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Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells
Vol. 111, No. 3, 1983 March
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
29, 1983
Pages
EFFECT OF HEMOLYSATE ON CALCIUM INHIBITION (Na+ + K+)-ATPase Douglas Department
and Michael
Wayne State
'Detroit, Received
February
OF THE
OF HUMAN RED BLOOD CELLS
R. Yingst
of Physiology,
970-979
J. Marcovitz
University
Michigan
School
of Medicine,
48201
14, 1983
The sensitivity of the (Nat + K+)-ATPase in human red cell membranes to inhibition by Ca*+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca*+ causing 50% inhibition of the (Nat + Kt)-ATPase is shifted from greater j?a;hz; uM free Cazt in the absence of hemolysate to less than 10 uM free Ca hemolysate diluted 1:6U compared to --in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Cazt. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the to Ca*+ in a dose-dependent fashion, causing over 80% (Nat f Kt)-ATPase inhibition of the (Nat + Kt)-ATPase at 10 $I free Ca*+ at the highest concentration of the extract tested. The active factor in this membrane because it had no effect on the (Nat + Kt)-ATPase extract is Ca*+-dependent, in the absence of Ca*+. Trypsin digestion prior to the assay destroyed the ability of this rotein extract to increase the sensitivity of the (Nat + Kf)-ATPase to Ca 8t .
It (1)
has been known
and the
(Nat
for
some time
t K+)-ATPase
(Nat
t Kt)-ATPase
from
free
Ca*'
50% inhibition
causing
has been estimated to the
concentration
other
cells
membranes
of the
reported
that
(Nat
from
inhibits
the
(Nat
cells,
+ K')-pump
100 IJM (5)
which
50% inhibition
human red cells
+ K+)-pump
(Nat
of resealed
of
is
970
intracellular
reasonably (Nat
close
+ Kt)-ATPase
More
recently
human red cell
ghosts
Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N'-Z-ethanesulfonic acid; EGTA, ethyleneglycol-bis(B-aminoethyl ether)N,N'-tetraacetic EDTA, ethylenediaminetetraacetic acid. 0006291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
does the
in human red cells
of the (2).
+ K')-pump
as it
The concentration
(3,4).
of Ca*+ causing
unsealed
Ca*'
of human red blood
to be approximately
in white
the
(2)
that
it
acid;
was
and
BIOCHEMICAL
Vol. 111, No. 3, 1983
containing
Ca2+- sensitive
the
cellular
Ca*+ is
free
approximately (Nat
ten
t K+)-pump
sensitivity cells
may simply
afforded
by the
does
explain
not
pump in
resealed
explain
this
showed
the
measuring if
the
Cazt
the
in
improved
the
difference
ghosts
it
broken
membranes
(Nat
contains
arsenazo
ghosts
+ K+)-ATPase that
had been washed activity. a factor
the Ca2+
compared
to
intact free
reasoning,
between of broken
the
Ca2+
however, (Nat
+ K+)-
membranes.
To
the
resealed
ghosts
cytoplasm
diluted
20 fold
free
The present which
either
higher
Similar
intra-
Ca2+ (6),
of intracellular
III.
to Ca*+ contained
+ K+)-ATPase
hemolysate
resealed detection
(Nat
for
The apparent
may be significant
sensitivity
10 PM free
in Ca*+ sensitivity
and the
to monitor
estimated
t K+)-ATPase.
t Kf)-pump
RESEARCH COMMUNICATIONS
ITI
than
previously
use of entrapped
difference
(Nat
on the
(Nat
arsenazo
50% by less thdn
(Nat
reflect
a higher
whereas
lower
or the
of the
chromophore
inhibited
fold
AND BIOPHYSICAL
increases
of cytoplasm study the
prior
was designed inhibitory
which
to to test effect
of
t K+)-ATPase.
