310 BJ Letters

Are the antibodies to a peptide 100 - p p I

complementary to angiotensin II useful to S 6I- 0

isolate the angiotensin II receptor?

50 -
In 1981, Biro reported a comparative computer analy-
sis on the specificity of protein interactions [1]. He
proposed that these specifically interacting proteins may (a)
1.-
be coded by nucleic acids containing complementary 20
0- --I

sequences. This molecular recognition theory was seri- 0

I
I I I
10 8 6 4
ously considered and expanded by Blalock & Smith [2]. 0, -log [Concentration (M)]
In the genetic code, codons for hydrophilic and hydro- C 100-
phobic amino acids on one strand of DNA are generally .C_
c
complemented by codons on the other strand. These m
would code for a different 'complementary' set of amino
acids. Moreover, they demonstrate that peptides specified 50-
by complementary RNA bind to each other with speci-
ficity and high affinity [3]. They suggested that the
complementary peptide would resemble the receptor 4% (b)
binding site. This hypothesis was well illustrated for 0-
ACTH [4], y-endorphin [5] and LHRH [6]. I~~
I
I
8I IX

More recently, a similar approach was used for insulin 10 8 6 4

[7] and for angiotensin II [8]. In this latter study, a -log [Concentration (M)]
peptide, termed IIA, specified by RNA complementary 1500 -
to the mRNA of angiotensin II (All) specifically inhibited 0,
the binding of radiolabelled AII to AII receptors on rat E
1000 -

