THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 24, Issue of August 25, pp.

11768-11775,1988
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Antigen-Antibody Interaction
SYNTHETICPEPTIDES DEFINE LINEAR ANTIGENIC DETERMINANTS RECOGNIZED BY MONOCLONAL
ANTIBODIES DIRECTED TO THE CYTOPLASMIC CARBOXYL TERMINUS OF RHODOPSIN*

(Received for publication, January 25, 1988)

Robert S. HodgesS, Robin J. Heaton, and J. M. Robert Parker

From the Department of Biochemistry and the Medical Research Council of Canada Group in Protein Structure andFunction,
University of Alberta, Edmonton, Alberta T6G 2H7,Canada

Laurie Molday and RobertS. Molday

From the Department of Biochemistry, The University of British Columbia, Vancouver, British Columbia V6T 1 W5

The specificities of four monoclonal antibodies rho nikov et al., 1982; Hargrave et al., 1983), a model of rhodopsin
1D4, lCS,3A6, and 3D6 prepared by immunization of structure has been proposed in which the polypeptide chain
rod outer segments containing rhodopsin have been spans the membrane lipid bilayer seven times. Labeling and
defined using synthetic peptides. All of these antibodies proteolysis studies (Molday and MacKenzie, 1983; Clark and
interact within the 18 residues at theCOOH terminus Molday, 1979) have indicated that the COOH terminus is
of rhodopsin and recognize linear antigenic determi- exposed on the cytoplasmic side of the disc membrane whereas
nants of 4-11 residues. Twenty-seven synthetic pep-
tide analogs of varying lengths of native sequence or the NH2 terminus is exposed on the lumen or interdisc surface.
containing single amino acid substitutions at each po- Four monoclonal antibodies to bovine rhodopsin have been
sition of the COOH-terminal18 residues have provided shown to bind to the COOH-terminal region (MacKenzie et
some insight into the mechanism of antigen-antibody al., 1984). These monoclonal antibodies would be ideal can-
binding. Our results clearly demonstratethat antibod- didates to study the interaction of antibody and antigensince
ies can be highlyspecific at key positions as shown by the known antigenic region is small (18 residues or less) and
the loss of binding on single amino acid substitutions synthetic peptides are able to compete with native protein for
in the bindingsite. In contrast singleamino acid sub- binding to anti-protein antibodies raised to rhodopsin-con-
stitutions at other positions in the binding site only taining disc segments. Although it is generally thought that
affect affinity for some antibodies. Ionic interactions monoclonal antibodies recognize discontinuous epitopes (van
candominate immunogenic determinants. Immuno- Regenmortel, 1986; Benjamin et al., 1984), we report that
genic determinants are not restricted to highly charged monoclonal antibodies to the COOH terminus of intact rho-
hydrophilic regionson the surface of a protein andmay dopsin-containing discs recognize residues confined to small
be dominated by hydrophobic interactions. Although linear epitopes ranging from 4 to 11 residues. In a previous
certain side chains can dominate the interactionof the study of antigen-antibody interaction, we reported that a
antigen with antibody, our results are in agreement
with the interpretation that the free energies of all the NH2-terminal acetylated residue was critical for the binding
contact points are additive and a certain free energy of antibodies to EDP208 pilin protein, and this binding was
must be present to achieve binding. Antibodies with restricted to a NH2-terminal pentapeptide (Worobec et al.,
different specificities directed to the same region of 1985). Similarly the NH,-terminal acetylated amino acid was
the protein antigen can be produced in an immune found to be essential for the binding of synthetic peptides to
response. Peptide antigens representing regions of a anti-cytochrome c antibodies (Paterson, 1985). In this report
protein antigen bind best to the anti-protein antibody we describe results of antigen-antibody interactions at the
when the sequence is shortened to contain only those COOH terminus of rhodopsin, the importance of the free
residues binding to thespecificity site in theantibody. COOH-terminal a-carboxyl group, the additive binding effect
Cross-reactivity between protein antigens can be ex- of hydrophobic and hydrophilic residues, and thatmonoclonal
plained by conservation of the critical residues in the antibodies with a variety of specificities, both ionic and hy-
combining site. drophobic, can recognize small linear determinants.
MATERIALS AND METHODS
Rod Outer Segment Membranes-Bovine rod outer segments were
Rhodopsin is the major membrane glycoprotein in rod outer purified by sucrose-density centrifugation under dim red light as
segment disc membranes of vertebrate retinalrod photorecep- previously described (Wong and Molday, 1986). Rod outer segment
tor cells. On the basis of protein sequence analysis (Ovchin- disc membranes used as a source of rhodopsin were obtained by
hypotonic lysis of rod outer segments followed by flotation on 5%
* This work was supported by the Medical Research Council of Ficoll according to themethod of Smith et al. (1975). Protein concen-
Canada (R. S. H. and R. S. M.), equipment grants from the Alberta tration was determined by the Lowry method using bovine serum
Heritage Foundation for Medical Research (to R. S. H.), and National albumin as a standard (Lowry et al., 1951).
Institutes of Health Grant EY-02422 (to R. S. M.). The costs of Antibodies-Antibovine rhodopsin monoclonal antibodies (rho
Publication of this article were defrayed in part by the payment of 1D4, 3.46, 1C5 and rho 3D6) were derived from culture fluids of
page charges. This article must therefore be hereby marked adver- hybridoma cell lines (MacKenzie et al., 1984; Hicks and Molday,
tisement in accordance with 18U.S.C. Section 1734 solelyto indicate 1986).
this fact. Goat antimouse immunoglobulin antibody used in indirect solid-
$ To whom correspondence should be addressed. phase radioimmune assays was iodinated with NalZ5Iby the chlor-