MATERIALS
AND METHODS
Membranes used in the experiments shown in Figures 1, 2, and 3 were prepared according to Muallem and Karlish (7). Two to four day old aircontaminated bank blood was washed 3 times in 10 volumes of cold 172 mM Tris-HCl (pti 7.6 at 22'C) by centrifuging for 1 minute at 12,000g at 4°C in a Sorvall RC-56 with an SS-34 rotor (Newtown, CT). The buffy coat was removed by aspiration after each wash. Cells were centrifuged at 12,DOOg for 5 minutes, the supernatant was removed, and one volume of packed. cells were hemolysed in 10 volumes of distilled water at 0°C and stirred for five minutes. This solution was centrifuged for 10 minutes at 45,OOOg, and the supernatant which constitutes the hemolysate was aspirated and frozen at -20°C for later experiments. The ghosts were washed and centrifuged three times in 20 volumes of ice cold 1 mM MgC12 and 2 mM Hepes-Tris (pH 7.4 at 22°C) at 45,000g for 5 minutes and then suspended in 25 volumes of a solution containing 2 mM Hepes-Tris and 5 mM EGTA-Tris (pti 7.4 at 22°C). This solution was frozen for 30 min in liquid nitrogen, thawed and incubated for 30 min at 37"C, then refrozen and reincubated, a procedure designed to remove proteins such as calmodulin which may bind to the membranes in a Ca-dependent fashion (7). The ghosts were then centrifuged, washed four times in the solution consisting of 1 mM MgC12 and 2 mM Hepes-Tris and frozen at -20°C for future experiments. Membranes used in Figures 4 and 5 were prepared by the Dodge procedure Two to four day old air-contaminated bank blood was washed 3 times in (8). equal volumes of 310 ideal mOsm sodium phosphate buffer, pH 7.4, by centrifuging for 1 min at 12,OOOg at 4°C. The buffy coat was removed by aspiration after each wash. After the last wash the cells were centrifuged for 5 minutes at 12,000g and all of the supernatant removed. Approximately 20 ml 971
Vol. 111, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of these packed cells were combined with 20 ml of the 310 mOsm buffer, mixed and then rapidly poured into 560 ml of ice cold 20 mOsm sodium phosphate buffer, pH 7.4. This solution was stirred on ice for 5 minutes and centrifuged 40 minutes at 40,OOOg at 4°C. The supernatant was decanted, the button on the bottom of the tube aspirated off, and the membranes washed two more tiIWS in 30 volumes of the 20 mOsm buffer. The resultant ghosts were combined into 4 centrifuge tubes and washed 2 more times in the 20 mOsm buffer centrifuging at 40,OOOg for 30 minutes. In order to remove proteins that bind to the membrane in a Ca-dependent manner, the membranes were suspended in 5 volumes of 0.1 mM EDTA, 1.0 mM Tris, pH 8.0 and incubated at 37°C for 30 minutes following the procedures of Mauldin and Roufogalis (9). The mixture was cooled for 5 minutes and centrifuged for 20 minutes at 40,OOOg. The supernatant was saved (EDTA extract see below) and the membranes resuspended to the original volume with 5.0 mM Tris:HCl, 15 mM NaCl, pH 7.2 and frozen at -20°C. Before an assay the ghosts were thawed and washed 3 times in 30 volumes of 5 mM Tris:HCl, 15 mM NaCl, pH 7.2, centrifuging at 30,OOOg for 5 minutes. The membranes were then suspended in the same buffer and added to the assay at the appropriate time. The supernatant obtained after the first centrifugation following the EDTA extraction was concentrated 6 fold on an Amicon PM 30 membrane under nitrogen pressure at 25 psi. It was then diluted 10 fold with 5 mM Tris:HCl, 15 mM NaCl, pH 7.2 and reconcentrated under the same conditions. This procedure was repeated twice more to assure that all the phosphate and EDTA was washed through the PM 30 membrane and out of the sample. The final protein concentration of the extract was approximately 1 mg/ml. Inorganic phosphate was analyzed by the method and protein according to Lowry (11). Free Ca2+ and the total concentration of EDTA, Ca2+, and Mg2+ (12) appropriate associations constants from Martell and 37°C and 0.17 M. All chemicals were reagent grade. muscle prepared to be low in Ca2+ and "vanadium free"
of Brotherus et al. (10) Mg2+ were calculated from using constants for the Smith (13) corrected for ATP was from equine (Sigma, St. Louis, MO).
