Proc. Natl. Acad. Sci. USAVol. 85, pp. 5927-5931, August 1988Botany

Characterization of a benzyladenine binding-site peptide isolatedfrom a wheat cytokinin-binding protein: Sequence analysis andidentification of a single affinity-labeled histidine residue bymass spectrometry

(photoaffinity labeling/azidobenzyladenine/wheat embryo/storage protein/laser photodissociation Fourier-transform mass spectrometry)

A. CHRIS BRINEGAR*t, GEOFFREY COOPER*, ANNE STEVENS*t, CHARLES R. HAUER§,JEFFREY SHABANOWITZ§, DONALD F. HUNT§, AND J. EUGENE Fox*¶*ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568; and §Department of Chemistry, University of Virginia, Charlottesville, VA 22901

Communicated by Folke Skoog, April 15, 1988 (received for review September 15, 1987)

ABSTRACT A wheat embryo cytokinin-binding proteinwas covalently modified with the radiolabeled photoaffinityligand 2-azido-N6-['4C]benzyladenine. A single labeled peptidewas obtained after proteolytic digestion and isolation byreversed-phase and anion-exchange HPLC. Sequencing byclassical Edman degradation identified 11 ofthe 12 residues butfailed to identify the labeled amino acid. Analysis by laserphotodissociation Fourier-transform mass spectrometry of 10pmol of the peptide independently confirmed the Edman dataand also demonstrated that the histidine residue nearest the Cterminus (underlined) was modified by the reagent in thesequence Ala-Phe-Leu-Gln-Pro-Ser-His-His-Asp-Ala-Asp-Glu.

Wheat embryo cytokinin-binding factor 1 (CBF-1) is anembryo-specific protein from wheat having a high affinity forseveral active cytokinin derivatives (1-3). Originally isolatedfrom preparations of wheat germ (4, 5), CBF-1 has recentlybeen shown to have characteristics commonly associatedwith seed storage proteins, including high nitrogen content (5,6), structural similarities to the vicilin-type storage proteins(7), rapid accumulation to high levels during the filling stageof embryogenesis (7-9), and localization in membrane-boundprotein bodies in the tissues surrounding the embryonic axis(6, 10). Although the role of CBF-1 in regulating cytokininfunction in vivo has not been demonstrated, one hypothesisis that it functions as a storage protein that also sequesterscytokinins during the filling stage of embryogenesis (6, 10).A concern shared by those who study plant hormone-

binding proteins is whether these molecules participate inhormonal regulation or just coincidentally interact nonspe-cifically with hormones in vitro (11). Of major difficulty ininterpreting binding data is the relatively low binding affini-ties of these proteins for plant hormones (as compared withanimal hormone-receptor systems), which often obscuresdetection of specific and nonspecific binding sites. Oneapproach to characterizing specificity of ligand binding is byuse of photoaffinity analogues (12). A previous study (13)showed the potential of labeling the benzyladenine (BzlAde)binding site of CBF-1 with 2-azido-N6-[14C]benzyladenine([14C]N3BzlAde), which covalently interacts with the singleBzlAde binding site.

In this report we characterized the interaction ofN3BzlAdewith CBF-1 by the isolation and sequence analysis of anN3BzlAde-modified peptide from CBF-1. By using the clas-sical Edman method and a new mass spectral method ofpeptide sequencing (14) we demonstrated the very high

specificity of this interaction, proving that a single histidineresidue in the protein was modified by N3BzlAde.

MATERIALS AND METHODS

Photoaffinity Probe Synthesis and Isolation. [Methylene-"4C]2-azido-N6-benzyladenine (["4C]BzlAde) was synthe-sized by the method ofTheiler et al. (15) as modified by Keimand Fox (13). [Methylene-'4C]2-chloro-6-benzylaminopurinewas first prepared from 2,6-dichloropurine (Aldrich) and[7-1'4C]benzylamine (Amersham, 51 mCi/mmol diluted to 4mCi/mmol with cold benzylamine; 1 Ci = 37 GBq). The finalproduct was synthesized by reaction of the 2-chlorinatedcompound with hydrazine followed by treatment with sodiumnitrite. [14C]N3BzlAde was purified by reversed-phase chro-matography on a PepRPC HR 5/5 column (Pharmacia) witha gradient of water (solvent A) and methanol (solvent B) (0-62% solvent B for 36 min, 62% solvent B for 8 min, 62-80%solvent B for 10 min at 0.3 ml/min). The azido derivative wasthe last component to elute [at 65-70% (vol/vol) methanol]and was distinguished by a UV-labile absorbance peak at 235nm. The compound was dried and redissolved in methanol to-1 mM with a specific activity of 4 mCi/mmol. Other thanworking in a reasonably darkened area, no special precau-tions were required to prevent photolysis of the azido group.

