THE JOURNAL OF BIOLOGICAL CHEMISTRY
13362-13366,1986
Q 1986 by The American Society of Biological Chemists, Inc
Structure of the Spectrin-Actin Binding Site of Erythrocyte
Protein 4.1*
Isabel Correas, David W. Speicher, and Vincent T. MarchesiS
From the Yale University School of Medicine, Department of Pathology, New Haven, Connecticut 06510
The complete primary structure of the functional site
of erythrocyte protein 4.1 involved in spectrin-actin
associations has been determined. The sequence of this
domain, which contains 67 amino acids and has a mo-
lecular mass of 8045 daltons, has been obtained by
NH2-terminal sequence analysis of an 8-kDa chymo-
tryptic peptide, three endoproteinase lysine C-cleaved
peptides and two peptides obtained by Staphylococcus
aureus protease V8 cleavage. All peptides including
the 8-kDa domain peptide were purified by reverse-
phase high performance liquid chromatography. Anti-
bodies against two different synthetic peptides of the
8-kDa domain are able to inhibit the association be-
tween protein 4.l, spectrin, and F-actin, corroborating
that the 8-kDa domain is responsible for the formation
of a ternary complex.
A computer search of the 8-kDa sequence with the
National Biomedical Research Foundation database
did not detect any significant homologies to known
sequences. Protein 4.1 is not related to any known
proteins and may represent a new protein superfamily.
The red cell membrane has been chosen as a model for
studying membrane-cytoskeleton interactions because of its
apparent simplicity relative to other cells. The membrane
skeleton is composed of a two-dimensional protein network
of which spectrin, a long, rod-like molecule composed of two
non-identicalsubunits, is the major component. Spectrin
dimers further self-associate into tetramers andoligomers (1)
and interact with protein 4.1 and actin (2-5) to form the
submembranous lattice. This anastomosing network is linked
to the membrane by two proteins. One, designated band 4.1
based on its position on SDS’ gels, binds to theend of spectrin
distal to the self-association site but proximal to thebinding
site for filamentous actin ( 5 , 6); this protein attaches the
spectrin-actin network to the cytoplasmic face of the mem-
brane through specific associations with at least two different
transmembrane glycoproteins (7, 8). The other membrane
linking protein, protein 2.1 (ankyrin) (9), binds to a part of
the @ subunit near the self-association end of the spectrin
dimer (10) and links it to the cytoplasmic domain of band 3
(11). In addition to linking the spectrin-actin complex to the
membrane, protein 4.1 greatly enhances the affinity of spec-
* This research was supported by National Institutes of Health
Grants GM21714-12 and AM27932-06. The costs of publication of
this article were defrayed in part by the payment of page charges.
This article must therefore be hereby marked “advertisement” in
accordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
$ To whom all correspondence should be addressed.
The abbreviations used are: SDS-PAGE,sodium dodecyl sulfate-
polyacrylamide gel electrophoresis; EL, endoproteinase lysine-C; SV8,
S. aureus protease V8; KLH, keyhole limpet hemocyanine; HPLC,
high performance liquid chromatography.
13362
Vol. 261, No. 28. Issue of October 5, pp.
Printed in U.S. A.
(Received for publication, May 8, 1986)
trin for F-actin, forming stable ternary complexes as demon-
strated by several different methods (2-5). Although the pre-
cise nature of the ternary complex is not yet well understood,
it is likely to be of general significance since proteins immu-
nologically related to erythrocyte spectrin and protein 4.1
have been described in numerous non-erythroid cells (for
reviews, see Refs. 12 and 13).
Previous work in this laboratory has identified a peptide of
erythrocyte protein 4.1 that mimics the capacity of the 4.1
molecule to enhance the association between spectrin and
actin. This fragment is incorporated into theternary complex
in approximately stoichiometric amounts and its activity is
comparable to thatof the intact4.1 molecule on a molar basis
(14). This 8-kDa peptide has been located within the 10-kDa
region of erythrocyte protein 4.1 which had previously been
shown to contain a CAMP-dependent phosphorylation site
(15, 16).
This report describes the further characterization of the 8-
kDa fragment including the purification and analysis of pep-
tides derived from two protease cleavages of the 8-kDa peptide
and thecomplete sequence of this segment. Antibodies raised
against two synthetic peptides whose sequences derived from
the 8-kDa peptide sequence are able to inhibit the interaction
between protein 4.1, spectrin, and actin.
MATERIALS ANDMETHODS
Isohtion of a Complex-promoting Peptide of Protein4.1”Two
different methods have been followed for the isolation of the 8-kDa
