C. O.

Bewaji

Biokemistri Vol. 1, No. 1, June, 1991 0795-8080/94 $3.00 + 0.00

Printed in Nigeria Klobex Academic Publishers

IMMUNOLOGICAL CROSS-REACTIVITY BETWEEN THE Ca2+-ATPases

FROM HUMAN AND PORCINE ERYTHROCYTE MEMBRANES

Clement O. Bewaji*
Department of Biochemistry, University of Ibadan, Nigeria
and Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland

(Received June 30, 1989)

ABSTRACT: Antibodies against the purified porcine erythrocyte Ca2+-

pumping ATPase (ATP phosphohydrolase, EC 3.6.1.3) were raised in rabbits.
In tests of immunological cross-reactivity, using immunoblotting techniques,
these antibodies cross-reacted with the Ca2+-ATPase from human and porcine
erythrocyte membranes, suggesting that the two enzymes have similar
antigenic sites. The enzyme from the two species also have identical molecular
weight of about 140,000 as determined by sodium dodecyl sulphate
polyacrylamide gel electrophoresis. The specific activity of the porcine
erythrocyte Ca2+-ATPase in the native membrane, however, is significantly
higher that that of the human enzyme. It is suggested that porcine erythrocytes
might be a better source of the Ca2+-ATPase for further molecular studies.

INTRODUCTION
Studies on the enzyme in intact
The existence of a calcium-pumping
erythrocyte ghosts were made difficult by
ATPase (Ca2+-ATPase) has been
the presence of another ATP-splitting
demonstrated in a variety of plasma
enzyme, the Mg2+-ATPase. Attempts to
membranes where it pumps out Ca2+
purify the Ca2+-ATPase by conventional
from the cytosol. The activity of the
techniques were also frustrated because
enzyme is now believed to be
the enzyme is present in minute amounts
responsible for the maintenance of a
in the human erythrocyte membrane, and
10,000 fold Ca2+ gradient across the
it co-eluted with band 3 – one of the
plasma membrane [1].
most abundant proteins in the erythrocyte
The properties of the enzyme has been membrane. The enzyme was also
traditionally studied in the erythrocyte extremely labile in the solubilized state.
membrane, particularly human However, it has been successfully
erythrocytes, and it was thought that the purified in a functional state on a
enzyme was typical of non-excitable calmodulin affinity column [6,7].
cells. However, the enzyme has now
The major goal of understanding the
been isolated from the plasma membrane
molecular events produced by various
of excitable tissues such as heart [2],
effectors on the erythrocyte and other
synaptosomes [3], squid nerve [4] and
plasma membrane Ca2+-ATPases, as well
adipocytes [5].
as the overall mechanism of plasma
membrane Ca2+ transport, would require
*Present Address: Department of Biochemistry,
a knowledge of the primary structure of
University of Ilorin, Ilorin, Nigeria. the enzyme. Such studies have been