11768
 Antigen-Antibody Interaction 11769
amine-T method (Hunter and Greenwood, 1962) and had a specific
activity of 1-2 X 10 dpm/pg.
Solid-phase Radioimmune Assays-For radioimmune assays, 96-
well flexvinyl microtiter plates were coated with rhodopsin by drying
down 25 plof Triton X-100-solubilizedrod outer segment membranes.
The plates were rinsed in phosphate-buffered saline (0.01 M sodium
phosphate, 0.15 M NaC1) containing 1%bovine serum albumin prior
to use. Competition assays were carried out by incubating 25 p1 of
serial dilutions of the peptide antigen with 25 plof hybridoma culture
fluid a t a dilution which gave 80-90% antibody saturation as meas-
ured in standard solid-phase radioimmune assays (MacKenzie and
Molday, 1982). The concentration of all peptides were determined by
amino acid analysis of stock solutions, which has an error of +-5%,or
by weight which when compared to amino acid analysis had an
accuracy of &lo%. After 30-60 min at 25 C, 25 p1 from each well
was transferred to therhodopsin-coated microtiter plates. The micro-
titer plates were incubated for 30 min, washed in phosphate-buffered
saline, and treated with 25 11 of 261-labeledgoat antimouse immu-
noglobulin antibody (10 pg/ml; 1-2 X 10 dpm/pg). After 30-60 min,
the plates were again washed in phosphate-buffered saline, cut, and
counted in a Beckman 8000 gamma counter. Variations in the 160
values (peptide concentration required to obtain half-maximum in-
hibition of antibody binding to rhodopsin) were observed for different
stock hybridoma cell culture fluids and rod outer segment prepara-
tions. In repetitive assays, however, 160 values for a given peptide
agreed within -C50%. Variations in hybridoma supernatant are due to
the quantity of monoclonal antibody secreted per ml of fluid and the
dilution used in the competition assay. Differences in 160values were
used to get around this difference. When different hybridoma cell
culture fluids and rod outer segment preparations were used the I I I I
native peptide sequence was always used as a control to ensure the 10 20
relative accuracy of the peptide analog results. A variation in 160
values between different peptide analogs of 2-fold or greater is con-
sidered significant.
Peptide Synthesis-Unless otherwise stated,all chemicals used ELUTION TIME ( m i d
were reagent-grade. Diisopropylethylamine, dichloromethane FIG. 1. Representative reversed-phase chromatographic
(DCM), trifluoroacetic acid, and dimethylformamide were obtained elution profiles on analytical columns of the crude synthetic
from General Intermediates of Canada. Diisopropylethylamine, peptides. Panel A, peptide 5, Fig. 1 [Ac(Ala2)R(1-12)-OH]; Col-
DCM, and trifluoroacetic acid were redistilled before use. HPLC- umn, SynChropak RP-8, 4.1 mm inner diameter X 150 mm, 6.5 pm,
grade acetonitrile was obtained from either Fischer Scientific, or 300 A, (2-8. Panel B , peptide 1, Fig. 1 [AcR(1-18)-OH]; Column,
Caledon Laboratories. Double-distilled water was purified by passage Pharmacia PEP RPC, 5 mm inner diameter x 50 mm, 5 pm, 100 A,
througha Milli-Q water purification system. t-Butyloxycarbonyl- C-18. The solvent system used for both columns was a linear AB
amino acids were obtained from Institut Armand Frappier (Laval, gradient where A = 0.05% trifluoroacetic acid/HzO and B = 0.05%
Quebec), Protein Research Foundation (Osaka, Japan), andBachem trifluoroacetic acid/acetonitrile. Gradient rate was 1%B/min with
Fine Chemicals. Co-poly (styrene, 1%divinylbenzene) chloromethyl flow rates of 1 ml/min. The crude peptides were purified as outlined
resin (0.9 mmol Cl/g resin) was obtained from Pierce Chemical Co. under Materials and Methods.
Co-poly (styrene, 1%divinylbenzene) benzhydrylamine-HC1 resin
(0.9 mmolNHz/g resin)was obtained from Institut Armand Frappier.
Twenty-seven analogs were synthesized using the general proce- described previously and acetylated in asolution of toluene, pyridine,
dure for solid-phase peptide synthesis described by Merrifield (Stew- and acetic anhydride (3:3:1) for 1 h. The programs used for attach-
art and Young,1984; Erickson and Merrifield, 1976) on either a ment of each aminoacid using the Beckman 990 synthesizer were the
Beckman 990 Peptide Synthesizer, or an Applied Biosystems 430A same as previously described (Worobec et al., 1985; Parker and
Peptide Synthesizer. All peptides with a free carboxyl terminus were Hodges, 1985). Coupling procedures for the Applied Biosystems syn-
initiated by esterification of the cesium salt of the COOH-terminal thesizer have been described (Kent andLewis, 1985).
amino acid to co-poly (styrene, 1%divinylbenzene) chloromethyl Peptides werecleaved from the resin with HF (20 ml/g resin)
resin (Stewart and Young, 1984). Substitutions of approximately 0.9 containing anisole (2 ml/g resin), and ethanedithiol(20drops/g resin)
mmol of amino acid/g resin were obtained. For peptides with an a t 4 Cfor 45 min. The solvents were removed under reduced pressure
amide COOH terminus, co-poly (styrene, 1%divinylbenzene) benz- a t 4 Cfor at least 3h. The resin was washed with ether, and extracted
hydrylamine-HC1was neutralized with 5% diisopropylethylamine for with 3 X 10 ml trifluoroacetic acid. The trifluoroacetic acid was
1 h and theCOOH-terminal aminoacid was coupled to theresin with evaporated and thepeptide redissolved in water and lyophilized.
dicyclohexylcarbodiimide for 1 h (1:l equivalent, protected amino Purification by HPLC-The crude peptides were purified using
acid/dicyclohexylcarbodiimide). reversed-phase chromatography on either a SynChropak RP-P C-18,
Side-chain protecting groups for t-butyloxycarbonyl-aminoacids 300 A, 6.5pm column (250 X IO mm inner diameter) or a Rainin
used were: Lys(2-chlorobenzyloxycarbonyl), Asp(0-benzyl), Glu(0- Dynamax Macro C-18, 300 A, 1 2 pm column (250 X 21.4 mm inner
benzyl), Ser(benzyl), and Thr(benzy1). Removal of the t-butyloxycar- diameter) with a linear AB gradient where A = 0.05% trifluoroacetic
bony1 protecting group at each cycle was done in 50% trifluoroacetic acid/HzO and B = 0.05% trifluoroacetic acid/acetonitrile. The gra-
acid/DCM for 20 min. This was followed by neutralization in 5% dientrate was 1%B/min with flow rates of 2 ml/min for the
diisopropylethylamine. All amino acids were coupled using DCC- SynChropak column and 10 ml/min for the Rainin column. Repre-
activated couplings (1:l equivalent, protected amino acid/DCC) in sentative HPLC elution profiles of the 12-residue and 18-residue
DCM, except Thr which was coupled as a symmetric anhydride (2:l crude synthetic peptides before purification are shown in Fig. 1.
equivalent, protected amino acid/DCC) in DCM, and Gln and Asn,
which were coupled in dimethylformamide as N-hydroxybenzotriazole RESULTS
active esters. Double couplings of 1 h each were performed at each Four antibovine rhodopsin monoclonal antibodies, rho1D4,
step of the synthesis. The completed peptides were deprotected as 3A6, 1C5, and 3D6, were previously shownto bind to bovine
The abbreviations used are: DCM, dichloromethane; DCC, dicy- rhodopsin in the COOH-terminal 18 residues, 1-18 where
clohexylcarbodiimide; HPLC, high performance liquid chromatogra- 1 is the COOH-terminalresidue(MacKenzie et al., 1984;
phy. Hicks and Molday, 1986). To localize further the antibody-
11770 Antigen-Antibody Interaction
binding sites and examine the importance of each residue in
this 18-residue peptide to antibody binding, the 27 peptides
shown in Fig. 2 were synthesized, purified, and examined in
a solid-phase competitive inhibition assay. To demonstrate
the effect of each amino acid side chain on antibody binding,
the following single amino acid substitutions of each side
chain were carried out; when the amino acid side chain in the
