THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 266, No. 36, Issue of December 25, pp.

24413-24419,1991
(0 1991 by The American Society for Biochemistry andMolecular Biology, Inc. Printed in U.S. A.

N-Acylation of Aplysia Egg-laying Hormone with Biotin

CHARACTERIZATION OF BIOACTIVE AND INACTIVEDERIVATIVES*

(Received for publication, May 23, 1991)

Susan L. Knock*, Brian T. Miller§, James E. Blankenship*§, Gregg T. NagleSQ,John S. Smithll, and
Alexander Kuroskyll 11
From the $Marine Biomedical Institute, the §Department of Anatomy and Neurosciences,and the VDepartment of Human
Biological Chemistry and Genetics, the University of Texas Medical Branch, Galveston, Texas 77550

Chemicalmodification of the egg-laying hormone abdominal ganglion of several species of the marine mollusc
(ELH) of Aplysia by reaction with the N-hydroxysuc- Aplysia (Chiu et al., 1979; Nambu and Scheller, 1986; Nagle
cinimide ester of biotin, which contained 6-aminohex- et al., 1988a). ELH is one of several peptides encoded by the
anoic acidas spacer, yielded seven distinct derivatives ELH gene that isprocessed from a37-kDa hormoneprecursor
that were readily separated by reversed-phase high (Scheller et al., 1983; Mahon et al., 1985; Newcomb and
performance liquid chromatography. The derivatives Scheller, 1987; Newcomb et al., 1988). When the bagcells
were chemically characterized by amino acid compo- undergo a prolongedafterdischarge of intense repetitivefiring,
sitional analysis, sequence analysis, and mass spec- ELH and other peptides coordinately
are released to influence
trometry. The seven derivatives resulted fromcombi- specific targets (Kupfermann and Kandel,1970; Stuart et al.,
nations of differential modification of the three amino 1980; Sigvardt et al., 1986). ELH acts asa nonsynaptic trans-

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groups in the ELH molecule located at Ile’ (a-NHZ), mitter within the abdominal ganglion to cause burst augmen-
Lyss, and Lys”. Of the seven derivatives formed,only
one, monobiotinyl LysS6-ELH, was biologically active tation of neuron R15 (Branton et al., 1978; Mayeri et al.,
in eliciting egg-laying activity and altering the electro- 1979b, 1985; Levitan et al., 1987) and prolonged excitation of
physiological activity of the abdominal ganglion neu- LB and LC cluster neurons (Mayeri et al., 1979b, 1985). ELH
ron R15 and LB and LC cluster neurons. In addition, also acts asa conventional neurohormone on distant targets,
evaluation of the time courseof biotinylation of ELH for example exciting neuronB16 in thebuccal ganglion (Kirk
revealed that the relative rate of amino group reactiv- et al., 1988; Kirk and Scheller, 1986), influencing locomotor-
ity was C - N H ~ - L ~>S e-NHz-Lyss
~‘ >> a-NHz-Ile’. The related neurons in thepedal and pleuralganglia (Mackey and
slow rate of reaction of the terminal a-amino group Carew, 1983; Stuart and Strumwasser, 1980), and inducing
suggested that it was relatively inaccessible to bioti- release of ripe oocytes from the gonad (ovotestis) (Kupfer-
nylation, possibly due to conformational factors or to mann, 1967; Rothman et al., 1983). The release, packaging,
ion-pairformationwith an unidentifiedcarboxyl and deposition of the eggs are key events that characterize
group. Lossof bioactivity of ELH monobiotinylated on the complex behavioral actof egg laying (Strumwasser et al.,
the a-amino group, coupled with the unusually low 1969; Arch and Smock, 1977; Cobbs andPinsker, 1982).
reactivity of the a-amino group, provided strong evi- Although the complete expression of the egg-laying process
dence for the importance of the a-amino group ELH in may involve the coordinated actions of other peptidesreleased
function. Furthermore, the development and availabil- by the bagcells, injectionof purified ELH alone, nevertheless,
ity of a bioactive ELH probe should greatly facilitate can cause egg laying in a mature animal (Chiu et al., 1979).
the isolation, characterization, and localization of the Because of the centralrole of ELH inegg laying, a great deal
ELH receptor. of study into thebiochemistry and physiology of the bag cell
system has been undertaken (for reviews, see Rothman et al.,
1985; Arch and Berry, 1989; Conn and Kaczmarek, 1989).
Egg-laying hormone (ELH)’ isa basic, amidated 36-amino However, to date, little is known about the ELH receptor
acid peptide produced by the neuroendocrine bagcells in the largely due to the fact thata suitably labeled ELH probe has
not been available.
* This investigation was supported by National Institutes of Health In order to pursue more
in detail the mechanismsof action
Grants NS 29261 (to A. K.) and NS 07185 (to W. D. Willis, Jr.), by of ELH on its various target tissues, to identify its precise
National Science Foundation Grant BBS 8711368 (to J. E. B.), by
Grant H-1190 from the Robert A. Welch Foundation (to A. K.), by a
cellular target(s) in the gonad andelsewhere, to characterize
Biomedical Research Support Grant from the University of Texas the pharmacological properties of this molecule, and to aid in
Medical Branch Graduate School of Biomedical Sciences (to A. K.), the identification and characterization of its receptor(s), it
and by an award from the University of Texas Medical Branch Small would be of major benefit to generate suitablylabeled ELH.
Grants Program (to B. T. M.). The Florence and Marie Hall Endow- We report here a method for labeling ELH with biotin that
ment for Programs of Excellence in Education in the Medical Sci- provides a high yield of a biologically active and chemically
ences provided financial support of the Marine Biomedical Institute defined analog that can used be as a probeto investigate some
computer facilities used in this project. The costs of publication of
this article were defrayed in part by the payment of page charges, of the relevantissues addressed above. Importantly, the chem-
This article must therefore be hereby marked ‘‘advertisement’’ in ical modification of ELH with biotin has also revealed signif-
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
11 To whom correspondence should be addressed Dept. of Human N-biotinyl 6-aminohexanoyl N-hydroxy(su1fo)succinimide ester;
Biological Chemistry and Genetics, University of Texas Medical NHS-cAhx-biotin, N-biotinyl 6-aminohexanoyl N-hydroxysuccinim-
Branch, Galveston, TX 77550. Tel.: 409-772-2771;Fax: 409-772-4865. ide ester; A-ELH, ELH-relatedpeptide encoded by the A gene; HPLC,
The abbreviations used are: ELH, egg-laying hormone; NHS- high performance liquid chromatography; FAB/MS, fast atom bom-
biotin, biotin N-hydroxysuccinimide ester; sulfo-NHS-cAhx-biotin, bardment mass spectrometry; Pth, phenylthiohydantoin.