adrenal membranes. Further experiments indicated that
IIA bound directly to All and not to the receptor.
Furthermore, a monospecific antibody against IIA cross-
reacted with the All receptor and was able to inhibit the
secretion of aldosterone from rat adrenal cells.
If correct, this approach would allow the purification
of the receptor in large amounts and consequently help
in the search for specific All antagonists [9]. For this
reason, the octapeptide specified by the RNA sequence
complementary to that for All and reported in [8] was
synthesized in our chemistry department by B. Riniker.
Its effects on the binding of radiolabelled AII to the
receptor were tested.
Despite strictly adhering to the method reported by
Elton et al. [8], using fresh rat adrenals, we were never
able to reproduce their results (Fig. 1 a). The comp-
lementary peptide IIA (0.01-100 aM) did not inhibit the
binding of '25l-AII to the receptor. IIA was also tested
according to our own standard assay procedures using
membranes prepared from human uterus, rat adrenals
and rat aorta smooth muscle cells (SMC). Our method
differed primarily in the membrane preparation technique
(Polytron homogenization of frozen tissue) and the
concentration of the radioactive ligand (0.35 nM). In
none of the tissues tested was IIA able to compete with
radiolabelled All. As the complementary peptide should
bind All, a further experiment was performed in which
unlabelled All was preincubated for 30 min at room
temperature with 10-5 M-IIA, before a further incubation
of 60 min at 25 °C with SMC membranes and 125I-AII.
However, despite the preincubation, the IC for All in
the presence of IIA was not different from the control
(0.75 and 0.76 nm respectively; Fig. lb). The slopes of the
curves were also identical (1.0 and 0.98 respectively).
Moreover, the two peptides were incubated for 60 min at
room temperature and submitted to reverse-phase t.l.c.
Only two spots were found, with RF values similar to
those of All and IIA alone, thus providing no evidence
for the formation of an All-IIA complex.
Despite our negative results, polyclonal antibodies
I*
0
'C 500Q-
m (c)
0 I I I .I I
0 2 4 6
Total All (nM)
Fig. 1. Effect of complementary peptide IIA on 125I-angiotensin
II binding
(a) Rat adrenal membranes were incubated with 0.01 mm-
1251I-AII and increasing concentrations of unlabelled AII
(0) or IIA (O). (b) Rat smooth muscle cell (SMC)
membranes were incubated with 0.35 nM-_25I-AII and
increasing concentrations of unlabelled AII which had
been preincubated with buffer only (0) or with IIA ().
(c) Rat SMC membranes, pretreated with buffer only (M),
0.1 tM-anti-IIA ( 0) or 0.33 ,#M-anti-IIA (A), were incu-
bated with 0.99 nM-2511-AII and increasing concentrations
of unlabelled All. Data were analysed and transformed
using the LIGAND program [11].
were raised against the two peptides. Each of these
antisera recognized specifically the peptide against which
they were induced, without detectable cross-reaction
with the other peptide. The titres of the anti-AII and
anti-IIA sera were 1: 30000 and 1: 10000 respectively as
analysed in a solid-phase e.l.i.s.a. using peptide-coated
plates and phosphatase-labelled goat anti-(rabbit Ig)
antibodies for development [10].
The effect of these antisera on the binding of radio-
labelled AII to rat SMC membranes was analysed after
preincubation of the membranes with 0.1 and 0.33 #M
anti-All or anti-IIA. Bound radioactivity increased dose-
dependently with anti-All. However, this result was also
obtained in the absence of membranes, demonstrating
that it was due to the radioactive antigen-antibody
complex remaining on the glass fibre filter used to
separate the membrane-bound from free ligand. In
1989
BJ Letters
contrast, there was no effect on the binding of radioactive
All when the membranes were preincubated with anti-
IIA (Bmax = 1422, 1386 and 1415 fmol/mg of protein
and Kd = 0.42, 0.41 and 0.43 for the control, 0.1 and
0.33#,M-anti-IIA respectively; Fig. 1c), indicating that
this antibody did not react with either iodinated angio-
tensin II or the receptor.
On the basis of the apparent interaction between the
complementary peptides All and IIA, it was claimed that
corresponding antisera would contain antibodies with
anti-idiotypic affinity for each other [8]. In our experi-
ments, however, the antibody preparations to AII and
IIA did not show any interaction as revealed by solid-
phase e.l.i.s.a. analysis.
The fact that we are unable to confirm any of the data
reported by Elton et al. [8] is perplexing. Our assay
methods are well established and high-titre antibodies
were used. Our results lend support to the suggestion of
Biro [12] that his initial hypothesis, that proteins which
specifically interact with a receptor-are coded for by
nucleic acids complementary to nucleotidsequences of
the nucleic acid coding for the receptor, is doubtful. Biro
found 16 complementary sequences between the mRNA
of human insulin and the human insulin receptor. How-
ever, he found 81 additional sequences when comparing
10 non-related nucleic acids with that of the receptor.
