 10 nM[3HJJH I in the presence (+- - -+) or absence (0-0) of 20 ,ug ofunlabeled JH L. The column was eluted at 40C with the standardbuffer in Materials and Methods. Ten-drop fractions were col-lected.

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2). Thin-layer chromatographic analysis of the material in thefirst radiolabeled peak indicated the presence of only intact JHI, whereas similar analysis indicated that the second peakcontained predominantly the acid diol and a small amount ofthe acid metabolite. This second peak represents the inclusionvolume of the column and contains only small molecules. Thematerial in the first peak was estimated to have a molecularweight of 85,000 from a linear regression analysis of the elutionprofile of standard proteins. Essentially similar results wereobtained when unlabeled cytosol was first separated by chro-matography on Sephadex G-100 and then assayed for hormonebinding by the DCC method. Highly variable results wereobtained unless the cytosol was first treated with DFP. It is notclear whether DFP acts by inhibiting proteolysis or hormone-degrading enzymes.

Similar results were obtained by the use of velocity sedi-mentation centrifugation (Fig. 3), in which the bound ligandentered the sucrose gradient and moved to a position close tothat of the bovine serum albumin standard (Mr 67,000). Un-bound hormone remained at the top of the gradient. Again itwas demonstrated that binding of labeled JH I could be pre-vented by competition with excess unlabeled hormone.The affinity of the cytosolic binding protein for JH I was

determined by both kinetic and equilibrium methods. It wasfirst necessary to determine the length of time required for thehormone to associate with the binding site. A second order re-action is evident from the plot of time versus amount bound(Fig. 4). The equation for the ligand-protein binding reac-tion,

kahormone (H) + binding site (B) complex (C),

kdleads to the rate equation for the formation of the complex:

dc-=ka [H] [B]-kd[C].dt aIntegration of this rate equation yields the following expres-sion:

= (HoBo Ce kat + In (HoBoCe Ce

10

0'-4

x

0

Mr

FractionFIG. 2. Sephadex G-100 (superfine, 65 cm X 1.5-cm inside di-

ameter) chromatography of Kc-cell cytosol (3.0 ml) incubated firstwith DFP (0.15 mM) for 30 min followed by [3H]JH 1 (20 nM) for 30min at 21°C. The column was eluted at 4°C with the standard buffer.Sixty-drop fractions were collected and monitored at 280 nm(0-0), and 100-,ul aliquots were removed for scintillation spec-trometry (+-+). EXC, exclusion volume; y-GL, gamma globulin;BSA, bovine serum albumin; OVA, ovalbumin; CHY, chymotrypsi-nogen.

0x

2.0 -

1.0

Bottom 5 10 15 20 25 30 TopFraction

FIG. 3. Velocity sedimentation centrifugation through a pre-formed (10-40%, wt:vol) continuous sucrose gradient in the standardbuffer. Cytosol (1.3 ml) was preincubated with labeled JH 1 (20 nM)for 20 min at 220C followed by 20 min at 40C. This sample was layeredonto the sucrose gradient and centrifuged at 65,000 rpm for 150 minat 40C (Sorvall TV-865 rotor). The solution was fractionated (6 dropsper fraction) and analyzed by scintillation spectrometry. Standardproteins were bovine serum albumin (BSA) and hen egg albumin(HEA).

in which Ho = initial concentration of unbound hormone; Bo= initial concentration of binding sites; Ce = equilibriumconcentration of hormone-binding site complex; Ct = amountof hormone-binding site complex at time t. Hence a plot of tversus

HoBoln Ce t

Ce - Ct

results in a line with a slope equal to

(HoBoC)kH00 Ce) k~aCe(see insert Fig. 4). Ce was calculated as the average of theamount bound at 100 and 120 min. Bo was determined inde-pendently by incubating cytosol with excess (1 ,M) JH I. Thevalue of ka, determined from the slope, was 1.29 X 106 4 0.09X 106 M-1 min-1. This value indicates a slow rate of associa-tion-slower than the rate of diffusion and, hence, not diffu-sion-controlled.The rate of dissociation of the hormone was also measured.

A first-order reaction was evident from the rate at which boundlabeled JH I was displaced by unlabeled JH I. A plot of timeversus In (bound labeled JH I) resulted in a line with a slopeequal to kd (Fig. 5). The dissociation rate constant was calcu-lated to be 1.34 X 10-2 4 0.16 X 10-2 min-1.An equilibrium dissociation constant, Kd, for the binding of

JH I to the binding site can be calculated from the ka and kdvalues:Kd = kd/ka = 1.03 X 10-8 + 0.21 X 10-8 M. The low value

of Kd indicates the presence of a high-affinity binding site forJH I in the cytosol of the Drosophila Kc cells.