24413
24414 Biotinylation of Aplysia Egg-laying Hormone
TABLE I
Bioactivity and stoichiometryof biotinylated E L H derivatives
Derivative mol biotin/ Egg-laying Residue
fraction" mol ELHb bioactivity modified'
1 0 + None
2 1.01 k 0.02 (1) +
-
Lys36
3 1.01 ? 0.01 (1) LysX
4 1.94 -t 0.13 (2) - Lys', Lys'G
5 1.09 ? 0.05 (1) - a-NH2
6 2.06 k 0.07 (2) - a-NH2,Lys'"
7 2.26 k 0.10 (2) NDd o(-NH2,Lys8
8 3.01 2 0.09 (3) - a-NH2, Lys', and Lysj6
8' 2.73 ? 0.20 (3) -
From Fig. 1.
*Established by compositional analysis; n = 3 for all analyses,
except fraction 7, which was a duplicate; fraction 2was also confirmed
by FAB/MS to contain one biotinyl group and fraction 1 to contain
none.
Deduced from compositional and sequence analyses of biotinyl-
ated derivatives (see Table 11) and from compositional analysis of
tryptic peptides (see Fig. 3, Table 111).
ND, not determined.
Fraction 8 was reacted with hydroxylamine and repurified by
HPLC.

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, . .
0 60 65 70 75 SO 85
Elutlon tlme (mm)
t 15 mln afterb~ELHadditton
0 )
FIG. 1. Results of HPLC analysis of thebiotinylation of ELH
a s a function of time. The reaction of NHS-tAhx-biotin with ELH
(15 nmol, each panel) was carried out at a 40:l molar ratio at 25 "C
for 0 min ( A ) , 5 min ( B ) , 15 min (C), and 30 min ( D ) . Unreacted
ELH (fraction 1 ) and seven peptide derivatives (fractions 2-8) were
consistently observed at the elution positions indicated. The aceto- FIG. 7. Biotinylated L ~ s ~ ~ - E mimics
LH effects of natural
nitrile gradient shown was the same for all HPLC analyses. ELH on identified neurons. Neuron R15 ( A ) and a representative
LC cluster neuron ( B ) were recorded simultaneously in normal arti-
icant informationregardingregions of structurethatare ficial seawater. R15 was bursting normally and the LC cell was silent
critical to function. A preliminary report of this work has (some small synaptic potentials can be seen in the flat LC cell base
line). C, after approximately 15 min in 1 GM biotinyl Lys"-ELH ( b -
been presented (Knocket al., 1990). E L H ) , R15's bursting pattern was intensified with more spikes per
burstand larger interburst hyperpolarizations. D, the LCcell is
EXPERIMENTALPROCEDURES AND RESULTS' beginning to fire spontaneously after addition of biotinyl ELH. Re-
cording calibrations for all panels are shown in B : uertical, 20 mV;
DISCUSSION horizontal, 2 min.

Preparation of a biologically active labeled ELH molecule

t o be used in pharmacological, anatomical, and receptor stud- 2C). There was a n obvious difference in the relative reactivi-
ies has in the past been an intractable problem. The devel- ties of the NHS-biotin reagent when compared to NHS-cAhx-
opment of abiotinylated ELH probe as describedherein biotin. Under identical conditions, the NHS-biotin was con-
provides, for the first time, the capability of producing high siderably more reactive (compare Figs. 2A and 2 0 ) . Thus, the
yields of a biologically active ELH derivative that can be biotinylation reagent containing the spacer arm may be the
conveniently used as a labeled probe. Furthermore, the de- more suitable reagentfor chemical modificationstudies, since
scribed modification studies with biotin have provided addi- its slower rate of reaction may allow better discrimination of
tional insight into the structural features of ELH that are reactivity between amino groups.
critical to itsegg-laying function. We had previously obtained evidence that in the case of
The Vydac C18 column usedto fractionate the biotinylated certainpeptides, e.g. gonadotropin-releasinghormone, the
ELH derivatives provided excellent resolution of the deriva- seryl and tyrosyl residues could be 0-acylated under similar
reactionconditionsas employed for ELH.3 However, the
tives (Fig. 1).Even the two monobiotinylated derivates, bio-
tiny1 LysRand biotinyl Lys", were well separated (Fig. l B , biotinylation of ELH under the conditionsdescribed gave no
fractions 3 and 2, respectively). Increasing the molar ratio of evidence of significant 0-acylation (Fig. 4, Table I).
Chemical characterization of the biotinylated ELHderiva-
reagent:peptide from 4:l to 40:l resulted in increased forma-
tion of derivatives with more biotinyl groups attached (Fig. tives by amino acid compositionalanalysis (Table I), sequence
analysis (Table 11), FAB/MS (Table I), and analysis of the
Portions of this paper (including"Experimental Procedures," tryptic peptides (Fig. 3, Table 111), established the positions
"Results," Figs. 2-6, and Tables I1 and 111) are presented in miniprint of attachment of biotinylmoieties to the ELH molecule.
a t the end of this paper. Miniprint is easily read with the aid of a
standard magnifying glass. Full size photocopies are included in the ' B. T. Miller, T. J. Collins, G. T. Nagle, and A. Kurosky, submitted
microfilm edition of the Journal thatis available from Waverly Press. for publication.
 Biotinylation Egg-laying
ofHormone
Aplysia 24415
were biotinylated on the a-amino terminus, automated se-
quence analysis could not be carried out. This problem was
solved by tryptic hydrolysis of the two fractions, followed by
HPLC fractionation and compositional analysis of the puri-
fied peptides (Fig. 3, Table 111). Thus, it was established that
fraction 6 was dibiotinylated on Ile' and Lys:'" and fraction 7
on Ile' and Lys'.
The results obtained from the time course study of ELH
biotinylation were especially striking.Clearly, the order of
amino group reactivity was t-NHz-Lys:'6> c-NH2-LysR>> a -
NH,-Ile' under the reaction conditions described (Fig. 5 C ) .
In 30 min, essentially all of the Lys:16and about 85% of Lys'
had reacted; however, the a-amino group was only about 27%
reacted. Although the molar excess of reagent to peptide was
40:l (or 13.3:l per amino group), the amino group reactivity
was likely limited due to reagent hydrolysis by water. The
relatively slow reactivity of the a-NH2group when compared
FIG. 8. Electrophysiological effects of ELH andbiotinyl with the t-NH2 groups was somewhat surprising since, typi-
Lys"-ELH on neuron R15. The left column of traces A-E are cally, a-NH2 groups have significantly lower pK values and
samples from a cont,inuous chart recording. The bursts marked by are usually more reactive than t-NH, groups. This difference
arrows are illustrated in the right column as expanded oscilloscope
tracings. A, control; B, 12 min after addition of synthetic ELH at a
in reactivity may be the result of a secondary structural or
bath concentration of 10 FM. Note the large and prolonged interburst conformational effect or may be due to the formation of an
hyperpolarizations and short but intense bursts. There is also en- ion pair between the a-NH, group and some as yet unidenti-
hanced background synaptic activity from interneurons. C, control fied carboxyl group. Additional support for the involvement