Photoaffinity Labeling of CBF-1. CBF-1 was isolated frommature durum wheat seeds (Triticum durum cv. Mexicali) asreported previously (7) and adjusted to 0.5 mg/ml in 0.2 Mammonium bicarbonate, pH 8.0. [14C]N3BzlAde was addeddropwise with gentle mixing to a final concentration of 12,uM. After 1-hr incubation on ice the solution was transferredto a 1-cm-pathlength quartz cuvette and irradiated from adistance of 1 cm with short wave UV light (500 ,uW/cm2measured at 254 nm; Mineralight Lamp model R-520; Ultra-violet Products, San Gabriel, CA).To measure incorporation of [14C]N3BzlAde into CBF-1,

aliquots containing 10 ,ug of protein were removed at inter-vals from 0-5 min of irradiation, precipitated in 20% (wt/vol)trichloroacetic acid on ice, washed twice with cold acetone,dissolved in 0.1 ml of 1% NaDodSO4, and the radioactivitywas measured by liquid scintillation counting. Tryptic digestswere performed by adding tosylphenylalanine chloromethyl

Abbreviations: CBF-1, wheat embryo cytokinin-binding factor 1; Bzl-Ade, N6-benzyladenine; N3BzlAde, 2-azido-N6-benzyladenine; [14C]-N3BzlAde, 2-azido-N6-["4C]benzyladenine.tTo whom reprint requests should be sent at present address:Department of Biological Sciences, San Jose State University, SanJose, CA 95192.tPresent address: Baylor College of Medicine, 1200 Moursund,Houston, TX 77030.Present address: Miles Inc., 1127 Myrtle Street, Elkhart, IN 46514.

5927

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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ketone-treated trypsin (United States Biochemical, Cleve-land, OH) immediately after 5 min of irradiation at a 1:10(wt/wt) ratio of trypsin to CBF-1. Aliquots were removed atintervals from 0-120 min at 370C and diluted with an equalvolume of double-strength NaDodSO4/PAGE sample buffer.After immediate boiling to inactivate the trypsin, the sampleswere analyzed by NaDodSO4/PAGE (16) and fluorography(17).

Proteolysis of [14CJN3BzIAde-Labeled CBF-1 and Isolationof the Labeled Peptide. After partial digestion with trypsin for1 hr and trichloroacetic acid precipitation as described above,the dried pellet (containing 2.5 mg of modified CBF-1) waswashed in cold acetone, dissolved in 0.13 ml of 8 M urea, anddiluted with 0.39 ml of 40 mM ammonium bicarbonate, pH8.0. Endoproteinase Glu-C (protease V8) (0.1 mg, BoehringerMannheim Biochemicals) was added and incubated for 20 hrat 370C. The pH was adjusted to 2.5 with 6 M HCl, and thesolution was filtered by centrifugation through a Centrex0.2-,m cellulose acetate membrane (Schleicher & Schuell).Peptide separations were performed using Pharmacia's fastprotein liquid chromatography (FPLC) instrument and col-umns with monitoring at 214 nm. Columns, eluents, andgradients are described in Fig. 3.Edman Sequencing and Amino Acid Analysis. Edman se-

quencing on 400 pmol of the purified [14C]N3BzlAde-labeledpeptide was done on an Applied Biosystems (Foster City,CA) model 470-A vapor-phase microsequencer. Two-thirdsof each Edman cycle was used for identification of thephenylthiohydantoin-amino acid using an Applied Biosys-tems model 120-A microbore phenylthiohydantoin-aminoacid analyzer. Radioactivity released with each cycle wasmeasured by liquid scintillation counting of the remainder.For amino acid analysis, 100 pmol of peptide was hydro-

lyzed in vacuo in 6 M HCI at 110°C for 18 hr. The hydrolysatewas derivatized with phenyl isothiocyanate, and the phenylisothiocyanate-amino acids were analyzed on a WatersAssociates Pico-Tag system according to the manufacturer.Mass Spectrometry. Mass spectra were recorded on a

tandem quadrupole Fourier-transform mass spectrometerequipped for laser photodissociation experiments with aLumonics ArF excimer laser. Operation of this instrumentfor both molecular weight determination (18) and amino acidsequence analysis (14) of oligopeptides has been described.