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0

'e3.0C C.) ~~~~~~~3.0

1.01.0 .

0 30 60j9Time, min

0 20 40 60 80 100 120Time, min

FIG. 4. Rate of association of JH I with cytoplasmic protein.[3H]JH I (10 nM) was incubated with a 50% solution of cytosol in thestandard buffer containing DFP. Parallel incubations were also car-ried out in which excess unlabeled JH I (10 ,uM) was added. Bindingwas determined by the DCC assay. Specific binding (the differencebetween tubes incubated with and without excess JH I) was deter-mined and designated as bound hormone. The line is a theoreticalcurve fit to the data points. (Inset) Conversion of the same data toa linear plot. (See the text for the equation of the line.) Ho, the initialconcentration of unbound hormone; Bo, the initial concentration ofbinding sites; Ce, the equilibrium concentration of the hormone-binding protein complex when saturated; Ct, the concentration ofhormone-binding protein complex at the designated time. A ka valuewas determined from the slope of the line, which was determined bya least-squares, linear-regression analysis of the data points.

The rates of association and dissociation also aid in estab-lishing the necessary conditions (incubation period of 3 hr).under which an equilibrium measurement for complex for-mation can be obtained. This study was performed again withthe DCC assay to separate bound from unbound hormone (Fig.6). Transformation of these data to a Scatchard plot (16) yieldeda straight line (Fig. 6 Inset), demonstrating a single type ofhigh-affinity binding site. From the slope of the line, a valuefor the equilibrium dissociation constant Kd was calculated: Kd= 1.56 X 10-8 L 0.55 X 10-8 M. This value lies within the limitsof experimental error of the value obtained by kinetic mea-

Time, min

FIG. 5. A semilog plot of the rate of dissociation of JH I from thecomplex with binding protein. [3H]JH I (10 nM) was incubated witha 50% solution of cytosol in the standard buffer for 3 hr, and thenexcess unlabeled JH I (10 AM) was added. At the designated times,aliquots were removed and assayed for bound hormone by means ofthe DCC assay. These values have been corrected for nonspecificbinding. A kd value was determined from the slope of the line. Theline was drawn by the method of least squares.

surements. The agreement of these two values is verificationof a high-affinity binding site for JH I. It is possible that onlyone stereoisomer of JH I is recognized by the binding protein,and consequently the determined values of the equilibriumconstants could be twice the true value because the racemicmixture of JH I was used.The x-axis intercept value on the Scatchard plot (Fig. 6), 2.06

nM, is a measure of the concentration of binding sites in thereaction mixture. By using that value and the calculated volumefor the Drosophila Kc cell in suspension as 2.56 X10-7 llpercell, 2500 binding sites per cell was estimated.

Specific binding could not be demonstrated when the acidand diol metabolites of [3H]JH I were assayed. The potentmolting hormone analog, ponasterone A, did not compete withjuvenile hormones for the binding macromolecule. In addition,parallel binding studies with cytosol of control cells (humanfibroblasts) showed a complete absence of JH binding (data notshown). Competition binding experiments were conducted withthe three unlabeled juvenile hormones competing with [3HIJHI. The amounts of unlabeled hormones required for 50% inhi-bition of the binding of labeled ligand were within an order ofmagnitude of each other.

DISCUSSIONIt has been demonstrated in numerous systems that both peptideand steroid hormones act via membrane or cytoplasmic re-ceptors (17, 18). JH is chemically distinct from these other twoclasses of hormones. This paper provides evidence for a cyto-plasmic binding macromolecule for JH, which would indicatethat JH action is mediated in a manner analogous to the steroidhormones. Initial binding of JH to membrane receptors has notbeen ruled out, nor has it yet been demonstrated that thecytoplasmic binding protein can enter the nucleus. Nonetheless,the Kc-cell cytoplasmic JH-binding protein can be presumedto be a receptor because of its ligand specificity, its saturability,and its ability to concentrate the native hormone from the ex-tracellular medium. It is of interest to note that although Dro-sophila Kc cells have been grown for many generations without

Free, nMFIG. 6. Amount of radioactive hormone bound as a function of

the amount of labeled hormone added to a constant amount of Kc-cellcytosol. [3H]JH I (2-20 nM) was incubated with 12.5 ,l of Kc-cellcytosol in a total volume of 100 ul for 3 hr at 23°C. The bound hor-mone was separated from the free hormone by using the DCC assay.Nonspecific binding at these low hormone concentrations amountedto less than 6% of the total binding, even at the highest hormoneconcentration. (Inset) Scatchard analysis of the same data AKd valueis determined from the slope of the line. The line was drawn by themethod of least squares. B, the concentration of bound hormone; Fand free, the concentration of unbound hormone.