RESULTS The effect the
presence
Figure
1.
ATPase,
activity
inhibits
absence (Ca2+ the
and absence The membranes
the
which
of Ca2+ on the ATPase
(Nat
of ouabain
types
the
In the
of Ca2+ stimulates 1).
contain
activity
Mg2+ - ATPase.
(Fig.
from
the
Ca*+,
increases
ATPase,
and increases
in the
(2)
the
human red blood of ATPase
measured
(Nat
in
presence presence
+ Mg2+)-ATPase
inhibitory
is
activity:
effect
shown
(1)
mixture
the the
in the and (3)
(Nat
+ K+)10,
of Ca2+ on the
(Ca*+
(Nat
minus
concentration
containing
of Ca2+ on the
in
of ouabain
of Ca2+ and ouabain increasing
in
Mg2+-
activity
of ouabain;
and inhibits
to assay
effect
cells
+ K+)-ATPase,
of hemolysate
stimulating
membranes
of Ca2+ and presence
in the
hemolysate the
of human red cell
absence
activity
absence
(Cazt
Adding
UM free
three
t K+)-ATPase;
minus
t Mg2+)-ATPase,
ATPase
of hemolysate
measured
the
activity
25 and 50 t Mg*+)-
+ K+)-ATPase
Vol.
111,
No.
BIOCHEMICAL
1983
3,
Ouob co*+ Herno,
Fig.
(Fig.
1.
Kf)-ATPase
0 IJM free
or
the
component
of
free
the
of the
- + 2525 - -
-t 00 ++
- + 5050 - -
(Nat
and
-+ 1010 ++
-+ 2525 ++
-+ 5050 tt
(PM)
hemolysate
on
is
maximal
of
the
hemolysate
to
10
The
increased
by
(Nat free
the
inhibition similar
shift
in caused
to
that
effect
only
the
1 is
shown
and
is
Cazt-inhibition
+ K+)-ATPase Ca2+
no
on
either
the
(Na+
At
0 uM
1).
+ K+)-ATPase
inhibition
is
(Fig,
has
Figure
evidenced
Ca2+
hemolysate
in
2).
$I
the
presented
is
experiment
- + 10 10 - -
-ATPase
data
hemolysate
this
Mg 2i-ATPose
Ca2+
(Fig.
in
0
Ca
Mg*+
hemolysate
increase
(No'+ K+)-ATPose (Co*++Mg*')-ATPose
- + 0 0 - -
At
effect
Ca2+
m m
RESEARCH COMMUNICATIONS
The effect of Ca2+ and hemolysate on the (Nat + K+)-ATPase, (Ca2+ + Mg2+)-ATPase, and Mg2+-ATPase in human red cell membranes. The assay was conducted by first preincubating the membranes for 10 minutes at 37°C in the assay mixture. The reaction was initiated by the addition of ATP to a final concentration of 1 I#!. Fifteen minutes later the reaction was stopped and the concentration of inorganic phosphate analyzed. The final assay volume after the addition of the ATP was 0.6 ml and contained 204 ua membrane orotein. 18 nF1 NaCl, 30 ai KCl, 112 mM choline Cl, 20 at! Hepes-Tris, 5 n@lEDTA, 0.2% bovine serum albumin, pH 7.3, and where a propriate, 5.5 mM Mg (0 free Ca2+), 1.84 mM Ca2+ and 3.68 mM f4g$+ (10 uM free Ca2+, 500 LIM fr e Mg2+), 2.99 p Ca2+ and 2.56 r&l Mg2+ (25 PM free Ca2+, 500 PM M#+), 3.79 mM Ca +, 1.79 mM Mg2+ (50 UM free Ca2+, 500 UM free Mg +I, 0.5 mM ouabain, and 0.1 ml hemolysate, representing a 60 fold dilution of the original cellular contents. The rate of ATP hydrolysis was calculated as phosphate liberated per mg membrane protein per minute after subtracting off less than 1.3% of the ATP found hydrolyzed in the absence of membranes. Mean values with standard deviations are given for triplicate samples. These results from one experiment are representative of 4 others.