Downloaded from www.jbc.org by on August 14, 2006

period 29 min after starting wash out. The cell has returned to a of the a-aminogroup in some significantstructural motif was
firing pattern similar to that in panel A , although the bursting is indicated by the later elutionof the monobiotinyl a-NH,-Ile'
slightly more intense.L), 20 min after addition of hiotinyl Lys:''-ELH
at a bath concentration of 10 FM. The effect was indistinguishahle derivative (fraction 5 ) relative to the othertwo monobiotiny-
from that causedby native ELH (compare oscilloscope tracings in R lated derivatives in fractions 2 and 3. A reasonable explana-
and D). E , 1 h after starting washout of biotinyl Lys:"'-ELH. Recording tion for the significantly laterretention of the fraction 5
calibrations are as shown in panel D.Chart recordings: uertical line, derivative is that biotinylation of the ol-amino group would
20 mV; horizontal line, 10 s. Oscilloscope tracings: verticalline, 20 disrupt ion pair formation and/or other structural elements,
mV; horizo,nta:l line, 500 ms. The straight line referen,ce in the oscil-
loscope trac:in gs was arbitrarily set a t -36 mV.
resulting in a more unfolded peptide that would elute later
during HPLC. The atypical a-NH, group reactivity and pro-
jected involvement in conformationor ion pairing may explain
1 the importanceof the amino-terminal region to thebiological
activity of ELH. Our results demonstrated that all biotinyl-
ated ELH derivatives modified at the amino terminus (also
Lys') were not bioactive in the egg-laying assay (Table I). In
addition, it was reported that removal of the terminal Ile of
ELH togenerate ELH-(2-36) also results in loss of egg-laying
activity (Strumwasser et al., 1987; Strumwasser, 1988). There-
fore, both chemical modification and residue deletion experi-
ment,s underscore the import.ance of the amino-terminal re-
4 10 min alter b-ELH addltlon gion for ELH activity. Our results emphasized specifically the
importance of the a-aminogroup itself.Taken together, these
observations should prove helpful in interpreting the three-
dimensional structure of ELH when it becomes available.
Of the seven biotinylated ELH derivatives obtained in this
study, only the Lys:'" derivative was biologically active. The
bioactivity was essentially identical with that of ELH, as
evidence by egg-laying activity, as well as by measurement of
."
electrophysiological activity using LB andLC cluster neurons
+ 10 mln after ELH additlon 2 and neuron R15 (Figs. 7-9). From these results we concluded
that theLysti6 sidechain was not critical to the bioactivity of
FIG.9. Intracellular electrophysiological recordings of ELH. Thisconclusion was supported by the fact that A-ELH-
neuron L8 following addition of ELH and biotinylated ELH. like peptides and the caudodorsal cell hormone, which are
Neuron L8 exhibited a moderate level of spontaneous activity that ELH homologs, all contain leucine at position 36 (Nagle et
was significantly increased in the presence of either 2 ~ L Mbiotinyl
Lys:'"-ELH ( b - E L H )( A ) or ELH ( B ) .The latency to onset of effect al., 1989). Moreoever, experiments involving residue deletion
for either peptide was less than 10 min, and the effect persisted for at the carboxyl terminus using synthetic peptidesdemon-
over 1h for each peptide.The records, fromtop to bottom, are samples strated that ELH-(1-35) amide and -(1-34) amide caused egg
taken from a continuous recordingof over 3 h. The calibrations inR laying (Strumwasser, 1988).
also apply to A: vertical, 20 mV; horizontal, 1 min. Finally, these studiesunderscore the utilityof biotinylation
reagents as excellentchemicalmodificationreagents. This
Initially, there was some uncertainty concerning which lysyl point was emphasized inthe observed relative rates of reaction
residue was specifically modified in the case of the two dibi- of the three amino groups contained in ELH. The reactivity
otinylated fractions, 6 and 7. Since both of these fractions of NHS-cAhx-biotin appears to be sufficiently slow to allow
24416 Biotinylation Egg-laying
ofHormone
Aplysia
differentiation of relative reactivities within a reasonable time Mayeri, E., Brownell, P., and Branton,W. D. (1979a) J. Neurophysiol.
frame and reaction conditions. Further experiments are un- 42, 1185-1197
Mayeri, E., Brownell, P., Branton, W. D., and Simon, S. B. (1979b)
derwaytodocumentobservedreactionrateconstants of J. Neurosphysiol. 4 2 , 1165-1184
amino groups using these reagents. Mayeri, E., Rothman, B. S., Brownell, P. H., Branton, W.D., and
Padgett, L. (1985) J. Neurosci. 5 , 2060-2077
Acknowledgments-We wish to thank Dr. ShermanPernia for Merrifield, R. B. (1963) J. Am. Chem. SOC.85, 2149-2154
peptide synthesis, Terrell Stamps for computer graphics, and Ange- Nagle, G. T., Painter, S. D., Blankenship, J. E., Choate, J. V. A,, and
lina Mouton for preparation of the manuscript. We are especially Kurosky, A. (1988a) Peptides 9,867-872
grateful to Drs. K. Biemann and I. A. Papayannopoulos of the MIT Nagle, G. T., Painter, S. D., Blankenship, J. E., and Kurosky, A.
(1988b) J. Biol. Chem. 263. 9223-9237
Mass Spectrometry Facility for FAB/MS spectra. Nagle, G. T., Painter, S. D., and Blankenship, J. E. (1989) Biol. Bull.
(Woods Hole) 177,210-217
REFERENCES Nambu, J. R., and Scheller, R. H. (1986) J. Neurosci. 6,2026-2036
Arch, S., and Berry, R. W. (1989) Brain Res. Reu. 14, 181-201 Newcomb, R., and Scheller, R. H. (1987) J.Neurosci. 7,854-863
Arch, S., and Smock, T. (1977) Behau. Biol. 19,45-54 Newcomb. R.. Fisher. J. M.. and Scheller. R. H. (1988)
. , J. Biol. Chem.
Barber, M., Bordoli, R. S., Sedgwich, R. D., and Tyler, A. N. (1981) 263,12514-12521'
J . Chem. SOC. Chem. Commun.7, 325-327 Painter. S. D.. Kalman. V. K.. Nanle. G. T.. Zuckerman. R. A.. and
Blankenship, J. E. (1985) J.'Mor>hbl. 1 8 6 , 167-194 '
Branton, W.D., Arch, S., Smock, T., and Mayeri, E. (1978) Proc. Rock, M. K., Shope, S. B., Blankenship, J . E., and Schlesinger, D. H.
Natl. Acud. Sci. U. S. A. 7 5 , 5732-5736 (1986) J. Neurobiol. 1 7 , 273-290
Chiu, A.Y., Hunkapillar, M. W., Heller, E., Stuart, D. K., Hood, L. Rothman, B. S., Weir, G., and Dudek, F. E. (1983) Gen. Comp.
E., and Strumwasser, F. (1979) Proc. Natl. Acad. Sci. U. S. A . 7 6 , Endocrinol. 52, 134-141
6656-6660 Rothman, B. S., Mayeri, E.,and Scheller, R. H. (1985) in Gene
Cobbs, J. S., and Pinsker, H. M. (1982) J. Comp. Physiol. A Sens. Expression in Brain (Zomzely-Neurath, C., and Walker, W. A., eds)
Neural Behau. Physiol. 1 4 7 , 523-535 pp. 235-274, John Wiley & Sons, New York
Conn, P. J., and Kaczmarek, L. K. (1989) Mol. Neurobiol. 3, 237-273 Scheller, R. H., Jackson, J. F., McAllister, L. B., Rothman, B. S.,
Dakshinamurti, K., Landman, A. D., Ramamurti, L., and Constable, Mayeri, E., and Axel, R. (1983) Cell 3 2 , 7-22
Sigvardt, K. A., Rothman, B. S., Brown, R. O., and Mayeri, E. (1986)
R. J. (1974) Anal. Biochem. 6 1 , 225-231