9
 Biokemistri Volume 1, No. 1 (1991)
hindered by the fact that the enzyme
represents only a minor fraction (less that
0.1%) of the total protein of the human
erythrocyte membrane. In a comparative
study of the activities of the membrane-
bound Ca2+-ATPase in mammalian
erythrocytes, it has been demonstrated
that the enzyme is more abundant in
porcine erythrocyte membranes [8,9]
This paper further characterizes the
porcine erythrocyte Ca2+-ATPase with
respect to some physical and
immunological properties compared with
those of the human enzyme.
MATERIALS AND METHODS
Prepatation of erythrocyte ghost
membranes. Haemoglobin-free ghost
membranes deficient in calmodulin were
prepared from human and porcine
erythrocytes by the recently developed
molecular filtration method using the
Millipore Pellicon Cassette System
[10,11]. The human erythrocytes used
were outdated cells obtained from the
Red Cross (Rotes Kreuz) Zurich. Fresh
pig blood was obtained from the Schweiz
Schlachthaus, Zurich. The blood was
stored in acid citrate dextrose buffer
(USP) at 4oC and processed within two
days. All steps of the membrane
preparation were carried out at 4oC.
Purification of calmodulin. Calmodulin
was purified from bovine brain by the
procedure described by Gopalakrishnan
and Anderson [12].
Purification of erythrocyte Ca2+-ATPase.
Erythrocyte Ca2+-ATPase was purified
on a calmodulin-Sepharose 4B affinity
column in the presence of phosphatidyl-
choline by the procedure of Niggli et al.
[6].
Determination of protein. Protein was
determined by a modification of the
procedure of Lowry et al. [13] described
10
by Markwell et al. [14], using bovine
serum albumin as standard.
Assay of Ca2+-ATPase activity. The
ATPase reaction was measured by the
pyruvate kinase, lactate dehydrogenase
coupled assay which monitors the
disappearance of NADH
spectrophotometrically [15]. Usually, 20
l of erythrocyte ghost membranes
(about 100-200 g protein) was added to
a cuvette containing 1 ml of the assay
buffer which contains, in final
concentrations, 130 mM KCl, 20 mM
HEPES, pH 7.4, 2 mM MgCl2, 10 M
free Ca2+, 1 IU pyruvate kinase/ml and 1
IU of lactate dehydrogenase/ml, and
thermostated at 37oC. When necessary,
about 3 g of calmodulin was added to
the reaction mixture. The absorbance
difference between 366 and 550 nm,
measured on an AMINCO Dual
Wavelength (DW-2) spectrophotometer,
was recorded for 4 minutes. EGTA was
then added to a final concentration of 0.1
mM and the recording was continued for
4 minutes. The Ca2+-ATPase activity was
calculated from the difference of the two
slopes.
Sodium dodecyl sulphate polyacrylamide
gel electrophoresis. Membrane proteins
and the purified Ca2+-ATPase were
subjected to electrophoresis using the
procedure of Weber and Osborn [16].
10% gels were routinely used. In some
cased, 12% gels according to Laemmli
[17] were used.
Production of antisera. One rabbit
(Chinchilla breed) was bled to obtain
about 2ml of pre-immune serum. The
rabbit was then immunized by
subcutaneous injection of 200 g of
purified porcine erythrocyte Ca2+-
ATPase mixed with 0.5 ml of Freund’s
complete adjuvant. Booster injections of
the same amount of antigen were
administered at intervals of two weeks.
Five days after the third dose, the rabbit
 C. O. Bewaji
was bled again and 5 ml of blood was
collected into a glass centrifuge tuble.
The blood was incubated at 37oC for 1 hr
then stored at 4oC for 16 hrs to allow the
clot to retract. The blood was then
centrifuged at 12,000 x g for 20 minutes
and the clear serum was stored at -80oC
until needed.
Immunoblotting. Purified Ca2+-ATPase
from human and porcine erythrocytes
were subjected to electrophoresis in one-
dimensional slab gels according to the
procedure of Laemmli [17]. The proteins
were then transferred to nitrocellulose
sheets essentially by the method of
Towbin et al. [18].
After the transfer, the nitrocellulose
sheet was soaked in 25 ml of phosphate
buffered saline (PBS) containing 10%
New Born Calf Serum (NBCS), and
incubated for 16 hr at 4oC. This step is to
block all non-specific antibody binding
sites on the nitrocellulose. The
nitrocellulose sheet was subsequently
incubated at room temperature for 1-3 hr
with a PBS solution containing 10%
NBCS and erythrocyte Ca2+-ATPase
antibody (0.1 mg IgG/ml). The
nitrocellulose sheet was then washed
three times (10 minutes each time) with
25 ml of PBS containing 10% NBCS,
and incubated for at least 2 hr with the
same solution into which a secondary
antibody, Goat anti-rabbit/IgG, has been
added. This was followed by three
washes of 10 min each with a PBS
solution, without NBCS.
The nitrocellulose sheet was finally
stained by adding 25 ml of PBS, 60 l of
a 10 mg/ml solution of o-dianisidine and
250l of 1% hydrogen peroxide, in that
order, until the blot became visible. The
sheet was then air-dried.
11
RESULTS
Table 1 shows that the specific activity
of the Ca2+-ATPase in unfractionated
porcine erythrocyte membranes is
significantly higher than in human
erythrocytes (p < 0.001). The two
enzymes respond similarly to stimulation
by calmodulin. The results of a typical
purification experiment for the porcine
erythrocyte Ca2+-ATPase is presented in
Figure 1. Figure 2 also shows Coomasie
Blue-stained polyacrylamide gels of the
enzyme at various stages of purification.
The purified enzyme shows a sharp band
corresponding to a molecular weight of
about 140,000 on the gel. The
experiment illustrated in Figure 3 shows
that the enzyme from the two species
react strongly on nitrocellulose sheets
with the antibodies raised against the
purified porcine erythrocyte Ca2+-
ATPase, suggesting that there are
immunological similarities between the
two proteins.
DISCUSSION
The results presented here show that
the Ca2+-ATPase purified from human
and porcine erythrocyte membranes are
similar in some important respects.
These include apparently identical
molecular weights of about 140,000;
stimulation by calmodulin and similar
immunological properties. The
immunological cross-reactivity of the
two enzymes suggests that they have
identical antigenic recognition sites. This
may mean that the amino acid sequence
in the two proteins are similar (if not
identical).
Verma et al. [19] have shown, using
the same immunological criteria, that
Ca2+-ATPases from plasma membrane
sources are structurally different from
skeletal muscle sarcoplasmic reticulum
Ca2+-ATPase. This finding has been
confirmed by Caroni et al. [20] who
 Biokemistri Volume 1, No. 1 (1991)