1).
The
AND BIOPHYSICAL
in
amount
from
50
the
presence by
the
found
973
in
not
Figure
2.
affected
by
produced of UM free of
hemolysate in
ouabain-sensitive
9 other
Ca2+
by
the
necessary
for
in
absence
Ca*+ hemolysate at
the
the (Fig.
10
separate
UM free
50%
2). Ca2+
experiments
of The in
t
Vol. 111, No. 3, 1983
BlOCHEMlCAL
0
Hemol. Fig.
using
2.
AND BIOPHYSICAL
0
10 IO
25 25
50 50
- +
- t
- t
- t
RESEARCH COMMUNICATIONS
(pM)
The effect of Ca2+ and hemolysate on the (Na+ + K+)-ATPase. The data are from Figure 1 and were calculated for a given set of conditions by taking the difference between randomly matched values plus and minus ouabain, and then calculating the mean and standard deviations of these differences. The standard deviations are larger for the conditions where both CaZ+ and hemolysate are present because the values are differences between two larger numbe s due to concomitant stfmulati n of the (Ca2+ + Mg2')-ATPase by Caht and hemolysate (Fig. 11. Cakt and the hemolysate interacted in a significant fashion (P < 0.051 to alter the activity of the (Nat + K+)dTPase, as shown by a two-way analysis of variance.
similar
concentrations
of hemolysate
under
comparable
conditions
(data
not shown. Boiling ability
to
(Fig. the
the
hemolysate
increase
the
3) and only (Ca*+
Mg2+)-ATPase of the
(Nat
In the of the
(Nat
as did
+ K+)-ATPase
(data
not
(data
its
not the
was carried
it
to the membranes
of Ca2+ on the ability
shown).
in place
hemolysate,
above experiments + K+)-ATPase
effect
reduced
calmodulin the
to adding
inhibitory
slightly
+ Mg2+)-ATPase
0.2 PM of purified
prior
of hemolysate
but
did
not
the
activity
experiments, simulated
increase
its
(Na+ t K+)-ATPase
to stimulate In other
destroyed
the
of
adding the
(Ca2+ +
Ca2+ inhibition
shown). effect
of the
out using 974
hemolysate red cell
on Ca2+ inhibition membranes
prepared
by
Vol.
111,
No.
6IOCHEMlCAL
3, 1983
AND BIOPHYSICAL
0 0 -tB
Hemol. Fig.
3.
a procedure were
prepared that
increase
the
that would Ca2+ deplete
the
lo lo 10 -tB
(PM)
The effect of boiled hemolysate on the (Nat + K+)-ATPase at 0 and 10 $4 free Cc?+. The experiment was carried out as described in the legend to Figure 1 except that in this case the preincubation was 15 minutes and 120 mM NaCl, 20 mM choline, and 232 ug membrane protein were substituted for the value of those constituents listed in Figure 1. The concentrations of both membranes and hemolysate are similar to that shown previously, but are from different preparations. The hemolysate sample marked "B" is from the same stock as that marked "t", but was boiled for 5 minutes prior to the assay, filtered on Whatman #2 paper, and then added in the same volume as "t". The ouabain-sensitive differences for each condition are between six values with ouabain randomly matched with six values without ouabain as described in Figure 2.
which
proteins
0
RESEARCH COMMUNICATIONS
depletes
in
this
might
removal
increase inhibition. membranes
in
bind
sensitivity of
the
manner also
Ca2+
membranes
such
of
an
attempt
the
membrane
of
the
(Na+
of
To
test
if
of
the
protein
these
calmodulin
to
to
components
probability
bound
prior
in
the an
procedures which
increases
975
other
The
(Nat effect
which
It
manner was
anticipated
+ Kt)-ATPase
assay
of
the
remove Ca*+
membranes
cytoplasmic
a Ca*+-dependent
+ K+)-ATPase. to
observing
remove
(7).
inhibition
hemolysate
and
on
calmodulin
also of
the
Vol. 111, No. 3, 1983
Fig.