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contact with insect hormones, they have functional receptorproteins for both ecdysteroids and JH.

There are indications that JH serves a physiological functionin both larva and adult Drosophila. Topical application of JHblocks metamorphosis of pupating flies (19) ada, in the mutantapterous4, a JH analog is able to mediate the synthesis andsubsequent sequestration of yolk protein by developing oocytes(20). Moreover, in isolated abdomens of normal adult females,a JH analog induces vitellogenesis (21), and JH inhibits ecdys-teroid-induced evagination of imaginal discs in vitro (22). Al-though JH has not been conclusively identified in D. melano-gaster, it has been shown to have an effect on Kc cells in vitro.In cultured Drosophila cells, the effects of ecdysterone on cellmorphology are partly reversed by JH (23), and the stimulatoryeffects on cell proliferation are inhibited by a JH analog(24).The existence of hemolymph carrier proteins for JH is well

documented (see ref. 1 for a review). These proteins may serveprimarily a protective function, rather than a mediatory one,although no adequate test of a mediatory function has yet beenmade (25). A recent report (26) has provided evidence for ahemolymph JH-carrier protein in larvae of D. hydei. The dis-sociation constant for JH I is 0.1 uM (26). If D. melanogasterhas a similar protein in the hemolymph, it could pass hormoneon to a protein of higher affinity on or in the cell. In order toclarify the role of the cytoplasmic JH-binding protein, it willbe necessary to follow its fate in the cell. So far, nuclear bindingof the hormone in insect cells has been demonstrated (8 andunpublished experiments) but the movement of bound hor-mone from cytoplasm to nuclei has not been shown. Exactlyhow these transfers would be accomplished is not clear.The absence of any competition by ecdysteroids indicates

that the observed antagonism between the molting hormoneand JH involves a mechanism other than simple competitionfor binding sites on the same receptor. Further elaboration ofthe binding specificity of the cytosol protein is of considerableinterest, for it may shed light on the mechanism of action ofboth hormones and analogs.The discovery of the cytosol hormone-binding protein also

places JH within the framework of the extensively studiedsteroid hormone system. Understanding the relationship be-tween this protein and others that bind JH in hemolymph andcell nuclei will advance considerably our understanding of themechanism of action of this unique and important insect hor-mone.

We thank Professor F. J. Kezdy for helpful discussions about treat-ment of kinetic and equilibrium binding data and Ms. Gwen Schoonfor assistance with the human fibroblast cells. Portions of this research

were supported by Grants PF-1529 and CD-12 from the AmericanCancer Society, Grants AM 05929 and GM 13863 trom the NationalInstitutes of Health, and Grant PCM-7421379 from the NationalScience Foundation.

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3. Yund, M. A., King, D. S. & Fristrom, J. W. (1978) Proc. Natl.Acad. Sci. USA 75,6039-6043.

4. Maroy, R., Dennis, R., Beckers, C., Sage, B. A. & O'Connor, J. D.(1978) Proc. Natl. Acad. Sci. USA 75,6035-6038.

5. Schmialek, P. (1973) Nature (London) 245, 267-268.6. Schmialek, P., Borowski, M., Geyer, A., Miosga, V., Nundel, M.,

Rosenberg, E & Zapf, B. (1973) Z. Naturforsch. 28, 453-456.7. Akamatsu, Y., Dunn, P. E., Kezdy, F. J., Kramer, K. J., Law, J.

H., Reibstein, D. & Sanburg, L. L. (1975) in Control Mechanismsin Development, eds. Meints, R. H. & Davies, E. (Plenum, NewYork), pp. 123-149.

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eds. Menn, J. J. & Beroza, M. (Academic, New York), pp. 155-176.

13. Munck, A. & Wira, C. (1975) Methods Enzymol. 36,255-264.14. Kramer, K. J., Dunn, P. E., Peterson, R. C., Seballos, H. L.,

Sanburg, L. L. & Law, J. H. (1976) J. Biol. Chem. 251, 4979-4985.

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