[14C]N3BzlAde-labeled peptide at the 30-pmol level wasdissolved in 3 A1l of 5% (vol/vol) acetic acid. For molecularweight determination, a mixture containing 0.5 Al of 1:1glycerol/monothioglycerol and a 0.5- to 1.0-,ul aliquot ofsample was exposed to a beam of 10 keV cesium ions for 4msec in the ion source of the mass spectrometer. Sample ionssputtered from the matrix were transferred to an elongatedion cyclotron resonance cell and caused to move coherentlyin large orbits at cyclotron frequencies characteristic of theirindividual masses after irradiation with a radio frequencypulse. Image currents produced by this motion on two of thecell walls were amplified, digitized, and Fourier transformed.Data from 20 to 50 such experiments were summed in <20 secto produce the mass spectrum.

Lyophilized labeled peptide (20-25 pmol) was converted tothe corresponding methyl ester with 30 ,ul of 2 M HCl inmethanol for 2 hr at room temperature. After the removal ofsolvent, the sample was dissolved in 2-2.5 ,p of 5% aceticacid. An aliquot corresponding to 10 pmol was used to obtainthe results shown in Fig. 4. To generate laser photodissocia-tion data, sample (M + H)+ ions stored in the ion cyclotronresonance cell were exposed to a single 10-nsec pulse ofirradiation at 193 nm from the ArF excimer laser. Massspectra were recorded as described above. Subtractive Ed-man degradation was done as described (19).

RESULTSIncorporation of ['4CJN3BzlAde into CBF-1. Preliminary

experiments using a 1:1 molar ratio of [14C]N3BzlAde toCBF-1 BzlAde-binding sites indicated that fewer than 20% ofthe sites were modified (based on our proposed model of oneBzlAde-binding site per native CBF-1 trimer). Increasing thelevel ofthe photoaffinity reagent to a 4-fold molar excess overBzlAde-binding sites resulted in a very reproducible modifi-cation of =85% of the sites (Fig. 1). Under the statedconditions, maximum incorporation was seen after 1 min ofUV irradiation. Because of the low specific activity of the[14C]N3BzlAde, maximum incorporation of radioactivity intoCBF-1 was no higher than 50 cpm/Ag. Identical treatment ofovalbumin or immunoglobulin G with [14C]N3BzlAde yielded<5% of the radioactivity incorporated into CBF-1.

Tryptic Digestion of [14C]N3BzlAde-Labeled CBF-1 andIdentification of the Labeled Fragments. Partial tryptic diges-tion of native CBF-1 after labeling with [14C]N3BzlAde (Fig.2A) indicated a domain of the protein that was extremelysusceptible to proteolytic attack. This primary cleavage ofthe 54-kDa subunit into polypeptide groups near 37 and 17kDa occurred very rapidly. In contrast, the further degrada-tion of the 37-kDa polypeptide group into a set near 23 kDawas very slow, requiring >1 hr even at a 1:10 ratio of enzymeto CBF-1. A very similar pattern of CBF-1 degradation wasseen in preparations from commercially milled wheat germ(8) and during wheat embryo germination (7), indicating thatthe same domains of CBF-1 are recognized by endogenousproteases. The 23-kDa tryptic fragments and the initial17-kDa polypeptide group were then remarkably resistant tofurther proteolysis. In fact, full BzlAde-binding activity wasmeasured after a 2-hr digestion (data not shown), suggestingthat the BzlAde-binding site was contained in one or both ofthese polypeptide groups. The resistance of these corefragments to trypsin must be due to conformational effectsbecause CBF-1 contains -12% arginine (6), and the additionof denaturants results in complete digestion and loss ofactivity (data not shown).The labeling of CBF-1 with the photoaffinity reagent had

no effect on the pattern or degree of trypsin digestion ascompared to the unlabeled protein. The size heterogeneityseen in each of the tryptic fragment groups may have resultedfrom endogenous exoproteinase activity during CBF-1 iso-lation or incomplete digestion by trypsin. Because seed

-

I.-

E

41

0.h.S

r

CInM

ocin

a

U'

1.0

0.8

0.6

0.4

0.2

Irradiation time, min.