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Kirk, M. D., and Scheller, R. H. (1986) Proc. Natl. Acud. Sci. U. S. Smith, J. S., Miller, B. T., Knock, S. L., and Kurosky, A. (1991a)
A. 83,3017-3021 Anal. Biochem. 197,247-253
Kirk, M. D., Taussig, R., and Scheller, R. H. (1988) J. Neurosci. 8 , Smith, J. S., Miller, B. T., and Kurosky, A. (1991b) Anal Biochem.
1181-1193 197,254-257
Knock, S. L., Miller, B. T., Kurosky, A., Nagle, G. T., and Blanken- Stewart, J. M., and Young, J. D. (1984) Solid Phase Peptide Synthesis,
ship, J. E. (1990) SOC.Neurosci. Abstr. 1 6 , 1030 2nd Ed., pp. 85-89, Pierce Chemical Co., Rockford, IL
Kupfermann, I. (1967) Nature 216,814-815 Strumwasser, F. (1988) Current Top. Neuroendocrinol. 9 , 105-122
Kupfermann, I., and Kandel, E. R. (1970) J . Neurophysiol. 33,865- Strumwasser, F., Jacklet, J. W., and Alvarez, R. B. (1969) Comp.
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Strumwasser, F., Schiller, D. L., andKent, S. B. H. (1987) SOC.
Levitan, E. S., Kramer, R. H., and Levitan, I. B. (1987) Proc. Natl. Neurosci. Abstr. 13, 38
Acad. Sci. U. S. A. 8 4 , 6307-6311 Stuart, D. K., and Strumwasser, F. (1980) J. Neurophysiol. 43, 499-
Mackey, S., and Carew, T. J. (1983) J . Neurosci. 3,1469-1477 519
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and Scheller, R. H. (1985) J. Neurosci 5, 1872-1880 iol. 43,488-498