domain of erythrocyteprotein 4.1. In the first, the 8-kDa active
peptide was sedimented as a complex with spectrin and F-actin.
Protein 4.1 (2 mg) wasdigested with a-chymotrypsin at 1:260 enzyme
to substrate ratio and incubated with spectrin (12 mg) and F-actin (8
mg) for 90 min at 0 “C. After centrifugation at 150,000 X g for 30 min
on a SW50.1 rotor, the 8-kDa peptide obtained in the pellet fraction
was chromatographed on gel filtration columns (two 30-cm Bio-Si1
TSK-400 columns, two 30-cm Bio-Si1TSK-250 columns, and one 30-
cm Bio-Si1 TSK-125 column, all in tandem, Bio-Rad) equilibrated in
8 M urea, 0.2 M Tris, 10mM 2-mercaptoethanol, pH 7.0. Effluent was
monitored a t 280 nm and flow rates of 0.3 ml/min were used. The 8-
kDa fraction was further purified by reverse-phase chromatography
on a 4.6 X 250-mm, C-4, RP-304 column (Bio-Rad) equilibrated with
0.1% trifluoroacetic acid. Elution of the peptide was achieved using a
linear gradient of0-60% acetonitrile in the same buffer. Peptides
were detected by absorbance a t 215 nm and by tryptophan fluores-
cence (excitation 280 nm, emission filter 370 nm).
A second method for isolation of the 8-kDa peptide involved an
initial size separation of the 4.1-derived peptides (8 mg) produced by
mild chymotrypsin digestion by HPLC gel filtration, followed by
further purification of the resulting 8-kDa peptide by reverse-phase
high performance liquid chromatography. The columns and buffer
conditions have been described above.
Lysine-specificProteolytic Cleavage-The 8-kDa peptide (10 nmol)
was incubated for 17 h at 37 “C in 200 mM Tris-HC1, 1 mM EDTA,
0.02% sodium azide, pH 7.8, with endoproteinase lysine-C (Boehrin-
ger Mannheim) using an enzyme to substrate ratio of 1:lOO. The
reaction was terminated by titrating the samples to pH 2.0 with
trifluoroacetic acid. The sample was analyzed by reverse-phase chro-
 Spectrin-Actin Binding Siteof Erythrocyte Protein4.1
matography under the conditions described above.
Staphylococcus aureus Protease V8 (SVS) Digestion-The 8-kDa
peptide (17 nmol) was incubated for 16 h a t 37 "C in 300 mM
T -r
NH,HCO,, 50 mM Tris, 5 mM EDTA, pH 7.8, with protease V8 using
an enzyme to substrateratio of 1:30. The reaction was terminated by
titrating the sample to pH 2.0 with trifluoroacetic acid. The sample
was then treated as described for the lysine-C digest.
Amino Acid Composition and Sequence Analysis-Peptides were
hydrolyzed a t 100 "C for 20 h in uacuo in 6 N HCl containing 0.2%
phenol. Amino acid analysis was performed on aDionex D-500 amino
acid analyzer using a ninhidrin detection system.
Automated sequence analysis of the peptides was performed on a
Beckman 890C Sequencer using the 0.1 M Quadrol program 030176.
3 mg of Polybrene were added to thespinning cup and a blank cycle
was run before addition of peptide samples. Conversion of the thia-
zolinone-derivatives to phenylthiohydantoin derivatives was carried
out in a Sequemat P-6 autoconverter using 1 N methanolic HCl.
Phenylthiohydantoin derivatives were identified by HPLC as previ-
ously described (17).
Nomenclature of Peptides-Peptides were named based on the type
of cleavage used endoprotease lysine-C (EL); S. aureus protease V8
(SV8). Each of these two sets of peptides have been numbered in the
order which they occur in the complete sequence starting at the
amino-terminal.
Preparation of Antibodies-Based on the 8-kDa peptide sequence
that we obtained, two synthetic peptides were made with additional
cysteine residues in their COOH-terminal ends (Peninsula Labora-
tories). Peptide A included amino acids 1-15 and peptide B amino
acids 46-59 (Fig. 4). Both peptides were independently coupled to B
KLH (keyhole limpet hemocyanine) using the bifunctional reagent
rn-maleimidobenzoylsulfosuccinimide esterin 50 mM phosphate
buffer, 1 mM EDTA, pH 7.0.New Zealand White rabbits were
immunized by subcutaneous and intramuscular injections of 1 mg of
KLH-peptide in complete Freund's adjuvant. They were boosted twice a3
at 4-week intervals using 0.5 mg of KLH-peptide with incomplete
adjuvant. IgG-enriched fractions were obtained from 40% saturation
ammonium sulfate precipitates. Antibodies against KLH were ad-
sorbed to Sepharose CL-4B beads to which KLH was linked via
IE
cyanogen bromide (18), andthe resulting unbound fraction was
purified further on a Sepharose CL-4B column to which hexanedi- wk0.2
amine was linked via cyanogen bromide (18) and activated with m-
maleimidobenzoylsulfosuccinimideto get the synthetic peptide cou-
pled. Antibodies were eluted from the column with 1M acetic acid in
0.15 M NaCl, pH 4.0, dialyzed against phosphate-buffered saline, and
concentrated.
Monovalent Fab antibodies were produced by cleavage of antibod-
ies with papain by the method described by Porter (19).
Sedimentation Assay-Protein 4.1, spectrin, and F-actin were in-
cubated and sedimented as earlier described (14). In the cases where
B 0.1