native sequence was alanine, aglycine residue was substituted;
all larger side chains were substituted by alanine except for
the acidic residues aspartic and glutamic acid (positions 8 ',
17', and 18', Fig. 2) which were substituted by the uncharged
isosteric residues, asparagine and glutamine, respectively
(peptides 11, 20, and 21, Fig. 2). These Asn and Gln substi-
tutions would examine the importance of ionic interactions
in antigen-antibody binding while leaving all other interac-
tions with that residue intact (van der Waals, hydrophobic or
hydrogen bonding). The two peptide chain-lengths of 1'-12'
or 1'-18' were selected based upon previous results where
competitive inhibition studies indicated that antibody rho
3A6 required a length 1'-12' and larger whereas antibody rho
1C5 required peptide 1'-18' (MacKenzie et al., 1984). Anti-
body rho 1D4 binding was not inhibitedby peptides 2'-13' or
3'-18', indicating that theCOOH-terminal alanine residue of
rhodopsin was required. Competition studies using COOH-
terminal peptides l"4' to 1'-18' were equally effective inhib-
itors of antibody rho 3D6 binding to immobilizedbovine
PEPTIDE PIPTIOE NAME
NUMBER
I Rc-Rrp-Glu-Rla-Ser-Tr-Thr-Val-Ser-Lyr-Thr-Glu-Thr-Ser-Gln-UaI-Rla-Pro-Rla-OH
5
6 RC"
7 Rc-Ual
FIG. 2. Amino acid sequence of 9 Rls-OH
synthetic peptide analogs of the 10
COOH-terminal AcR(l"18') re-
gion of bovine rhodopsin. The num- II
bering of the sequence begins from the 12
COOH-terminal end of the protein with
the COOH-terminal residue denoted 1'. 13
Nomenclature example, Ac(Ala13')R(1'- 14
18')-OH, synthetic NHz-terminal-acet-
ylated rhodopsin fragment, residues 1'- 15
18' with a COOH-terminal carboxyl 16
group and alanine substituted at position
13'. Amide denotes a COOH-terminal 17
amide. The circled residues denote the I8
position of the amino acid substitution.
19
20
21
22
23
24
25
26
27
rhodopsin (Hicksand Molday,1986). Therefore, synthetic
analogs of lengths l ' - l Z ' and 1'-18' seemed appropriate to
test antigen-antibody binding of these four monoclonal anti-
bodies. Based upon the results with these analogs, the mini-
mum lengths for maximum antibody binding would then be
synthesized.
All peptides representing internal regions of the rhodopsin
sequence were synthesized as the Ne-acetylated and COOH-
terminal amides to prevent the introduction of charged groups
near the antibody-binding site. This is of particular impor-
tance, as the minimum peptide chain length is synthesized
which maximizes antibody binding. Talbot andHodges (1981)
clearly demonstrated the importance of not introducing N"
or C" charged groups into a binding site peptide which origi-
nates from an internal sequence of protein molecule. These
workers determined the minimal inhibitory peptide of the
troponin I inhibitory sequence is 12 residues and that the N"-
amino groups or C"-carboxyl group significantly affected the
ability of this peptide to inhibit the actomyosin ATPase.
This precaution of N"-acetylation and C"-amides should
always becarried out since these groups are isosteric with the
peptide bonds at either end of the peptide. Although, in this
study the 150 values for Ne-acetylated 1'-12' or 1'-18' were
indistinguishable from the nonacetylated 1'-12' or 1'-18'
peptides when tested with rho 1D4 and rho 3A6, this precau-
tion was observed for all shorterpeptides.
SEOUENCE
1 8 ' 1 7 ' 1 6 ' 1 5 ' 1 4 1 3 ' 1 2 ' 1 1 ' 10' 9' 8' 7' 6' 5' 4' 3' 2' 1'
W
Rc-Ual Rls-OH
flr-Ual Rla-OH
Rc-UaI Rh-OH
Rla-OH
~c-Rsp-6lu-Rla-Ser-~r-Thr-Ual-Ser-Lyr-Tr-6lu-amide
Rc-Thr-Thr-Val-Ser-Lys-Thr-Glu-amide
Ac-Val-Ser-Lyr-Rr-Glu-amide
Ac-Thr-Glu-Thr-Ser-GIn-Ual-Ala-Pro-RIa-OH
Rc-Thr-Ser-Gln-Val-Ala-Pro-Ala-OH
Ac-Ual-Ala-Pro-Rla-OH
 Antigen-Antibody Interaction
Importance of the COOH-terminal a-Carboxyl Group to An-
tibody Binding-Synthetic peptides 2 and 3, AcR(1'-12')-OH
and AcR(1'-12')-amide, respectively, were compared in the
solid-phase competitive inhibition assay. The results shown
in Table I indicate that theCOOH-terminal a-carboxyl group
is essential for antibody binding to both rho 1D4 and rho
3D6. The Iso value increased from 0.9 to 158 p~ when com-
paring peptide 2 and 3, respectively, for antibody rho 1D4
binding and peptide 3 showed no binding to antibody rho 3D6
(Table I).In contrast antibody rho 3A6 bound equally well to
peptides 2 and 3 indicating that the a-carboxyl group of the
COOH-terminal alanine residue is not involved in binding to
this antibody.
Importance of Each Amino Acid Side C h i n to Antibody
Binding-Systematic replacement of each residue in the
COOH-terminal 18-residue sequence was made in either the
12- or 18-residue peptide. The substitutions for each of the
first 12 positions of the COOH-terminal sequence were made
in analogs of the 1'-12' sequence and are shown in Fig. 2,
peptides 4-15. The substitutions for each of the positions 13-
18were made in analogs of the 1'-18' sequence and areshown
in Fig. 2, peptides 16-21. All four antibodies used in this study
bound to the 18-residue peptide containing the COOH-ter-
minal residue of rhodopsin. In addition, three antibodies, rho
1D4,3A6, and 3D6 bound to the12-residue peptide containing
the COOH-terminal residue of rhodopsin.
Monoclonal Antibody rho 104 Binding Studies-As shown
in Fig. 3 and Table I, the region 1'-9' is important for peptide
binding to monoclonal antibody rho 1D4. These results sug-
gest that side chains of residues l', 10'-18' make only weak
or insignificant contributions to antibody rho 1D4 binding.
Substantial decreases in antibody binding, as reflected by the
150 values in Table I, were seen with peptides 5, 7, 9, 11, and
12, where proline 2', valine 4', serine 6', and threonine 9'
were replaced with alanine and glutamic acid 8' was replaced
11771
with glutamine (Iw values of2000, 63, 125, 10, and 6.3 pM,
respectively, when compared to peptide 2 of0.9 pM repre-
senting a7-2220-fold decrease). The most important residues
interacting with antibody rho 1D4 werealanine 3', glutamine
5', and threonine7' where no detectable binding was observed
when these positions were substituted by glycine 3', alanine
5', and 7', respectively. The results of the competitive inhi-
bition assays for monoclonal rho 1D4 antibody binding are
summarized graphically in Fig. 3 where the solid bars indicate
the importance of each residue to antibody binding.
The results shown in Fig. 3 suggested that the minimum
peptide sequence required for maximum antibody rho 1D4
binding was residues 1'-9'. This conclusion was verified by
the data shown in Table I1 where binding increased approxi-
mately 6-fold whencomparing peptide 25 (1'-9') with peptide
2 (1'-12'). It appears that the 1'-9' peptide reproducibly
serves as a more effective antigen than the 1'-12' peptide.
Interestingly, peptide 26 (1'-7') which contained the residues
demonstrated to be critical for binding (Fig, 3) showed no
detectable binding. It appears as if the loss of binding energy
from residues 8' and 9', although small, was substantial
enough to prevent binding from a peptide containing residues
1'-7'. MacKenzie et al. (1984) showed that peptide 1'4'
bound to antibody rho 1D4. Another possibility is the loss of
hydrogen bonds to thepeptide bonds on removing residues 8'
and 9'.
Monoclonal Antibody rho 3A6 Binding Studies-As shown
in Fig. 3 and TableI, the side chains in the region 8'-12' are
important for peptide binding to monoclonal antibody rho
3A6. These resultssuggest that theside chains of residues 1'-
7' and 13'"' make only weak or insignificant contributions
to antibody rho 3A6 binding. Substantial decreases in anti-
body binding, as reflected by the IsOvalues in Table I were
seen for peptides 12 and 15 where threonine 9' and valine 12'
were replaced with alanine (I50values of 1000 and 350 p ~ ,