FIG. 1. Incorporation of ["4C]N3BzlAde (inset) into CBF-1. A4-fold molar excess of radiolabeled N3BzlAde (4 mCi/mmol) overBzlAde binding sites was used to modify CBF-1. The extent ofbinding-site modification was calculated on the basis of trichloro-acetic acid-precipitable radioactivity after UV irradiation for theindicated times.

5928 Botany: Brinegar et al.

NH --L4CH2N N

"I \>N

IN3 H I

-1 -LD 1 2 4 5

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Proc. Natl. Acad. Sci. USA 85 (1988) 5929

1 2 3 4 5 6

A1 2 3 4 5 6

B

45--37

31 -

6- -5

FIG. 2. Analysis of radiolabeled tryptic fragments of CBF-1 aftermodification with ['4C]N3BzlAde. After incubation with trypsin forvarious times the digests were analyzed by NaDodSO4/PAGE, andthe polypeptides were visualized by Coomassie blue staining (A) andfluorography (B). Lanes: 1, undigested CBF-1; 2, 0.5 min; 3, 5 min;4, 20 min; 5, 60 min; and 6, 120 min. Molecular masses of standardproteins are shown at left, and estimated molecular weights of theCBF-1 fragments are shown at right.

storage proteins are often expressed from multigene families,heterogeneity may be inherent in CBF-1 polypeptides.Fluorography of the separated tryptic fragments of

[14C]N3BzlAde-labeled CBF-1 (Fig. 2B) showed that thephotoaffinity probe was coupled to the 37-kDa polypeptidegroup and its 23-kDa subfragments. The specific nature ofthis labeling is suggested by the absence of detectableradioactivity associated with the 17-kDa group of polypep-tides. The 17- and 23-kDa tryptic fragments could be sepa-rated by cation-exchange chromatography on CM-Sepharose4B (Pharmacia) in 6 M urea/20 mM sodium succinate, pH 6.0(data not shown), with the 17-kDa polypeptides passingthrough unbound and the 23-kDa polypeptides eluting withthe addition of 0.5 M NaCl. However, the removal of the17-kDa polypeptides from the tryptic digestion products wasfound unnecessary in the subsequent isolation of the labeledpeptide from the 23-kDa fragments.

Isolation of a [14C]N3BzlAde-Labeled Peptide from TrypticFragments. As mentioned above, further proteolysis of theCBF-1 fragments generated by a 1-hr trypsin digestion wasimpossible without the inclusion of a denaturant. Solubiliza-tion of trichloroacetic acid-precipitated 17- and 23-kDa tryp-tic polypeptides in 8 M urea (followed by dilution to 2 M) wasnecessary to obtain a more complete digestion with a secondenzyme. Several proteases, including chymotrypsin, trypsin,endoproteinase Lys-C, endoproteinase Glu-C, and elastase,were screened for their ability to generate a ['4C]N3BzlAde-labeled peptide so that a large percentage of the label couldbe recovered in one chromatographic peak. EndoproteinaseGlu-C digestion met this criterion and provided the bestseparation of peptides by reversed-phase HPLC (Fig. 3A).Approximately 50% ofthe recovered radioactivity was in twofractions eluting at 40-42 min. The remaining radioactivitywas scattered among several fractions; no single fractioncontained >5% of the total radioactivity. Without the initialtrypsin treatment, the endoproteinase Glu-C digestion wasnot so complete and resulted in two major radioactive peaks(data not shown) eluting at -41 and 46 min.

Further purification was performed by anion-exchangechromatography in the presence of 2 M urea (Fig. 3B). Thelabeled peptide was eluted at 0.3 M in a NaCl gradient (ustafter a broad peak of poorly resolved contaminants) and

to2

I0S2In a-

O 10 20 30 40 5o 60

Tim. mn.