the monovalent Fab antibodies were present, protein 4.1 (8 pg) and
the Fabfragment (15pg) werepreincubated overnight on ice. Samples
were processed as described elsewhere (14).
PAGE and Immumblotting-SDS-PAGE was carried out as de-
scribed (20).SDS-PAGE proteinswere transferred onto nitrocellulose
paper and immunoblotting was performed according to Towbin et al.
(21).
RESULTS AND DISCUSSION
Fig. 1 represents typical profiles of the reverse-phase puri-
fication of the 8-kDa domain of protein 4.1 following two
different approaches. In the first, the active peptide was
sedimented in a complex with spectrin and F-actin. An a-
chymotryptic digest of protein 4.1 wasincubated with spectrin
and F-actin as described under "Materials and Methods," to
form astableternary complex. The pellet fraction which
contained spectrin, F-actin, and the 8-kDa peptide was sepa-
rated by gel filtration. The 8-kDa peak was further purified
by HPLC reverse-phase chromatography and three peptide
components were identified, as indicated in Fig. lA. These
peptides are very similar in theiramino acid composition
(data not shown) and probably represent differing sites of
cleavage by chymotrypsin. The presence of all three peptides
in the sedimentable complex indicates that all three forms
retain complex-promoting activity. Peak 3 was used for the
0-
FIG. 1. Reverse-phase purificationof the 8-kDa domain of
protein 4.1. Samples were eluted from a 4.6 X 250-mm, C-4, RP-
304 column (Bio-Rad) equilibrated with 0.1% trifluoroacetic acid, pH
2.0 using a linear gradient of 0-60% acetonitrile in 0.1% trifluoroac-
etic acid, pH 2.0. The gradient shape is indicated. The differences
between the earliest steps of the purification procedure for panels A
and B are explained under "Materials and Methods." Peak 3 was
used for sequence analysis.
sequencing studies presentedbelow.
The second method for isolation of the 8-kDa peptide
involved an initial size separation of the 4.1-derived peptides
produced by mild a-chymotrypsin digestion by HPLC gel
filtration in the presence of 8 M urea, as previously described
(14). The resulting 8-kDa fraction was further purified by
reverse-phase chromatography, as indicated in Fig. 1B. The
peaks designated 1,2,and 3 correspond to thesimilarly labeled
peaks isolated by the complex-formation method. The identity
with peaks in Fig. lA is based on similar amino acid compo-
 13364 Spectrin-Actin
Binding
Site of Erythrocyte
Protein 4.1