EXPERIMENTAL PROCEDURES AND RESULTS Biorinylorion Reocrionr - For most of the expcrmcnts described. E L H was chemically modified with
NHS-rh-biotin. On O E C ~ S I O NHS-biotin
~, and rulfo-NHS-.Ah-biotin wcre also employed as dircurrd 10
the text. Sulfo-NHS-Ahx-biotin wasweighed and added to the reaction solution ar a dry powder whcreai
Mrrrerinlc - Tritluuroaeetie aad (HPLC Grade), sulfo-NHSt&-biotm, NHS-Ax-hiotin. NHS-biotin,
NHS-Aw-blotin and NHS-biotin were dissolved in Nfl-dimcthyllormamide just prior to addition to the
and avidiwagarorewere purchasedfrom Pierce ChemicalCo. Acetonitrile (HPLC Grads) and hydroxylamine
reaclion mixture. Reactions were lypically carried out in 0.05 M NaHCO,, p H 8.2, a1 ZS'C for 30 minor ar
hydrochloride(Certified Cradc) were obtained from Fisher Scientific. Vydae C18 (5 ym particle sire; 300A
specified. Rsaetion~were stopped by lowering the p H to 6.0 with the addition of 1 ml of 20% acstonilrile
pore diameter) reversed-phasecolumm (4.6 mm x 25 cm; phase 218Tp) and direct mnncct guard mlumm
in 0.1% TFA. T h e pH-adjusted reaetmn mixtures were subsequently injected directly onto the C18 HPLL
containmg Vydac CIS (5 pm particic size) eartrldges were purchased from Alltech Applied Science Labs
column equilibrated a1 23% acetonitrile in 0.1% TFA.
N,N-dimethyiformamidc.acctyicholinechloridc.and biatm were obtainedfromSigmaChemica1 Co.['*C]biotin A separatc set of experiments war conducted to observe the time-course of reaaion of E L H with NHS-
(40-60 mCi/mmol) was a product of Amersham. Reagents for amino acid mmporitional and wquenec
Ahx-biotin. The molar ratio of reagsnt:psplide was 401. Other reaction mnditionr were the Same as
analyses. includmg HPLC of Plh-amino acids. were from Applied Biosystems. [ne. r-Bulyloxyc'arbonyl amino
staled above.
acids and Other chemicals for peptide synthesis were purchased from Biosearch. Lyophilized trypsin war a
HPLC - Rearlion mixtures containing biotinylatcd E L H were applied to a Vydac C18 reversed-phue
product of Bwhringer Mannheim. Aptpie califomica (200-300 g) were obtained from Alacrily Marine
HPLC mlnmn and eluted with a linear gradient of ~olvcntA (0.1% 1"fluoroacctie acid) andsolvent B (100%
Biological S e ~ e e s(Redondo Beach. CA).
acetonitrile containing 0.1% trifluoroacetic acld) at a flow rate of 1.0 ml/min. The standard linear gradient
ELHSynrherir -Synthetic E L H was prepared in the Biosynthesis Laboratory a1 the Univcrslly of Teras
smploysd to fractionate E L H and 11s biotinylatcd derivatives was 2343% wkent B i n 100 min at Z'C. The
Medical Branch by solid-phase synthesis (Mernfield. 1963) using a fully automated synthesizer (Bioseareh
column eluate was monilored at 215 nm and 0.5-minfractions wsre collected. Fractions were pooled hared
Model 9600)and employing r-bulyloryearbonylchemistry. Reactive amino acld side chains were protected
on absorbance and were rvhjcctcd to amino acid compositional analysis. automated microJequsncs analysis.
ar follows: Asp and Glu with 0-benzyl ester; Scr and Thr wilh benzyl ester; Tyr with 2-
FAB/MS. and bioassays. A l l fractions were dried by vacuum centrifugationand stored at -2OC.
hromobenrylorycarbonylester; k g with 4-ralucnesulfonylester; andLys with 2-ehlorobcnzyloxyearbonyl
Amino Acrd Analysi$ . Sampler were applied directly to an Applied Bioryrtems
420H
errcr. T h e pepfrdc was r~multaneourlydeprorccted and cleaved from the resin by treamcm with H F (Stewart
(derivatizer/hydroly2er) amino acid analyzerwilh on-lme actd hydrolysisand pre-columnphcnylthmcarbamyi
and Young. 1984). Synthetic E L H was subsequently puriflsd on a Vydac C18 reversed-phase HPLC mlumn
derivatiration. T h e number of biotin groups ~ o v a l ~ n tbound
ly to pcptidcs war determined by amino acid
as de5crihcdbelow usmg a linear gradlent of 0.1% trifiuoroacctic aeldand aceLonitrile containing 0.1%
analysis of the 6-ammohexanaic acid spacer arm portion of the sulfonated and non-suifonated N H S - t h -
trifluoroacetic acid. E L H authenticity was confirmed by amino acid compar~tianal analysis
and
btotin rellgentr (Smith ef nl.. 1991a).
mmrorequencing (rcsuIts not shown) on an Applled Binsystems Modci 475A sequencer.
 Biotinylation of Aplysia Egg-laying Hormone
Ar,omurr.d Psptrde S q ~ r e r # c eAndyrir - Pumrry wucturdl mnlyr~swa\ drtermcncd urmg an Applied
Eio\y$rcms47% protcin/peptide mirrorequenrcr with an o n - l m M d d 120A mirrohore phenylthwhydantoin
amino a c d analyzer and a Model 900A drla prucerror as previously descrihed (Nagle ef ol., lY88hl. p -
,Ah*-hiotinyl-Lyr war ldentihed and quantified as described hy Smith el ul. (1991h).
Mars Sprcrrom~rry- Mars rpertromctrie analyses were performed at the Mass Spcctromclry Facil~ly.
Department of Chemistry.Mawachusettr Institute of Teechnolugy. Cambridge.Massachusetts. FABfMS
analysis of ELH and its hiottnylnted derivative was carred out in the first of two mass spectrometers of a
tandem high-resololution mass spectrometer (JEOL HXIIOfHXIIO) a$ prev~ourlydescrihed (Barher cf ul..
1981)
Hyhqlamme Rpocfion .Hydroxylamme hydrochloride ~ o l ~ t l o n
(0.5
s M in 0.2 M trimethylamine) were
adjusted to pH 9.0 with NaOH. HPLC-purified and lyophillzed. hiolmylated ELH derivatives were d ~ o l v c d
in 1M pl of 20% acetonitiil~and 50 +Iof thk m m u n was added 10 0.45 ml of buffered hydroxylamine
T h. e reaction was mired and allowcd to proceed for 4 h at 25°C. The p H was tested at the end of
~Iut~m
!hecncuhation period to verify that i t had rcmamcd unchanged. Trifluoroacetir acod (500 PI of 0.1%) was
then added to the reac~ionmixture prior TO >nlectlon onlo a C18 revendphase HPLC wlumn. For a
comparative control. !he remaming Sop1 of each biotmylatcd ELH derwative war chromatographcd withoul
hydrnxylamme treatment.
Tppyp,ic Hydrotym - ELH and hiotinylated-ELH dcrtvativer were hydrrblyzed with trypsin at an enzyme
to ruhitnle ratio of 1:lO (w/w) for 8 h at 3 7 T ~n2SU PI of 0.05 M trls-HCI buffer, pH X.1. After reaction
the sampler were acidified to pH-3 with lr!fluoroaeetic acid and appled directly 10 P C18 reuerxd.phase
HPLC column. HPLC conditions were a$ descrihed above and included a h e a r gradient of 8 4 5 % solvent
B o v a 135 mm followed hy 45.100% 101vent E for IO min Pooled fractions were lyophilized and subjected
to amino acid analysis.