FIG. 3. HPLC purification of a radiolabeled peptide from atrypsin/endoproteinase Glu-C digest of ['4C]N3BzlAde-modifiedCBF-1. (A) The filtered digestion products were separated on aPepRPC HR 5/5 reverse-phase column (Pharmacia) at 1 ml/min in0.1% trifluoroacetic acid with a water/acetonitrile gradient. (B)Fractions containing the major radiolabeled component from run Awere dried, dissolved in 2 M urea/20 mM Tris HCl, pH 8.0, andseparated on a Mono Q HR 5/5 anion exchange column (Pharmacia)at 0.5 ml/min with a NaCl gradient in the same buffer. (C) Fractionsfrom the radiolabeled component of run B were pooled and directlyinjected into a ProRPC HR 5/10 reversed-phase column (Pharmacia)and eluted under the same conditions as in run A.

contained =75% of the recovered radioactivity. The additionof urea to the buffers was necessary to increase the solubilityand yield ofthe peptide. Without urea the peptide eluted withsignificant contamination as a very broad tailing peak at 0.6-0.8 M NaCl. To remove the buffer, salt, and urea and toassess the purity of the peptide, the labeled peak fractionsfrom the anion-exchange separation were pooled and injecteddirectly into a reversed-phase column (Fig. 3C). A singlepeptide peak (=1 nmol) was eluted, which contained >95%of the recovered radioactivity. Only 10% of the initiallyincorporated radioactivity was recovered. As suggested byFig. 3A, approximately half of the loss was due to labeling oflow specificity (or nonspecific) sites and incompletely di-gested high specificity sites, whereas the remaining losseswere incurred during precipitation and resolubilization steps.

Sequence Analysis of the ['4CJN3BzlAde-Labeled Peptide byEdman Degradation. Gas-phase Edman analysis identified 11of the 12 residues in the peptide (Table 1). No phenylthio-hydantoin-amino acid was seen by HPLC analysis ofcycle 8.However, if an aliquot of the material from each cycle wastaken before HPLC injection and analyzed by liquid scintil-lation counting, over 70% of the recovered radioactivity wasfound in cycle 8 with the rest eluting as "carry over" in cycles9-11. This result indicated that the photoaffinity reagent wasspecifically attached to the amino acid at position 8 in thepeptide and that the identification of this phenylthiohydan-toin-amino acid by HPLC retention time was made impos-sible by the photoaffinity derivatization.Amino acid analysis was performed on the modified

peptide to determine if the covalent bond resulting from the

kDa92-

66-

I?

ECL

kDa-54

21 -

14 -

-23

8-

-17-12

u

w

xi

a

Ita

C15 1

0.2II ~~~~~~~~~~~~~4WII 3~~~~~~~~~~~~~~I

0.± -- -I-

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Table 1. Amino acid sequence of the [14C]N3BzlAde-labeledpeptide and recovery of radioactivity per Edman cycle

Cyclenumber

RadioactivityAmino acid released, cpm*

1 Alanine2 Phenylalanine3 Leucine4 Glutamine5 Proline6 Serine7 Histidine8 ND9 Aspartate10 Alanine11 Aspartate12 Glutamate

4224453603915

16115101869039

ND, not detected.*Corrected for a background 18 cpm.

reaction of the BzlAde nitrene radical and the residue atposition 8 in the peptide was labile to acid hydrolysis. If so,the analysis should yield the amino acid that was unidentifiedby sequencing or result in the doubling of one already seen inthe sequence. The bond was apparently acid labile becauseanalysis of the phenyl isothiocyanate-derivatized hydroly-sate of the labeled peptide (Table 2) indicated two residues ofhistidine per peptide instead of one as expected from thesequence. Because no other unexpected amino acids were

Y3376.1

.1

Table 2. Amino acid analysis of the ['4C]N3BzlAde-labeled peptide

Residues per peptide

Amino acid Measured* Expectedt

Aspartate 1.5 2Glutamate/glutamine 2.3 2Serine 1.3 1Histidine 2.0 1Alanine 1.9 2Proline 0.9 1Leucine 1.1 1Phenylalanine 1.1 1

*From amino acid analysis.tBased on Edman sequencing data (Table 1).

seen by amino acid analysis, the data indicated the histidineat position 8 in the peptide was modified by the photoaffinityreagent. The reason for the slightly low value ofone and a halfresidues of aspartate instead of the expected value of two isnot apparent. The sequencing results, however, conclusivelyidentified two aspartate residues in the peptide.Sequence Analysis of the [14C]N3BzlAde-Labeled Peptide by

Mass Spectrometry. The sequence of the peptide and theidentity of the N3BzlAde-modified residue were indepen-dently determined by laser photodissociation Fourier-transform mass spectrometry. Mass spectra recorded on thelabeled peptide and its corresponding methyl ester showed

Y4505.2HH+X

I513.2

.I.I if.