-
FIG. 2. Reverse-phase separation of 8-kDa peptides after
cleavage with endoproteinaselysine-C. 10 nmol of 8-kDa peptide
(peak 3, in Fig. 1) were digested as described under “Materials and
Methods.” The column and buffer conditions are similar to those
shown in Fig. 1. Peptides EL-1,EL-2, and EL-3 were used for
sequence analysis. The figure shown is a representative chromato-
gram.
MHms
FIG. 3. Reverse-phase separation of 8-kDa peptides after
cleavage with S. aureus protease V8. Approximately 17 nmol of
8-kDa peptide (peak 3, in Fig. 1) were digested as described under
“Materials and Methods” and separated under the conditions shown
in Fig. 1. Peptides SV8-1 and SV8-2 were used for sequence analysis.

TABLEI TABLE I1
Amino acid compositionof 8-kDa peptides cleaved with Amino acid composition of 8-kDa peptides cleaved with S. aurew V8
endoproteinase lysine-C protease
Amino acid compositions were determined as described under Amino acid compositions were determined as described under
“Materials and Methods” using 20 h hydrolysis. Numbers in paren. “Materials and Methods” using 20 h hydrolysis. Numbers in paren-
theses are number of residues indicated by the complete sequence. theses are number of residues indicated by the complete sequence.
EL-1 EL-2 EL-3 Peak1 SV8-1 SV8-2 Peak 1 Peak 2 Peak 3 Peak 4
CYs CYS
ASP 2.00 (2) 5.00 (5) Asp 1.12 (1) 1.19 (1) 5.12 (5) 5.10 (5) 2.13 (2)
Thr 1.01 (1) Thr 0.99 (1) 1.03 (1)
Ser 2.49 (2) 1.99 (2) 2.08 (2) 1.41 (1) 3.14 (3) Ser 4.56 (4) 2.47 (2) 4.05 (4) 1.92 (2) 3.87 (4)
Glu 1.38 (1) 3.16 (3) 3.30 (3) 3.45 (4) Glu 1.30 (1) 2.45 (2) 6.58 (6) 7.35 (7) 1.07 (1) 3.60 (3)
Pro 2.93 (3) 0.88 (1) Pro 3.74 (4) 3.76 (4)
GlY 1.33 (1) G~Y 1.32 (1) 1.39 (1)
Ala 1.00 (1) 1.07 (1) Ala 1.20 (1) 1.07 (1)
Val 1.51 (1) Val 2.31 (1) 1.05 (1)
Met 0.78 (1) 0.51 (1) Met 1.00 (1) 0.90 (1) 0.93 (1) 0.82 (1)
Ile 0.76 (1) 1.95 (2) 1.24 (2) Ile 1.48 (2) 2.83 (4) 1.45 (2)
Leu 0.89 (1) 1.00 (1) 3.48 (4) 0.93 (1) Leu 1.03 (1) 1.18 (1) 4.00 (4) 3.96 (4) 2.00 (2)
TYr 0.82 (1) TYr 0.80 (1) 0.70 (1)
Phe 0.95 (1) 1.01 (1) Phe 0.92 (1) 0.94 (1) 1.77 (2)
His 1.97 (2) 1.01 (1) 1.06 (1) 2.47 (2) His 1.00 (1) 1.08 (1) 2.96 (3) 2.00 (2) 0.89 (1)
LYs 2.20 (3) 0.88 (I) 3.37 (4) 3.99 (4) Lys 1.87 (2) 1.18 (1) 3.76 (4) 5.28 (6) 1.68 (2) 2.60 (3)
Arg 0.96 (1) 0.97 (1) 2.52 (3) -4% 1.67 (2) 2.58 (3) 2.55 (3) 1.75 (2)
Trp NA“ (1) Trp NA” (1) NA” (1)
Total 11 16 8 26 17 Total
20 7 30 40 10 27