Avidin Bhdlng Arrq - To establish whether or not hiotinylated ELH derivatives would hmd avidin.
we employed a mmpetitive binding assay wich ['%]biotin smilac Is that deswbed hy Vskshmamurti el el
(1974). Avidin-eoatedagarose berdr W C ~ Fadded 10 0.01 M phosphate bufferedsaline. p H 7.5, and mcuhated
with EU1-Lyr-3kAhr-biotin. unmodified ELH. or excess hioun for 5 min at 2YC. ['Tlhiotin (-1.4x I(P
dpm) WPI then added and incubated for IO min. Thhe beads were mllscted by vacuum filtration onto glasz
filten and washed er.tensively with phosphate buffered saline. After a final wash with 100% ethanol, howd
radiomtivily was determined m a Tracor Analytic Scintdlalian Counter. Significanl differences in bindmg
between groups were determined by analysis of variance.
Egs-LayingAsray - Aplpcacdifmnco were maintained inlarge aquariaconta~nmgrccirculaiingarrificial
seawater at 14 f 2°C for several days before experimentation. A l l of the animals were sexually mature and
could lay eggr ~n rcrpanre to m p i o n r of atrial gland extract within 2 h (Painter el d.,1985). Lyophilized
pepttdcs were fmst dissolved in 50 4 of 2090 ethanol. dduted with arlhcial scawatcr Io 0.5 ml, andthen
injected through the neck rnm the hemocoel. As a con~rolmeaswe, animals that did not lay eggs within 2
h were then injected with atrial gland extract.
ElKtmphyrlolo@cd Effmfs on Idenrlfied Newom - Individual abdominal ganglia were pinned. dona1side
up, in a 300 PI Sylgaid-mated chamkr. The avedying connective sheath was gently removed vrith a razor
blade sliver and the exposed ganglion was superfused at 2S'C with artificial seawater at a rate of ahout 1
ml/min. The raft war often slwed or stopped for apprormatsly 9-15 mi" at the time Of peptide application
and then resumed. No protanare inhibitors were utilized. Neuron RIS and for one of the LB or LC clu~tcr
neurons ~n the left Iowcr quandrant of the ganglion were penetrated for intrarrllular recording using
procedures prcviourly described (Rock el u l , 1986). L B / K neurons were identified by their topography, size.
color. rpantancaur aelivily, and m some cases. response IO applied acetylcholine. ELH sampler in artificial
seawater (25 to 250 4 ) were popelled into the hathlng medium directly ahhove the ganglion. giving a finalbath
concentration of IOb M 10 10' M.
RESULTS
Biolinylorwn of ELH - T h e lime-course reaction of ELH with NHS-ch-biotm was evaluated by HPLC
as shown in Figurc 1. Multoplc peptide derivatives were wmitiltsntly observed at the elution poritiom
illustrated durlng the chromatography of many different hiatinylatmn reaction mklurer carried out under
valmus reaetmn condniom. T h e relative amount of each derivative formed.however. was dependent on
biotinylation reaaion mnditionr. Amino acid wmposilional analysis indicated that eachpeak contained a
pcptide identical in campition to the origlnal E L H Compasitionalanalyses of fractions 2 through 8 gavc
positive wnfirmalion for the presence of bmtm whereas fracnon I. whwh eluted with the Same retention time
as the unmodified synthetic ELH. did not Contain biotin (Table I). Within the first 5 min of the reaction,
denvativcr in franions 2, 3, and 4 occurred on relatively large amounts (Fig. LB). By I5 mi& derivatives in
fractions 4. 6, and 8 had Increased and there was a concomitant decrease in fractions 2 and 3 (Fig. IC).
A comparative series of reactions of ELH with NHS-&-biotin at dtfercnt molar ratios of
reagcn1:pcptide (+I, 8 1 . and 401) resulted in the HPLC derivative distribution rho- in Figure 2. The
reanion of E L H with NHS-biotin (without <Ah*) under ldsntical condmons as for NHS-dmbiatin (Fig. 2.4)
resulted in a greater degree of hiorinylatwn as evidenced by the increase in relatwe yields of later eluting
ELH reaction products (Fig. W ) . The predominantderivatives obtained were dihiotinylared (Lys'and Ly?;
fraction 4) and tribtalinylalcd (e-NH, Lyra, and Ly?: fraction 8). There results Indicated that the NHS-
hmin reagent was more reactive than the NHS-Ahx-biotin reagent andfor less rusceptihlc to hydrolysis by
water. On the other hand, biotinylation prof~lesusing sulfo-NHS-Ah-blotin were very similar to those using
the "an-sulfonated reagent. NHS-rAhx-hiotc (rcsullr nut ihown).
24417
M~~ Speom,,wnc Anvlyru. HPLC-purifwl synthetic ELH and hiutinylatcd ELH (fracrlans I and 2.
8)were anslyzedby FABfMS. The d8aina molecular ion clusters observed in these
re\pectively: Fig. 1
analyses embitshed rhe nurnher uf hmtin moieties present in these Two fractions (Fig. 18). The spectNm uf
syntheuc ELH indicated a protonated mole~uleat m/z 4385.1. consonant wth the SlNClUrC of ELH (results
not shown) Similarly, the ipcctrum of fraction 2 indicated a major ion at m/r 4724.5which w u in agreement
wth the calculated value for monobiotinylated ELH.
Ammo Acld Comporltionol and sequence Andyscs. T h e stoichiometry ofhmtinyl groups bound per mol
ELH dsriwttvc was quantitated hy analysis of 6-ammoheranoic acidin fractions 1-8 (Fig. LB:Table 1) using
pre-column denvatizauon ammo and analysis (Smith el "1.. 1P91a). Compos~ttonalanalysis gave identical
results as that obtained from F4B/MS for fractions 1 and 2 which provided solid evidenrr that fraction 2
eontamed monobiotinylated ELH (Table I).Microsequence analyss demonstrated that this derivative was
monohiotinylatcd at Lyi" (Table 11). Tho peptide was not blocked at the amino ternnus and N6-Ahx-
biolinyl Pth-Lyb wa5 not detected at cycle 8 hut was detected at qcle 36 (Smith el d..1991b).
Curnpor~tionalannlyr~rof the hialinylatcd ELH derivative in fraction 3 (Ftg. Is) indicated that this
fraction wa5 a l m monohlotinylated(Tahlc I). Microsequence analysisdcmonslrared that this derlvative was
bmtinylrted at Lyr'(Tablc 11). Similar analys~sof the hiotinylated derivative I" fraction 4 (Fig. 18)indicated
that this fraamn was dihminylated (Table I).
Microsequence analysis revealed that
it was unblocked and wa-
hiut~nylatedI I Lyse
and Lys" (Table ll). The hiaunylaled derivative I" fraction 5 (Fcg. IS) w&%
munuhiotinylated at the amino terminus since microsequence analysis of 550 pmol indicated only 8.7 pmol
uf I l e confirming that lhls derivative war hlocked. Comporitional analysis of fraction 5 milcared one hiotln
momty (Table I).Similarly. the dihiotinylated derivatives m fractmns 6 and 7 (Fig. LB: Table I) were also
blocked ai ihc ammo lcrmmus (II2 pmol and 393 pmnl \cqucnred, rerpectiuely; 2 8 pmol and 23 pmol lie