TABLE I
Effect of single amino acid substitutions on peptidebinding to anti-rhodopsinmonoclonal antibodies
Monoclonal antibody
Peptide Peptide Group altered 1D4
1C5 3A6 3D6
no. region from -+ to ~ -
IW" LogIMl-LogI"50b 154 LogI~O-LogI"50
2 l"12' None 0.9 0 28 0
3 l"12' COOH + amide 158 2.3 28 0
4 l"12' 1'Ala + Gly 2.5 0.5 25 -0.05
5 l"12' 2'Pro + Ala 2000 3.4 35 0.09
6 l"12' 3'Ala + Gly - - 35 0.09
7 l"12' 4'Val+ Ala 63 1.8 56 0.30
8 l"12' 5'Gln + Ala - - 24 -0.07
9 1'-12' 6'Ser + Ala 125 2.2 35 0.09
10 1'-12' 7'Thr + Ala - - 31 0.04
11 l"12' 8'Glu + Gln 6.3 0.9 - -
12 l"12' 9'Thr + Ala 10.0 1.1 1000 1.55
13 l"12' 1O'Lys + Ala 1.0 0.1 - -
14 l"12' 11'Ser + Ala 0.5 -0.3 - -
15 l"12' 12'Val- Ala 0.3 -0.5 350 1.09