Position 33-43
27-43
1-26
60-67
44-59 Position
41-67
31-40
1-401-30
48-64
41-47
NA, not analyzed. a NA, not analyzed.

sitionsandretentiontime on the reverse-phase column.
Amino acid compositions of the rest of the peaks in Fig. 1B
indicated that they were fragments derived from the basic 30-
kDa domain of protein 4.1 (data not given). The absence of
these 30-kDa derived peptides in Fig. L4 further demonstrated
the specificity of the sedimentation assay. Peak 3 (Fig. 1 B )
was also used for the sequencing studies since it was identical
to thecorresponding peptide isolated by the alternatemethod
described above.
The intact 8-kDa domain (peak 3, Fig. L 4 ) was subjected
to automated Edman degradation. These results are summa-
rized in Fig. 4. Approximately half of the total sequence of
the domain was determined by sequence analysis of the intact
domain. The sequence of this region of the domain was
verified by sequence analysis of peak 3 in Fig. 1B (data not
shown). The NHz-terminal sequence of these two fractions
(peaks 3 in Fig. 1, A and B ) further confirmed their identity
as suggested above based on amino acid composition and
retention time on the reverse-phase column.
The reverse-phase separation of peptides obtained from the
8-kDa peptide following cleavagewith endoproteinase lysine-
C is shown in Fig. 2 and amino acid compositions are listed
in Table I. Peptides whose amino acid compositions fit the
known sequence (NHz-terminal sequence of the intact do-
main) were not treated further while those whose structures
were needed to document the complete sequence of the 8-kDa
peptide were sequenced to the COOH-terminal amino acid.
Only three of the peaks (EL-1, EL-2, and EL-3) were needed
for further sequencing analysis and theyresolved the complete
sequence of the 8-kDa peptide (Fig. 4). It was possible to
deduce the alignment of these peptides since EL-1 overlapped
the NHz-terminal sequence of the intact domain and EL-3
did not contain a lysine indicating that it derived from the
COOH-terminal end of the 8-kDa domain. Amino acid anal-
ysis showed that peak 1 in Fig. 2 corresponded to a peptide
containing residues 1-26 and peak 2 residues 27-43of the
domain sequence (Table I and Fig. 4).
The reverse-phase chromatogram of peptides obtained from
 Spectrin-Actin Binding Site of Erythrocyte
Protein 4.1 13365
10 20
Lys-Lys-Lys-Arg-Glu-Arg-Leu-AspGly-Glu-AIle-Tyr-Ile-Arg-His-Ser-~n-~t-

FIG. 4. The complete amino acid

sequence and peptide overlaps of 67
residues from the 8-kDa domain of 30 40
protein 4.1. Peptides obtained by
cleavage with endoproteinase lysine-C
~111-AspLeu-AspLys-Ser-Gl~l~l~Ile-Lys-Lys-His-~s-~a-Ser-Ile-Ser-Gl~
""_"
(EL) and S. aureus protease V8 (SV8)
are shown. Residues identified by auto-
mated Edman degradation are indicated
as follows: solid line, unambiguous iden-
tification of phenylthiohydantoin deriv-
ative; dashed line, tentative identificz-
tion. Residues are numbered consecu-
tively from the NH2-terminal end of the
8-kDa peptide.
-
I
50
Leu-Lys-Lys-Asn-Phe-Met-Glu-Ser-Val-~~l~~~Arg-~~r~l~~~pLys-Arg
5 2 -- 60