recovered, respectively). S~ncewe were unable to unamhiguuusly distinguish whichof thc lysyl residues w a
rpeciflcally hmtinylalcd m fractions 6 and 7 by sequence analysis, lheac two derivatives were ruhscquently
subjected 10 trypuc hydrolyss and fraaionation by HPLC (Fig. 3). Compositional analyrls of the wlatcd
peptides cunflrmed fhal fraction 6 war dihmmylated on llc' and Lys" whereas fraelwn 7 was dlhrutmylated
on Ile' and Lys'(Tah1e Ill) Finally. compositional analysis ofthe derivative tn fraction 8 (Fig 18) Indicated
that this der8Yatwe war rribiulinylated (Table I).Mtcrosequence analysis confirmed that it was blocked due
10 hiotinylation of the amino tarmmus (136 pmol sequenced,4.7 pmol Ile rccmcrcd).
Hydrolylrrmlne Tr.?mmenr of Blotinylufed ELH Detivoriver. To verify thatthe hiotinyl moieties were
attached v u amide linkagesrather than by ester linkages to hydroxyamino acids. as shorn for gonadotropin-
Downloaded from www.jbc.org by on August 14, 2006
a mlxlure of hmbnylated ELH (Ftg 4 4 ) was reacted with hydro+mtne
releasing hormone (Miller e, d.'),
at alkaline p H and then whlected to chromatography by HPLC (Rg. 48). Additionally. HPLC purified
derivatives 2-5 and 8 were individually reacted with hydroxylamine and rschromatographcd (asan example.
see Fig. 4C). There experiments demamtrated that none of the h~otmylatedderivatives in these fractionswere
hydroxylamine-lah$lcindicating that thew was no significant 0-acylation of Ser' or Ty? under the reacfmn
conditiom employed.
Time-course of BiorrnylorlonofELH HPLC analysis of the reaction products of ELH biotinylation with
ume indicated a rapid decrease ~n ELK with a concomitant rapid increasc 1" monobiottnylat~onforming
derivatives 2. 3 and 5 (Rg. SA). At later time points, the recovery of these dewatives dccreavd somewhat
as they were converted to the dihiotinylated derivatives 4.6.and 7. and finally lo the tribiotinylated derivative
x (Fig. 58). The tom1 HPLC recovery of 811 modtfied and unmodofied ELH spccier decreasedsomewhat
(-7.5%) with lnercarlng reaction time (Fig. SA). Ths reduction I" recovery after HPLC was likely due IO
the increase ~n peptide hydrophobicity P I more hiotinyl groups were added to the ELH moieculc. The
combined recovery of hiutmylated ELH modified at Ly? Lyra,or the amino Icrminus is Shawn in Figure SC
and demonhtrateda relatirely rapid hiotinylamn of Lys" and to a lesser ~ x t e n tLyra. The bootmylationof the
u s rurprwngly low and was ahout27% that of Lyr' after 30 mi"
ammo f ~ r m ~ n was Thhe Iummalion rccoverlec
for biotinyl Lys-36 and biotinyl Lyr-8 shown in Figure 5C did not include fractions 6 and 7: however. as
lndicatcd m F~gure58, the 0vcraII contnhution of these fiaFtionS was relatively small (<5%1 Derivatives
m fractions 6 and 7 were moslly converted Io the trib~otmylatcdform after 15-20 min of reactlon.
Avidin 8~nd;ngRrsq. The ahilily of hiotinyl Lyr-36-ELH to bind to avidin and compete with biotin for
binding to avldin is shown in Figure 6 . Excess cold biotin and biotinyl Lyr.36.ELH rignifieantly reduced the
ahmy of ["Clhmtin IO hind to awlmagarose beads (p<O.OS), whereas unmodified ELH did not inhibit
["C]biotin binding.
Egg-Loymg Bioauq. Synthetic ELH was found to he equipotent to native ELH in an egg-laying
bioassay. Biatinylatsd derivatives 2-6 and 8 obtained by HPLC purification of products of the reaction of
NHS-Ahx-biotin with ELH (Fig. ZB)were tested for egg-layingaclivlly by inlection of 1.5 m o l into recipient
animals. The E L H derivative in fraction 2 consistently elicited egg laying (n =3) whereas all other derivatives
were inactive at the same dow (n=3). Subsequently. fraction 2 andsynthettc ELH were mmparativcly
bioawaycd using 0 5. 1.0. and I 5 n m d of peptide Both produced tg laying at 1 0 and 1.5 nmol but no1 a!
0 5 "mol indlcaringthat derivative 2 wa equipotent lo synthetic ELH. Two days later. ind~ndual
animals that
were injected with fraction 2 were tested with the same dose of synthetic ELH and thore mnjected with
synthetic ELH were thentested with fraction 2. Under these mnditions. fraction 2 and synthetic ELH
produced an egg-laymng rcrponse at the same dose (1.0 nmol). A l l peptide concentrations were determined
hy ammo acid analysis.
Electroplyslolo~colEffects on Identified Neumnr. The comparative hioacuvily of hiotinyl Lys-36-ELH
and ELH was also lcrled eleelrophyriologically.Peptideswere applied ID the bath surrounding theahdommal
ganglion whlle recording from identified neurons whose response to ELH has heen well documented (Mayeri
el d 1979a,h, 19x5). Peptide\. soluhllmcd in 1U% ethanol and diluted with artiIiCia1seawnl~r,were applied
24418 Biotinylation Egg-laying
ofHormone
Aplysia
ID the bath by automalcc plpeucr; concentrationsgiven were final for thebath. Vehicle alone (artificial
seaya~erand ethanol) had no effecl on the neurons. Blotmy1 Lys-36-ELH had effects on cell RIS and LB
and LC cluster neurons indistinguishablefrom those causedby a bag cell afterdischargeor by ELH. As show
in Flgure 7. biolinylatcd ELH (1 r M ) produced more inlenle firing of Fel RIS and induced finng in an LC
c l u s w neuron. The number of spikes per hunt and the interbunt hyperpolarizationincreased in RLS. and
the previously silent LC cell began to fire steadily. There c f f e a psrrirtcd for aver I h. Figure 8 illustrates
results from another experiment i n which a comparison was made of the effects of ELH and biolinyl Lyr-
36.ELH on cell R1S In this case. IO ELH and biotmyl Lyr.36-ELH eachcaused comparableand strong
madhcations in the firing of lhir cell. AI there concenlratms each peplade caused a greatly accentuated
intorburst hyperpolarinatton and the cffsetr perrizted for prolonged pciiodr. Cell L8. Icprcscntative of the
LB cluster. was a150 equivalently rtmulatcd by equal concentratiem of either bmtinyl Lyr-36-ELH or E L H
(Fig. 9). In this experiment neuron LE displayed moderately hbgh sponlancour. irregular firing that w s often
inlcrruplcd by interneuron activiry. After several minuter m the prcsencc of 2 rM biotinyl Lyr-36-ELH or
synthetic ELH. Ihe ftring pnltern was changed to prolonged b u m of interne firing that continued for aver
1 h after washout Thus, comparable results with ELH and biotinyl Lys-36-ELHon LB/LC nCUrON and R1S
were seen in three Of three experiments