Similarly, Rasmussen & Hesch [13] using this approach
for parathyroid hormone were unable to find any experi-
mental indication that the hormone binds to a synthetic
peptide derived from the antisense RNA sequence. These
authors also questioned the general applicability of this
approach.
In conclusion, there is accumulating evidence to sug-
gest that the concept of using antibodies against comp-
lementary peptides as tools to isolate the corresponding
receptors cannot be applied in all cases.
Marc DE GASPARO, Steven WHITEBREAD,
Karin EINSLE and Christoph HEUSSER
Research Department, Pharmaceuticals Division, Ciba-Geigy
Ltd., Basle, Switzerland
1. Biro, J. C. (1981) Med. Hypotheses 7, 969-1007
2. Blalock, J. E. & Smith, E. M. (1984) Biochem. Biophys.
Res. Commun. 121, 203-207
3. Blalock, J. E. & Bost, K. L. (1986) Biochem. J. 234,679-683
4. Bost, K. L., Smith, E. M. & Blalock, J. E. (1985) Proc.
Natl. Acad. Sci. U.S.A. 82, 1372-1375
5. Carr, D. J. J., Bost, K. L. & Blalock, J. E. (1986) J.
Neuroimmunol. 12, 329-337
6. Mulchahey, J. J., Neill, J. D., Dion, L. D., Bost, K. L. &
Blalock, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,
9714-9718
7. Knutson, V. P. (1988) J. Biol. Chem. 263, 14146-14151
8. Elton, T. S., Dion, L. D., Bost, K. L., Oparil, S. & Blalock,
J. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2518-2522
9. Soffer, R. L., Bandyopadhyay, S., Rosenberg, E., Hoe-
prich, P., Teitelbaum, A., Brunck, T., Colby, C. B. & Gloff,
C. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 9219-9222
10. Enguall, E. &Perlmann, P. (1972)J. Immunol. 109,129-135
11. Munson, P. & Rodbard, D. (1980) Anal. Biochem. 107,
220-239
12. Segersteen, U., Nordgren, H. & Biro, J. C. (1986) Biochem.
Biophys. Res. Commun. 139, 94-101
13. Rasmussen, U. B. & Hesch, R. D. (1987) Biochem. Bio-
phys. Res. Commun. 149, 930-938
Received 3 April 1989
Vol. 261
311
'Complementary peptides': a response
After careful perusal of the letters by Guillemette et al.
[1] and de Gasparo et al. [2] we could find no readily
apparent reason other than the different systems em-
ployed to explain their inability to reproduce our findings
with the IIA peptide [3]. Perhaps the most perplexing
aspect of the present studies was the lack of interference
of IIA in the 125I-AII radioreceptor assay. This is
particularly puzzling since our findings of blockage of
the AII radioreceptor assay were in agreement with a
prior publication by another group of investigators.
Soffer et al. [4].
de Gasparo et al. [1] suggested that we had " seriously
considered and expanded" a theory by Biro. In fact, we
were initially unaware of it. When we became aware of
this hypothesis, it had been retracted. His retraction and
sequence analysis pointed out that, for instance, when he
searched for complementary sequences, he erroneously
did not consider reading frames. In spite of this, we were
surprised that de Gasparo et al. considered these papers
by Biro, which also postulated that reverse translation
of protein sequences was the mechanism for formation of
antibodies; the validity of the clonal selection theory of
antibody formation had been verified many years before
Biro's 1981 paper.
With regard to the statement of Guillemette et al. [1]
that "Our results question the validity of the hypothesis
that there is a molecular recognition code in which
peptide ligands and their receptor binding sites can be
encoded by complementary nucleotide sequences." it
should be noted that the paucity of negative results
contrasts with a rapid accumulation of reports that
support the validity of the theory [3-44]. These results
include the demonstration of the interaction of twelve
different peptide pairs specified by complementary
nucleotide sequences [3-14] as well as the purification of
the receptors for ACTH [5,18], opioids [19,23,24,38,44],
luteinizing hormone releasing hormone [7,22], AII [3],
fibronectin [10], arginine vasopressin [27,40], and sub-
stance P (D. W. Pascual, J. E. Blalock & K. L. Bost,
unpublished work). It is also worth noting that these
publications were from nine different groups.
J. Edwin BLALOCK,* Terry S. ELTONt and
Suzanne OPARIL
*Department of Physiology and Biophysics and tDepartment
of Medicine, University of Alabama at Birmingham, Birming-
ham, AL 35294, U.S.A.
1. Guillemette, G., Boulay, G., Gagnon, S., Bosse, R. &
Escher, E. (1989) Biochem. J. 261, 309
2. de Gasparo, M., Whitebread, S., Einsle, K. & Heusser, C.
(1989) Biochem. J. 261, 310-311
3. Elton, T. S., Dion, L. D., Bost, K. L., Oparil, S. & Blalock,
J. E. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 2518-
2522
4. Soffer, R. L., Bandyopadhyay, S., Rosenberg, E., Hoe-
prich, P. D., Teitelbaum, A., Brunck, T. K., Colby, C. B. &
Gloff, C. A. (1987) Proc. Natl. Acad. Sci. U.S.A. 84,
9219-9222
5. Bost, K. L., Smith, E. M. & Blalock, J. E. (1985) Proc.
Natl. Acad. Sci. U.S.A. 82, 1372-1375
6. Blalock, J. E. & Bost, K. L. (1986) Biochem. J. 234, 679-
683
7. Mulchahey, J. J., Neill, J. D., Dion, L. D., Bost, K. L. &
Blalock, J. E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,
9714-9718