1 1'"' None 0.8 0 4 0 2.5

16 l"18' 13'Thr + Ala 1.3 0.21 10 0.4 50
17 '"'1 14'Thr + Ala 1.2 0.18 101.5 0.4
18 l"l8' 15'Ser + Ala 1.2 0.18 4 0 4.2
19 l"l8' 16'Ala + Gly 1.2 0.18 4 0 28
20 l"l8' 17'Glu + Gln 1.2 0.18 10 0.4 8.0
21 l"l8' 18'Asp + Asn0.24 1.4 10 0.4 6.3
Iw, competitor concentration (PM)required for 50% inhibition in the competitive radioimmune assay.
b160", is the I
W value for either the N"-acetylated 1'-12' peptide [AcR(1'-12')-OH, peptide 21 or the No-
acetylated 1'-18' peptide [AcR(1'-18')-OH, peptide 11 with each monoclonal antibody. The value LogIW-LogIso"
is used for graphicalcomparisons of the effect of single amino acid substitutions. The dash indicates no measureable
binding to the monoclonal antibody.
11772 Antigen-Antibody Interaction
A 1 WW BB
3.4

0
Ov)
U
FIG. 3. Panel A , effects of single
amino acid substitutions inthe synthetic 0"
-1
2.4

peptide AcR(l'-18')-OH or AcR(1'-

I
12')-OH representing the bovine rho-
dopsin COOH-terminal sequence on
M
5:
binding to anti-rhodopsin monoclonal 1.4
m
antibodies. The hatched bars denote the 0
-1
results of competition assays with mono-
clonal rho 3A6 and the solid bars with
monoclonal rho 1D4. See Fig. 2 for the
0.4
amino acid sequence of all synthetic an-
alogs, Table I, and textfor details. Panel
B, carboxyl-terminal sequence of bovine
rhodopsin showing the location of the
linear antigenic determinants for the
various anti-rhodopsin monoclonal an- Native AC 18' 17' 1 6 ' 15' 1 4 13' 12' 1 1 ' 1 0 ' 9' 8' 7' 6' 5' 4 3' 2' I' OH
tibodies on the basis of synthetic peptide Sequence Ac-D-E-A-S-T-T-V-S-K-T-E-T-S-Q-V-A-P-A-OH
inhibition studies. Solid line represents
segment which givesmaximum antibody Substitution
. . . . .A .A .A . A .A . A . A . Q . A . A . A . A . G . A . G . amide
NH; N Q G
binding; dashed line represents smallest
observed segment which exhibits anti-
body binding. 6
I 3A6
"""

104
"""" - ""

18' 12' 9' 8' 1'

-Asp-Glu-Ala-Ser-Thr-Thr-Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala-COOH
"""-
I
"""""