Table 1: Effect of calmodulin on the Ca2+-ATPase activity of human and porcine

erythrocyte membranes

Species Mg2+-ATPase Ca2+-ATPase

 calmodulin + calmodulin

Human 0.32 ± 0.01 0.36 ± 0.02* 0.77 ± 0.02

Porcine 0.85 ± 0.05 2.86 ± 0.06* 4.15 ± 0.35

Values are expressed in mole/mg. protein/hour ± S. D.

*t = 68.46, n = 4, p < 0.001 (Student’s t-test)

Fig. 1: Affinity chromatography of Triton-X100 solubilized ghosts from porcine

erythrocytes on a calmodulin-Sepharose 4B column. The ghosts were
solubilized and loaded on the column at the rate of 30ml/hr. The column was
washed at the same rate with a buffer containing 100 M CaCl2 and the Ca2+-
ATPase was eluted with a buffer containing 2mM EDTA at the points indicated
by the arrows. 0.5 ml fractions were collected and the absorbance at 280 nm
() and ATPase activities () of each fraction was checked.

12
 C. O. Bewaji

Fig. 2: 10% SDS polyacrylamide gel electrophoresis of human (lanes 1-4, left panels)
and porcine (lanes 1-4, right panels) erythrocyte Ca2+-ATPase at various stages
of purification. The lanes correspond to thevarious stages outlined in the legends
to Fig. 1. Intact ghost membranes (lane 1), Triton X-100 solubilized ghosts (lane
2), Peak eluted with Ca2+ buffer (lane 3), Peak eluted with EDTA buffer (lane 4).

Fig. 3: Cross-reactivity of antibodies raised against the purified porcine erythrocyte

Ca2+-ATPase with (A) porcine and (B) human erythrocyte Ca2+-ATPase.