Leu-Ser-?hr-His-Ser-PFhe

- 5 3 *

1 3 R A r; R
1 2 3 4 5
A C A C A C A

3 ^ Spectrin
=
". -w - 0 -80
56
50
4 .I
34

28

-8

cT/4.1 o 1/400 Moo *A00 1/50

FIG. 5. Characterization of antibodies raised against syn-
thetic peptides of the 8-kDa domain. Protein 4.1 was digested
with a-chymotrypsin a t 0 "C for 30 min a t enzyme to substrateratios
( C T / 4 . I )ranging from 1:400 to 1:50. Proteins were run on a 10-15%
SDS-PAGE and electrophoretically transferred onto nitrocellulose
paper as described by Towbin (21). A mixture of antibody A and
antibody B was used to immunoblot the nitrocellulose. C, Coomassie
Blue stained proteins. A , autoradiogram of immunoblots. Numbers
designate the molecular weights of a-chymotrypticfragmentsin
kiladaltons. Red blood cell ghostmembranes (lunes I) were run
without digestion.
the 8-kDa peptide following cleavage with S. aureus protease
V8 is given in Fig. 3. This alternative cleavage method was
used to verify the alignment of the lysine-cleaved peptides
and confirm that these sequences were contiguous. Amino
acid compositions of the peaks labeled in Fig. 3 are summa-
rized inTable 11. Two peptides (SV8-1 and SV8-2) were
sequenced to establish the overlaps of the lysine-cleaved pep-
tides. The results of these sequence determinations are sum-
marized in Fig. 4. The amino acid analysis of theother
peptides from V8 cleavage indicated that:peak 1 corresponded
to residues 1-30; peak 2, residues 1-40; peak 3, residues 31-
40; and peak 4, residues 41-67.
The sequence summarized in Fig. 4 represents the entire8-
kDa complex-promoting domain of erythrocyte protein 4.1
and includes several interesting features. The peptide has a
highly charged NH2-terminal region; the first 6 residues and
8 of the first 10 are charged amino acids. Another cluster of
charged amino acids involves residues 29-35 which includes
FIG. 6. Inhibition of the ternary complex formation by an-
tibodies A and B. Protein 4.1 (8 pg) was incubated overnight a t
0 "C with monovalent Fab antibody (15 pg) or intact antibodies (24
pg of each antibody, A and B ) . Spectrin (33 pg) and F-actin (30 pg)
were added and incubated for an additional 45 min a t 0 "C and the
mixture was sedimented as previously described (14). Equivalent
samples of supernatant (S) and pellets (P) were electrophoresed on a
7-15% acrylamide gradient SDS gel. Activity was assessed based on
the distribution of spectrin between the supernatant and thepellet.
Spectrin and actin alone (lanes I); protein 4.1, spectrin and actin
(lanes 2); monovalent Fab-A antibody, protein 4.1, spectrin and actin
(lanes 3);monovalent Fab-B antibody, protein 4.1, spectrin and actin
(lanes 4 ) ; monovalent Fab-A and Fab-B antibodies, protein 4.1,
spectrin and actin (lanes 5);intact antibodies A and B, protein 4.1,
spectrin and actin (lanes 6 ) .The band migrating above actin in lane
6s is IgG-heavy chain.
6 charges in 7 positions. Overall nearly half of the residues in
this domain are charged residues (31 of 67) suggesting that
this entire segment may be exposed on the surface of the
intact molecule. Such an exposed configuration wouldbe
consistent with the role of the 8-kDa peptide as a promoter
of interaction between spectrinand F-actin. This domain
must bind to at least one other polypeptide chain in the
complex and could conceivably bind to asmany as threeother
polypeptides simultaneously (both spectrin subunits and ac-
tin).
Although we have no direct evidence that the8-kDa peptide
is phosphorylated, this domain has been located within the
10-kDa region of protein 4.1 which has previously been shown
to contain one or more sites for CAMP-dependent phospho-
rylation (15, 16). Previous work by Krebs and his associates
(for areview, see Ref. 22) has shown that thecatalytic subunit