1C5 I I 306
I

TABLE I1 lysine lo', and serine 11' where no detectable binding was
Effects of chain length on peptide binding to anti-rhodopsin observed, when these positions were substituted by glutamine
monoclonal antibodies 8', alanine lo', and ll',respectively. The importance of serine
Monoclonal Peptide Peptide region 11' andthreonine 9' also has been suggested in previous
no. or ROS" I,*
antibody studies indicating that kinase-catalyzed phosphorylation of
PM rhodopsin inhibits the binding of antibody rho 3A6 to rho-
l"12' 1D4 2 6.3 dopsin (Molday and MacKenzie, 1985). The results of the
25 1'-9' 1.3
26 l"7' -
competitive inhibition assays for monoclonal rho 3A6 anti-
24 8"12' - body binding are summarized graphically in Fig. 3 where the
ROS 0.02 hatched bars indicate the importance of each residue to anti-
3A6 2 l"12' 59' body binding.
8"18' 22 1.38 The results shown in Fig. 3 suggestedthat minimum peptide
23 8"14' 3.58
8"12' 24 19.60
sequence required for maximum antibody rho 3A6 binding is
ROS 0.08 residues 8'-12'. This conclusion wasverified by the data
1C5 1 l"18' 0.40 shown in Table I1 and Fig. 4. Fig. 4 is representative of the
2 l"12' - data obtained by solid-phase radioimmune competitive inhi-
22 8'-18' 0.21 bition assays displayed in Tables I and 11. Binding increased
23 8"14' 263
24 8"12' >loo0
approximately 3-fold when peptide 24 (8'-12') is compared
ROS 0.03 with peptide 2 (1'-12'). However, as the8'-12' sequence was
l"12'3D6 2 140 extended to 8'-14' (peptide 23) and 8"'' (peptide 22) bind-
l"4' 27 140 ing increased by 5- and 14-fold, respectively, when compared
ROS 0.06 to peptide 24 (8'-12') (Table 11). Interestingly, peptide 1 (1'-
ROS denotes rhodopsin outer segment disc membranes. 18') binds 7 times better than peptide 2 (1'-12') (Table I)
The dash (-) indicates no measurable binding to themonoclonal while peptide 22 (8'"') binds 43 times better than peptide
antibody. 2 (1'-12') (Table 11). These results suggest that, although
The IW values determined vary slightly with the preparation of
ROS, and themonoclonal antibody containing hybridoma cell culture extension of the 1'-12' peptide to 1'"' increases the binding
fluid. For example the I50 value for peptide 1'-12' was 28 / r (Table
~ affinity to monoclonal rho 3A6, the removal of unnecessary
I) and 59 ~ I M(Table 11). For this reason all peptides are compared to residues in the 1'-7' region results in a further substantial
the native sequences 1'-12' or 1'-18' in each assay. increase in binding affinity.
Monoclonal Antibody rho1C5 Binding Studies-In a similar
respectively, when compared to peptide 2 of 28 pM represent- fashion to the monoclonal binding studies with rho 3A6, the
ing a 36- and 13-fold decrease). The most important residues binding region for antibody rho 1C5 was localized to the 8'-
in interacting with antibody rho 3A6 were glutamic acid 8', 18' region (Fig. 3B, Table 11) and the importance of residues
 Interaction
Antigen-Antibody 11773
respectively, in peptide 2 (1-12). The contributions of all
other side chains in the COOH-terminal sequence were insig-
nificant to antibody rho 3D6 binding. These results suggest
8 that the minimum sequence required for maximal antibody
binding is 1-4. This conclusion is in agreement with the
3 results of Hicks and Molday (1986) who showedthat peptides
0 6 of lengths 1-4 to 1-18 were equally effective inhibitors of
X
0 rho 3D6 antibody binding to immobilized bovine rhodopsin.
a
a 4
No inhibition was observed with the 1-2 peptide or with
peptide 2-13. Our results clearly show that removal of either
2n the ionized carboxyl group by substitution with an isosteric
amide group or removal of the @-carbon of alanine 1 by
2 replacement with glycine completely removed peptide binding
to antibody rho 3D6.
A very interesting observation can explain the cross-reac-
0 tivity between rho 3D6 and opsin from cone cells (Hicks and
-3 -2 -1 0 1 2
Molday, 1986). The sequence of the COOH terminus of rod
Log Antigon Concentration ( I (Hargrave and Fong, 1977) and green and red cone protein
FIG.4. Competitive inhibition of monoclonal antibody rho (Nathans et al., 1986) is shown in Fig. 5. Absolute identity in
3 A 6 binding to rhodopsin by synthetic peptide analogs of the sequence is observed for the residues suggested in this study
COOH-terminal region of rhodopsin. The closed circles denote to be important for rho 3D6 binding, that is, the side chains
peptides 2, AcR(1-12)-OH; the open triangles, peptide 24, AcR(8- of 4, 2, and 1 and the a-COOH groups of 1.The sequence
12)amide; the open squares,peptide 23, AcR(8-14) amide,and the
closed triangles, peptide 22, AcR(8-18) amide.
differences occur at positions 3 and 5 which were shown to
be unimportant for peptide binding to the antibody rho 3D6.
Obviously increase in the size of the side chain at position 3
1
from alanine in rhodopsin to serine in opsin is not able to
Rod
0 0 0
Thr - Ser - G l n - Val - Ala - Pro - Ala - COOH
interfere with antibody binding, suggesting that this side
chain may not be in the antibody-binding site.