Retenllon Tlms (rnlnl

of lypllc hydrnlyrir of HPLC fractions 6 and 7 (Fig. 1). A) Hydrolysis

F& 3. Summary 01 RSUIIS
produrn were fractionated by C18 reversed-phase HPLC and identified by amino acid analysis. Potential

klr""' cleavage sitcs arc indicated by vertical arrows and theoretical resulting peptides are designated "a"through
"g". Actual tryptic peptidcr obtained from hydrolyzates of HPLC fractions I. 6, 7, and 8 are as indicated.
Dipeptides corresponding to regions "c", '"d".and "g" were not idenltfied and likely eluted early with (he salt
fraction. HPLC fractions 1 (uobiotinyhtcd) and 8 (sibiotinylatcd) served as controls to establish reternion
times and mnfirm susccptibllily to trypsin hydrolysis. B) Comporlre of observed retention times of tryptlc

Downloaded from www.jbc.org by on August 14, 2006

peptides shown in (A). Relative rCCovCries were normalized to pcpride '"e"which was identical in comporillon
for all four HPLC fraclionr. Identical rctcntion timesand mmpritianal analyses (see Table 111) were
obJerved for Ihc follawingtryplicpcplidcs: Lg=7g: Lc=6e=7e=&;6g=8g; ib-6b: and 7atm=8a+8b.

,. . . . .
0 60 65 70 75 80 85
Elutlon time(min)

F@ 2 R~rullrof blollnyl~llm
ofELH at vwlaur reagent:pcplldc ntlos. HPLC analyses of the reaetion
(30 min. 292) of NHS-rh-biotin with ELH at molar ratios of: A) 4:); 8 ) SI; C) 401: and D) HPLC
analysis of thc rcactlon (30 min; 25°C) of NHS-biotin with ELH at a 4:l molar ratio yielded primarily two
pcptidc derivatives as shown.
 Biotinylation Egg-laying
ofHormone
Aplysia 24419

Fraction 4

YlJ 1334 lUl5

504 916 687
IS1 336 270
429 773 6W
120 626 531
328 603 480
288 333 374
367 550 470
311910 11/56l 01494
376 528 513
31Y 464 443
11 Thr 11>1 30x 253
12 A\p I83 242 240
13 hkl ?1S 4117 355
- l J . . . . . . 14 1.C" ?23 343 319
15 1.C" 2115 357 395
16 lhr 1117 177 192
17 GI" u
lx 1x1 is5
IS GI" 12Y 215 201

bzz
1v Ile I26 211 162
Illy I19 148
75 YY 86
Ill3 120 160
X8 146 11Y
I4 I24 150
S2 153 131
Y3 135 ill
71 135 123
2 62 95 84
66 ~ox 97
54 874 103
0 51 Y4 74
;-11 : : : : : : 3s 71 91
0 5 10 20 15 25 30 IJ 20 17

Downloaded from www.jbc.org by on August 14, 2006

Reaction Time (min) 2x 17 21
8 I2 Y
I117 410 0/10
Fig, 5. Tlrnesourse or moverier of biotinylalrd ELH anrr mac1ion with NHS-tAhr-biolin. Fractions
defined in Figure 1 arc shown as PI-F8. A) unmodified ELH. monobiotinylatedderivatives, and total ELH-
related peptide recovered: B) dl- and rribiotinylated derivatives; and C) ~ u m a t i o n
recovery of unmodified
ELH and ELH modified at L p n , Lyr', and amino terminus for all derivatives.

Amlno a c d 64 6b 6e 6g 7ai7h 7g

0 11.1
0.9 ( I ) 1.1 (I)
0 0.1
nl 0.2
n 0
11 0
0 0. I
O 0.1
n 0
n n
0 0
Methionme 0 0 0
6-aminohexanoicrcd' 1.0 ( I ) 1.0 ( I ) 0.1
lP"le"Clne 1.9 (2) 0 0. I
Leucine 1.1 (I) 2.0 (2) 2.1 ( 2 )
Phenylalanine 0 0 0
Lysine 1.0 (I) 1.0 (1) 1.0 ( I )

Control Blotin ELH bELH Total 8 11 6 4 19 4

Fig, 6 Blnding of blotinyl LyrJ6ELH l o avidin. Mearurement of competitive inhibition of binding of
["Clbiotin to avidin-caaIed agarose beads in the presence of no competitor (control), unlabeled biotin.
unmodified ELH. and biotinyl Lys-36-ELH (b-ELH).