400 450 500 0 60550 600. . .

650 700

87

781.4I L

Y5+X880.4

Y6+X1017.5

,l A

o00 750 800 850 900 950 1000

C14

4Y7+XB!85+6XS 1201.7 1B9+X y9+X 356.8

1050 1100 1150 1200 1250 1300 1350

(M+H)"7 -4 1661.1

K4

I442.8 1485.9

1401 50 1500........ ..7.........1 400 1 450 1 S00 1 550

MASS IN A. M. U.

FIG. 4. Laser photodissociation Fourier-transform mass spectrum of methylatedN3BzlAde-labeled peptide from CBF-1. B and Yrefer to types of peptide ion fragmentationpatterns (see text) with subscripts indicating thenumber of residues in each ion. X represents theattached BzlAde nitrene, and other letters arethe one-letter abbreviations of amino acids. Theasterisk indicates a peptide ion in which ammo-nia has been lost from the N-terminal glutamine.

0OC'J7

z 0-.z

FQJ

>.-JL&J

4ML --LAL-" -uk --kW-.L" -A.Lk-mkd AAlLj iM- I LMMUL, AA AJA JLLLML.L

t-li . I . . . I . . . . . . . . . . . . . . . .

I. _s-JiA A ji- -- u.6&hAh-ALAL - AIL -L -.. Lji I -11 i -L By . Al

5930 Botany: Brinegar et al.

I A, I i L1 -I A .1 II i

. . . . . .

1600 1650 1700

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Proc. Natl. Acad. Sci. USA 85 (1988) 5931

\IV \\

Asp Ala Asp

OMe OMe

boo 'et-P -A ,,

Glu-OMe

OMe

FIG. 5. Sequence of the methylated N3BzlAde-labeled peptidededuced from its photodissociation mass spectrum. X represents theattached BzlAde nitrene. Masses of the theoretical B- and Y-typepeptide ions are shown along the top and bottom, respectively, of thesequence. Masses of peptide ions found in the spectrum (Fig. 4) arewithin 0.2 mass unit of these values.

abundant (M + H) + ions at m/z 1605.1 and 1661.1, respec-tively. The observed mass shift of 56 Da indicated thepresence of four carboxyl groups and, therefore, three acidicamino acids in the molecule. Shown in Fig. 4 is the laserphotodissociation mass spectrum recorded on 10 pmol of thepeptide methyl ester. Fragment ions at m/z 70.1, 110.1, and120.1 indicated the presence of histidine, proline, and phen-ylalanine, respectively, in the peptide (19).A search for a series of fragment ions having m/z values

corresponding to the loss of one or more residues from the Cterminus of the peptide, ions of type B (19, 20), was facilitatedby knowledge that the labeled peptide was generated from theprotein by endoproteinase Glu-C digestion and thereforeshould contain a glutamic acid residue at the C terminus.Loss of glutamic acid afforded the fragment ion at m/z1485.9. Additional B-type fragment ions corresponding to theloss of aspartic acid, alanine, and aspartic acid from the Cterminus were seen at m/z 1356.8, 1285.7, and 1156.6,respectively. Additional sequence information was obtainedfrom a search for fragment ions derived from the parentmolecule by the loss of one or more amino acid residues fromthe N terminus, ions of type Y (18, 19). The last two of thesefour ions, Y3 and Y4, yielded strong signals at m/z 376.1 and505.2, respectively, in the spectrum. The next ion in theseries appeared at m/z 880.4 and corresponded to the ad-dition of the modified histidine residue. The modified histi-dine (with the typical loss of the a-carbonyl group) alsoappeared by itself at m/z 348.1. Additional Y-type ionsappeared at m/z 1017.5, 1104.6, 1201.7, 1312.7, and 1442.8and specified the addition of histidine, serine, proline, glu-tamine, and leucine or isoleucine (the latter two cannot bedistinguished) to the parent ion in the C-terminal to N-terminal direction.The remaining mass difference (218 Da) between the last

observed Y-type ion and the (M + H) + ion can correspond toonly one of two dipeptide combinations, Met-Ser or Ala-Phe.To differentiate these possibilities, one cycle of Edmandegradation was performed on 10 pmol of sample. Becausethe (M + H) + ion derived from the peptide (after shorteningby one residue) had a m/z value 71 Da lower than the parentmolecule, the N-terminal residue was assigned alanine,thereby specifying the N-terminal dipeptide as Ala-Phe.The complete sequence, as deduced above, is shown in

Fig. 5 and confirms the sequence proposed from the Edmanand amino acid analysis data.