Cone @- Ser -@-Val -@-Pro - Ala - COOH

DISCUSSION
Thisstudy was carried outas part of this laboratorys
FIG.5. The amino acid sequence of the COOH-terminal re- investigation of the specificity of antibodies produced to a
gion of bovine rhodopsin from rod cells and bovine opsin from protein immunogen. The results of the present study suggest
human red and green cone cells. The circled residuesdenote that linear immunogenic determinants on native proteinsmay
sequence differences.
not only be abundant but are generally small in length and
in the 13-18 region determined (Table I). Both antibodies vary from 4-11 amino acid residues (Fig. 3B). These results
rho 3A6 and rho 1C5 show maximal binding to the peptide also suggest that linear immunogenic determinants on the
containing the residues 8-18. Antibody rho 1C5 binds 2-fold surface of native proteins can be considerably smaller than
more tightly to peptide 22 (8-18) than peptide 1 (1-18) the minimum of 7 residues suggested by Lerner et al. (1981).
(Table 11). These results again demonstrate that shortening The linear determinant for antibody rho 3D6 is the smallest
the chain length to remove residues unimportant to antibody native protein immunogenic determinant yet to be reported
binding (1-7 region) results in an increase in binding affinity (contained within 4 residues and involves only three of the
for the peptide. Although antibodies rho 1C5 and rho 3A6 four side chains). Recently, Worobec et al. (1985) reported a
bind to the 8-18 region, different residues are involved in linear immunogenic determinant at the NH2 terminus of a
binding. For example, alanine 16 has no effect on antibody pilin protein (contained within 5 residues). In fact this deter-
rho 3A6 binding but is of major importance to antibody rho minant was the immunodominant region of the pilin protein
1C5 binding when replaced by glycineresidue at 16 (I5ovalues (11,500 daltons),anda 5-residue peptide was capable of
of 28 p~ for peptide 19 [Ac-(Gly)l6R(1-18)-OH] compared titrating 80% of the polyclonal antibodies produced to the
to 2.5 p~ (11-folddecrease) for peptide 1containing the native native protein. Similarly, only three of five side chains were
sequence 1-18, Table I). Similarly, threonine 13 substituted of major importance for antibody binding. Interestingly, these
by alanine in the 1-18 peptide results in a 20-fold decrease two small determinants involved the NH2 or COOH terminus
in antibody rho 1C5 binding compared to a 2.5-fold decrease of the protein. The 4-residue determinant on rhodopsin for
for antibody rho 3A6 (compare peptides 1 and 16, Table I). binding antibody rho 3D6 involvesthe ionized a-COOH group
Threonine 14 is unimportant for antibody rho 1C5 binding, at the COOH terminus of the protein and the 5-residue
but a 2.5-fold decrease in binding is observed for rho 3A6, determinant for EDP208 pilus-specific antibodies involved
when peptide 17, Ac(Alal4)R(1-18)-OH is compared to the Ne-acetyl group at theNH2 terminus.
peptide 1. This study has allowed us to accurately delineate the func-
Monoclonal Antibody rho 306 Binding Studies-As shown tional groups involved in the antigen-antibody interactionfor
in Fig. 3B, Tables I and 11, the region 1-4 is important for three anti-rhodopsin monoclonal antibodies, rho 1D4, 3A6,
peptide binding to monoclonal antibody rho 3D6. The effect and 3D6. The following general observations can be made:
of single amino acid substitutions on peptide binding to anti- Ionic Interactions-In this report binding through ionic
rhodopsin monoclonal antibody rho 3D6 is shown in Table I. interactions has been shown to be very important in antigen-
The most important side chains in antibody interaction are antibody interactions at the COOH terminus of a protein.
alanine l, proline 2, and valine 4 where no detectable The binding of monoclonal antibodies rho 1D4 and rho 3D6
binding was observed when these positions were substituted to synthetic peptides was dramatically reduced or prevented
by glycine at position 1 and alanine at positions 2 and 4, when the free a-carboxyl group of the COOH-terminal Ala is
11774 Interaction Antigen-Antibody
replaced by an amide group. Landsteiner and Van der Scheer
(1934) were the first to suggest that thedistal part of a hapten
is immunodominant. Using dipeptides as haptens, these re-
searchers demonstrated that the COOH-terminal amino acid
provided the major binding energy in antigen-antibody inter-
action. Schechter et al. (1971) showed that the free carboxyl
terminus is critical in the binding of antipolyalanyl peptide
antibodies. This binding was abolished if peptides containing
COOH-terminal amides were used. Similar results have been
reported by Gras-Masse et al. (1985) in raising antipeptide
antibodies to streptococcal M protein peptides.
Ionic interactions can also dominate immunogenic deter-
minants at sites other than the COOH terminus. This was
observed for antibody rho 3A6 where the peptide antigen does
not bind when glutamic acid 8 or lysine 10 are replaced by
glutamine and alanine,respectively. Getzoff et al. (1987) have
described a recognition, induced-fit, and binding model for
antibody binding to myohemerythrin where the critical ionic
recognition step is followedby interaction with previously
buried residues, leading to binding.
Hydrophobic Interactions-Immunogenic determinants are
not restricted to highly charged hydrophilic regions on the
surface of a protein. For example, when any one of the 3
residues (alanine 3, glutamine 5, and threonine 7) of the
peptide antigen for rho 1D4 is replaced by glycine at 3 or
alanine at 5 and 7, binding of peptide antigen to antibody
is totally prevented(Fig. 3).Of the 9 residues involved in this
determinant (1-9) only the a-COOH group and the side
chain of glutamic acid 8 would be charged. The loss of the
charged groups on Glu-8 results in only a 7-fold decrease in
antibody binding whereas replacement of the COOH-terminal
carboxyl group by an amide group in the peptide antigen
causes a 175-fold decrease. Similarly, the 3 side chains in-
volved in antigen binding to antibody rho 3D6 are all hydro-
phobic (Val-4, Pro-2, and Ala-l, Table I) with the only
hydrophilic residue being the charged a-carboxyl group to
residue 1. Immunogenic determinants may be dominated by
hydrophobic interactions as is the case with the rho 3D6
determinant described above. This result agrees with Worobec
et al. (1985) where the threemost important functionalgroups
to antigen binding were the N-acetyl methyl group and two
leucine side chains at positions 3 and 4 of the 5-residue NH2-
terminal determinant (replacement of the acetyl group by a
formyl group or either leucine residues by a glycine residue
resulted in a 8,300-, 6,400-, and 20,000-fold decrease in bind-
ing of the peptide antigen to antibody, respectively). Thus,
the most stable contact points in the antigen-antibody com-
plex may be hydrophobic. This is reminiscent of observations
by Lemieux (1982) who showed that hydrophobic interactions
are important in theinteraction of carbohydrates with anti-
bodies and lectins. Also, Atassi (Atassi and Webster, 1983)