13366 Spectrin-Actin Binding Site of Erythrocyte Protein 4.1
of cyclic AMP-dependent protein kinase recognizes sites of
phosphorylation on substrates that contain at least one posi-
tively charged residue followedby one or two intervening
residues between a serine or threonine, both of which can be
phosphorylated. Based on this criterion, serines in positions
17, 55, and 62 arepotentialsites for phosphorylation by
CAMP-dependent protein kinase. Serine in position 27 might
also be a candidate (23).
The 8-kDa peptide sequence has been compared to the
National Biomedical Research Foundation database of 3182
protein sequences (May 1985 version) and no significant
relationships to other known proteins have been detected.
Protein 4.1 may represent a new protein class or superfamily
and this report presents the first sequence information for
protein 4.1.
In order to furtherinvestigate the nature of the interaction
of the 8-kDa peptide with spectrin and F-actin, antibodies
against two synthetic peptides whose sequences corresponded
to residues 1-15 (antibody A) and residues 46-59 (antibody
B) of the 8-kDa peptide (Fig. 4) have been raised. The speci-
ficity of these antibodiesis demonstrated in Fig. 5. It has been
established that mild proteolysis of protein 4.1 by a-chymo-
trypsin cleaves the molecule mainly in three central regions
generating fragments of 30-, 16-, lo-, and 22/24-kDa, respec-
tively (15). The 8-kDa domain is located within the 10-kDa
region (14). Antibody A and antibody B (used as a mixture)
recognized the intact protein 4.1 and those a-chymotryptic
fragments of protein 4.1 which contained the 8-kDa peptide,
+ +
as 56-kDa (30 + 16 10-kDa), 50-kDa (16 10 + 22/24-
kDa), 32/34-kDa (10 + 22/24-kDa), 26-kDa (16 + 10-kDa),
and the8-kDa peptide itself (Fig. 5).
Monovalent Fab antibodieswere produced and their ability
to inhibit the interaction of protein 4.1 with spectrin and
actin investigated. In the experiment shown in Fig. 6,spectrin
and F-actin were incubated alone (lanes 1 ) or with protein 4.1
(lanes 2) or with 4.1 inthe presence of monovalent Fab
antibodies (lunes 3-5) or with 4.1 in the presence of intact
antibodies (lunes 6 ) . Either Fab-A (lanes 3)) or Fab-B (lanes
4 ) fragments or both fragments added together (lanes 5) are
capable of inhibiting the association of protein 4.1 with spec-
trin andF-actin. A mixtureof intact antibody A and antibody
B also inhibits the formation of the ternary complex (lanes
6). The fact that either Fab-A or Fab-B fragments areable to
prevent the interaction between protein 4.1, spectrin, and
actin may suggest that the entire 8-kDa peptide is required
for such interaction.Alternatively, one or both Fab fragments
might sterically block a binding site that is only part of the
8-kDa piece.
Analogs of protein 4.1 have been identified in a variety of
non-erythroid cells (12, 13),butthey have not yet been
characterized functionally. Avian erythrocytes contain mul-
tiple isoforms of protein 4.1 not found in red cell precursors
of chick embryos at different stages of development (24). This
suggests that different isoforms may play different functions
related to their role during cell development. It would be of
interest to determine which of these forms contain the 8-kDa
domain andto what degree this peptide is conserved in
different cells and tissues.
Acknowledgments-We thank Dr. William C. Horne for his helpful
discussions, Drs. Barbara-Jean Bormann and TakashiTobe for their
advice in the production of antibodies and Drs. Thomas L. Let0 and
Richard A. Anderson for their comments. We gratefully acknowledge
the expert technical assistance of Gary Davis, Raymond DeAngelis,
and Anthony Lanzetti. We thank Vicki Rosenweig for assistance in
typing this manuscript.
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