13
 Biokemistri Volume 1, No. 1 (1991)
showed immunological dissimilarities
betwwen the heart sarcolemma and
muscle sarcoplasmic reticulum Ca2+-
ATPases.
Attempts have been made in some
laboratories to determine the amino acid
sequence of the human erythrocyte Ca2+-
ATPase [21, Carafoli, 1988 personal
communication]. However, these efforts
have met with a number of difficulties,
the most formidable of which are: (i) the
enzyme is present in very minute
amounts in the membrane, hence it is
difficult to obtain sufficient quantity for
amino acid sequence determination [11],
and (ii) the amino terminal of the
ATPase appears to be blocked (probably
acylated) thus no signal is obtained from
the sequenator which uses the Edman’s
degradation procedure [21].
Since porcine erythrocytes appear to
contain a lot more of the enzyme than
human erythrocytes [8, 9, 22], there is
the possibility of obtaining enough of the
enzyme for further molecular studies.
The immunological experiment reported
here provides indirect evidence that
human and porcine erythrocyte Ca2+-
ATPases may have identical primary
structures. If the amino terminal of the
porcine enzyme is also blocked,
proteolytic enzymes could be used as
tools to obtain a small polypeptide, from
the ATPase, whose amino acid sequence
could be easily determined. Trypsin has
been successfully used, in this respect, to
fragment the heart sarcolemma Ca2+-
ATPase [20], and human erythrocyte
Ca2+-ATPase [21].
ACKNOWLEDGEMENTS
This work was supported, in part, by a
study grant from the University of Ilorin,
Nigeria. The author wishes to thank
Professor Ernesto Carafoli (ETH,
Zurich) for laboratory facilities, advice
and encouragement.
14
REFERENCES
1. Carafoli, E. and Zurini, M. (1982) The Ca2+-
pumping ATPase of plasma membranes.
Purification, reconstitution and properties.
Biochim. Biophys. Acta 683, 279-301.
2. Caroni, P. and Carafoli, E. (1981) The Ca2+-
pumping ATPase of heart sarcolemma.
Characterization, calmodulin dependence,
and partial purification. J. Biol. Chem. 256,
3263-3270.
3. Hakim, G., Itano, T., Verma, A.K. and
Penniston, J.T. (1982) Purification of the
Ca2+ and Mg2+-requiring ATPase from rat
brain synaptic plasma membrane. Biochem.
J. 207, 225-231.
4. Beauge, L., Dipolo, R., Osses, L., Barnola, F.
and Campos, M. (1981) A Ca2+, Mg2+-
ATPase activity in plasma membrane
fragments isolated from squid nerves.
Biochim. Biophys. Acta 644, 147-152.
5. Pershadsingh, H.A.; Landt, M. and
McDonald, J.M. (1980) High affinity ATP-
dependent Ca2+ transport in adipocyte plasma
membranes: stimulation by calmodulin. Fed.
Proc. 39, 958 (Abstract).
6. Niggli, V.; Penniston, J.T. and Carafoli, E.
(1979) Purification of the (Ca2+ + Mg2+)-
ATPase from human erythrocyte membranes
using a calmodulin affinity column. J. Biol.
Chem. 254, 9955-9958.
7. Gietzen, K.; Tejcka, M. and Wolf, H.U.
(1980) Calmodulin affinity chromatography
yields a functional purified erythrocyte (Ca2+
+ Mg2+)-dependent adenosine triphosphatase.
Biochem. J. 189, 81-88.
8. Bewaji, C. O. and Bababunmi, E.A. (1987)
Abundance of the Ca2+-pumping ATPase in
pig erythrocyte membranes. Biochem. J. 248,
297-299.
9. Bewaji, C.O.; Olorunsogo, O.O. and
Bababunmi, E.A. (1985) Comparison of the
membrane-bound (Ca2+ + Mg2+)-ATPase in
erythrocyte ghosts from some mammalian
species. Comp. Biochem. Physiol. 82B, 117
– 122.
10. Rosenbery, T.L.; Chen, J.F.; Lee, M.L.;
Moulton, T.A. and Onigman, P. (1981)
Large-scale isolation of human erythrocyte
membranes by high volume molecular
filtration. J. Biochem. Biophys. Methods 4,
39 – 48.
11. Gietzen, K. and Kolandt, J. (1982) Large-
scale isolation of human erythrocyte Ca2+-
transport ATPase. Biochem. J. 207, 155 –
159.
12. Gopalakrishnan, R. and Anderson, W.B.
(1982) Ca2+-induced hydrophobic site on
calmodulin: application for purification of
 C. O. Bewaji
calmodulin by phenyl-Sepharose affinity
chromatography. Biochem. Biophys. Res.
Commun. 104, 830 – 836.
13. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.
and Randall, R.J. (1951) Protein
measurement with the Folin phenol reagent.
J. Biol. Chem. 193, 265 – 275.
14. Markwell, M.K.; Haas, S.M.; Tolbert, N.E.
and Bierber, L.L. (1981) Protein
determination in membrane and lipoprotein
samples: manual and automated procedures.
Methods Enzymol. 72, 296 – 303.
15. Yeh, L-A.; Ling, L.; English, L. and Cantley,
L. (1983) Phosphorylation of the (Na,K)-
ATPase by a plasma membrane-bound
protein kinase in Friend erythroleukemia
cells. J. Biol. Chem. 258, 6567 – 6574.
16. Weber, K. and Osborn, M. (1969) The
reliability of molecular weight determination
by dodecyl sulphate polyacrylamide gel
electrophoresis. J. Biol. Chem. 244, 4406 –
4412.
17. Laemmli, U.K. (1970) Cleavage of structural
proteins during the assembly of the head of
bacteriophage T4. Nature (London) 227, 680
– 685.
18. Towbin, H.; Staehelin, T. and Gordon, J.
(1979) Electrophoretic transfer of proteins
15
from polyacrylamide to nitrocellulose sheets:
Procedure and some applications. Proc. Natl.
Acad. Sci. (USA) 76, 4350 – 4354.
19. Verma, A.K.; Gorski, J.P. and Penniston, J.T.
(1982) Antibodies directed against human
erythrocyte Ca2+-ATPase: Effect on enzyme
function; immunoreactivity of Ca2+-ATPase
from other sources. Arch. Biochem. Biophys.
215, 345 – 354.
20. Caroni, P.; Zurini, M.; Clark, A. and
Carafoli, E. (1983) Further characterization
and reconstitution of the purified Ca2+-
pumping ATPase of heart sarcolemma. J.
Biol. Chem. 258, 7305 – 7310.
21. Emelyanenko, E.I.; Shakhparonov, M.I. and
Modyanov, N.N. (1985) Limited proteolysis
of human erythrocyte Ca2+-ATPase in
membrane-bound form. Identification of
calmodulin-binding fragments. Biochem.
Biophys. Res. Commun. 126, 214 – 219.
22. Bewaji, C.O. (1985) The isolation and
characterization of porcine erythrocyte Ca2+-
adenosine triphosphatase. Ph.D. Thesis,
University of Ibadan, Nigeria. 202 pp.