has reported that antigenic sites are not necessarily hydro-
philic and that hydrophobic interactions are very important
in binding interactions. In order for hydrophobic determi-
nants to exist, they must be at or near the surface, and this
is usually accomplished by a group of nearby charged and
noncharged hydrophilic residues which bring this hydropho-
bic region to the surface. For the antibody rho 3D6 determi-
nant, thisis accomplished by the hydrophilicity of the COOH-
terminal region 5-18 where the only hydrophobes are resi-
dues valine 12 and Ala-16 (Fig. 3).
Additive Effect-The observations with antibodies rho 3A6
and rho 1D4 (Fig. 3) suggest that although certain side chains
can dominate the interaction of the antigen with antibody,
the free energies of all the contact points are
additive (Schech-
ter, 1970) and a certain minimum free energy must be present
to achieve binding (Eisen, 1980). For example, whereas pep-
tide 25 (1-9) binds most tightly with antibody rho 1D4,
peptide 26 (l7) missing 2 residues does not bind (Table 11).
These side chains aremuch less important than the a-COOH
group of residue 1 and side chains of residues 2-7 (Fig. 3).
Similarly, peptide 22 (8-18) binds most tightly with anti-
body rho 3A6. In this case the loss of residues 15-18 results
in a decrease binding affinity of 1.7-fold (peptide 23, 8-14,
Table 11)while the loss of residues 13-18 results ina further
decrease in binding of 4.0-fold (peptide 24, 8-12 versus
peptide 23,8-14, Table 11). Withthisdeterminant the
minimum binding energy still has not been reached with the
peptide antigen 8-12 which is only 5 residues. These obser-
vations strongly suggest a greater specificity with antibody
rho 3D6 or rho 1D4 than antibody rho 3A6 and mutations
anywhere in theregion of the protein antigen 13-18 may be
accommodated with only a small decrease in binding affinity
with antibody rho 3A6.
Specificity and Affinity-Antibodies with different specific-
ities directed to thesame region of the protein antigen canbe
produced in an immune response to a protein antigen. Anti-
bodies rho 1C5 and rho 3A6 bind most tightly to the same
peptide antigen 8 yet their specificities are different. For
example the side chain of alanine 16 is important for rho
1C5binding but makes no contribution to binding to antibody
rho 3A6 (Table I). Similarly, the side chain of alanine 1 is
critical for antibody rho 3D6 binding (this antibody does not
bind to peptide 4 with a glycine at position l, Table I) yet
unimportant for rho 1D4 binding (the Iso values are 2.5 and
0.9 p ~ Table
, I, for peptides 4 and 2, respectively). Even
though both rho 1D4 and rho 3D6 are directed to theCOOH-
terminal carboxyl group, rho 1D4 is specific for Pro-2, Ala-
3, Gln-5, and Thr-7, whereas rho 3D6 is specific for Ala-
l,Pro-Z, and Val-4.
Interestingly, the combination of binding sites for the four
monoclonal antibodies used in thisstudy, each with their own
specificity, cover the complete 1-18 sequence which is ac-
cessible on rhodopsin.
The resultsinthis study clearly demonstrate the high
specificity of antibodies. The removal of a single methyl group
in the antigenic determinant can totally prevent antibody
binding (replacement of alanine 3 by glycine in the peptide
antigen for antibody rho 1D4(Fig. 3) or replacement of
alanine 1by glycine in the peptide antigen for antibody rho
3D6 (Table I)).
Synthetic peptide antigens may contain the entirespecific-
ity site for the protein antigens interaction with antibody
and yet the protein antigen may bind more strongly to the
antibody. This is shown in Table I1 where the antirhodopsin
monoclonal antibodies prepared from native rhodopsin in
disc membranes bound better to native rhodopsin (discs) than
the synthetic peptides. The differences ISo values for the most
efficient peptide binding and native rhodopsin binding to
monoclonals varied from 7-fold weaker in the case of rho 1C5
to 17-, 65-, and 2,330-fold in the case of rho 3A6, rho 1D4,
and rho 3D6 (Table 11).
Peptide antigens representing regions of a protein antigen
bind best to the anti-protein antibody when the sequence is
shortened to contain only those residues binding to thespec-
ificity site in the antibody. These results are shown in Table
I1 where maximal peptide antigen binding was obtained with
peptides 1-9 for antibody rho 1D4, 8-18 for antibodies
rho 3A6 and rho 1C5 and 1-4 for antibody rho 3D6. This
can be explained by an increase in peptide flexibility as the
peptide is shortened which allows it to fold more easily into
the proper orientation for binding in theantibody combining
 Interaction Antigen-Antibody
site. In contrast, the decrease in binding affinity for peptides
compared to thenative protein can be explained by the protein
antigen removing the flexible interference and fixing the
region in the exact orientation required for binding. This
interpretation is supportedby the x-ray diffraction studies of
the lysozyme-Fab, protein antigen-antibodycomplex (Amit et
al., 1986) where the classical lock and key is an adequate
simplification to describe this interaction. It should be noted
thatthestructure of the complex between antibody and
influenza virus neuraminidase show featuresinconsistent
with a rigid lock and key model for antigen-antibody inter-
actions and that conformational changes can be induced in
the antigen by antibody (Colman et al., 1987). However, both
x-ray studies involve discontinuous epitopes where the inter-
face between antigen and antibody involves a minimum of 16
residues on the antigen surface. The native monoclonals
described in this report recognize small linear determinants
between 4 and 11 residues. It will be interesting to compare
the x-ray diffraction results of an antigen-antibody complex
involving a linear epitode on the surface of a protein antigen.
Cross-reactivity-Cross-reactivity between protein antigens
can be explained by conservation of the critical residues in
the combining site (see Fig. 3 and Table I for the results of
antibody rho 3D6).
Many workers have reported using monoclonal antibodies
to locate or define antigenic sites. An ideal approach to the
problem of formulating a binding mechanism is to study the
effect of each residue inthe antigenic site. This is best
achieved by the chemical synthesis of antigenic analogs which
allow us to study sequences that do not occur naturally. We
feel that thestudy presented in thispaper will provide a useful
approach for delineating antigenic determinants and exam-
ining the molecular basis of antigen-antibody interactions.
For example information on the amino acid residues of rho-
dopsin which are important for monoclonal antibody binding
has enabled Oprian et al. (1987) to purify functionally active
rhodopsin from COS cells expressing the synthetic gene for
bovine rhodopsin using antibody rho 1D4 and a synthetic
COOH-terminal peptide in conjunction with immunoaffinity
chromatography. The results summarized in Fig. 3 show that
antibodies with a variety of specificities, both ionic and hy-
drophobic, can recognize small linear determinants.
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