DISCUSSIONIn this work, we sought to minimize nonspecific labelingcommonly seen with photoactivated azido derivatives (21) bykeeping the protein concentration low and using the lowestpossible ratio of ligand to protein. The fact that >80% of thehigh-affinity BzlAde binding sites in CBF-1 could be modifiedwith only a 4-fold molar excess of N3BzlAde is evidence fora high degree of specific labeling. Longer exposure to UVlight did not result in more labeling; therefore, the excessBzlAde nitrene was probably inactivated by reaction with

solvent. Under identical conditions, several proteins that donot bind BzlAde were only very marginally labeled.CBF-1 appears to have a domain that is extremely suscep-

tible to proteolysis. Incubation of the protein with low levelsof trypsin for a few seconds resulted in cleavage of the 54-kDapolypeptide into groups of 37- and 17-kDa fragments. Trypticdigestion of CBF-1 labeled with [14C]N3BzlAde showed thatthe modified sequence resided in the 37-kDa set of polypep-tides and that further digestion slowly converted these to a setof labeled 23-kDa fragments.

Previous studies have shown that CBF-1 isolated fromwheat germ is similarly "nicked" by endogenous embryoproteases (8). However, if isolated under nondenaturingconditions the protein retains full BzlAde-binding activitydespite being protease "nicked." The 37- or 23-kDa frag-ments by themselves do not bind cytokinins, nor have we

been able to reconstitute binding activity by mixing 37- or23-kDa fragments with the 17-kDa peptides.The reaction of N3BzLAde with CBF-1 is remarkably

specific-only a single histidine residue in the protein ismodified. For such specificity to occur, the ligand must beoriented in the binding site in a relatively fixed position suchthat the photoactivated nitrene radical at the 2-position of thepurine ring can come into contact only with the histidineimidazole ring. Previously we demonstrated (7) that CBF-1 iscomposed of three apparently identical subunits but bindsonly a single cytokinin molecule per molecule of protein. Theresults presented here suggest that the subunits interact in theCBF-1 trimer to form a single binding site.

The authors thank Dr. Gary Hathaway (Biotechnology Instrumen-tation Facility, University of California, Riverside) for gas-phaseEdman sequencing, Dr. Audry Fowler (Department of BiologicalChemistry, University of California at Los Angeles School ofMedicine) for micro-amino acid analysis, and Dr. John Steffens(Department of Plant Breeding, Cornell University) for suggestingthe collaboration between our laboratories.

1. Keim, P., Erion, J. L. & Fox, J. E. (1981) in Metabolism andMolecular Activities ofCytokinins, eds. Guern, J. & Pdaud-Lenoel,J. (Springer, Berlin), pp. 179-190.

2. Polya, G. M. & Bowman, J. A. (1979) Plant Physiol. 64, 387-392.3. Moore, F. H., III (1979) Plant Physiol. 64, 594-599.4. Fox, J. E. & Erion, J. L. (1975) Biochem. Biophys. Res. Commun.

64, 694-700.5. Erion, J. L. & Fox, J. E. (1981) Plant Physiol. 67, 156-162.6. Brinegar, A. C. & Fox, J. E. (1985) in Current Topics in Plant

Biochemistry and Physiology, eds. Randall, D. D., Blevins, D. G.& Larson, R. L. (Univ. of Missouri, Columbia), Vol. 4, pp. 91-100.

7. Brinegar, A. C., Stevens, A. & Fox, J. E. (1985) Plant Physiol. 79,706-710.

8. Brinegar, A. C. & Fox, J. E. (1985) Biol. Plant. 27, 100-104.9. Brinegar, A. C. & Fox, J. E. (1985) in Plant Genetics, ed. Freeling,

M. (Liss, New York), Vol. 35, pp. 147-155.10. Brinegar, A. C. & Fox, J. E. (1987) in Plant Hormone Receptors,

North Atlantic Treaty Organization ASI Series, ed. Klambt, D.(Springer, Berlin), Vol. H10, pp. 177-184.

11. Kende, H. & Gardner, G. (1976) Annu. Rev. Plant Physiol. 27, 267-290.

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