(Na'
4.
resultant
Methods). ATPase
at
increased up
to
extract
had
Dodge protein the
effect
0 and
10
uM free
percent
in
membranes
mixture
Then
the 80%
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The effect of proteins from the membranes of Dodge ghosts on the (Na+ + Kf)-ATPase at 0 and 10 uM free Ca*+. Each panel shows one of four similar experiments with each point representing the mean and standard deviation of three values. The protein extract contains proteins that were removed from Dodge ghosts incubated at 37'C in 0.1 mM EDTA, 1.0 mM Tris:HCl, pH 8.0 and subsequently separated from the membranes and concentrated on an Amicon PM 30 membrane, as described in the Materials and Methods. The assay ghosts are those from which the proteins were extracted. Note, the protein extract has no effect on the (Na+ + K+)-ATPase measured at D $I free Ca*+, whereas it inhibits the (Na+ + K+)-ATPase in a dose dependent manner when 10 uM free Ca is present in the assay. This suggests that the extract increased Ca-induced inhibition of the (Na+ + K+)-ATPase. The assay was cdrried out under the conditions specified in the legend to Figure 1, except that in this case the incubation time was 40 minutes and 20 mM NaCl, 10 mM KCl, 89 mM choline Cl, 20 mM Pipes:Tris, and 120 to 160 &ml membrane protein were substituted for the value of those constituents listed in Fig. 1. In all four experiments the extract and the Ca*+ interacted in a si nificant fashion (P < 0.05) to alter the rate of the (Na 9 + Kf)-ATPase, as demonstrated in a two-way analysis of variance.
+ Kt)-ATPase,
the
BIOCHEMICAL
of
concentrated
and
sensitivity
of
indicating
that
on
washed the the
concentrated
the
extract (Fig.
inhibition
effect
at
(Nat
on
(Nat an
The
uM free
(Fig.
in
an
Amicon
PM 30
measured
on
4).
(see
+ K+)extract
Cazt
from
less
than
20
percent
absence
of
Ca2+
the
In
inhibition
976
filter
the
membrane
from
and
that
XM 100
distinct
(Na+
(9)
show
(Fig.
to
the
EDTA
data
+ Kt)-ATPase
Amicon
is
extracted on
was
10
+ Kt)-ATPase
component
first
4).
manner the
were
was
Ca2+
a dose-dependent no
(8)
the 4). had by
the
Similar no
Ca*+
non-calmodulin
effect (data
extracts on not
the shown),
activator
BIOCHEMICAL
111, No. 3, 1983
Vol.
cQ+
of
the If
f
the
the
Mg*+)-ATPase
to
which
concentrated
Ca2+
is
stays
extract
its
membranes,
ATPase
-
The rate of (Na+ + K+)-ATPase activity at 0 and 10 $i free Ca*+ in the presence and absence of the membrane extract, and in the presence of extract that had been digested with trypsin. The protein was digested by adding 1 Pg of trypsin/25 pg protein and incubating at 37°C for 12 hours.The (Nat+ K+)-ATPase was carried out as described in Figure 1 under the conditions of Figure 4, except that the membranes had a final concentration of 211 Kg/ml. Each value is the mean of the difference between 3 samples without The bars ouabain minus 3 randomly matched values with ouabain. This experiment are the standard deviations of these differences. was repeated four other times with similar results.
(Ca*+
(9). to
5.
RESEARCH COMMUNICATIONS
0 m
Extraa Trypein Fig.
AND BIOPHYSICAL
ability
is to
destroyed
on top digested
alter
(Fig.
of
the
an Amicon
with
XM 100
trypsin
sensitivity
prior of
the
membrane to
(Na+
adding t
Kf)-
5). DISCUSSION
The than
above
calmodulin
binds
to
the
sensitivity favor boiling Ca*+
results that
is
membrane of
of
could
the
present in
is
the
hemolysate
sensitivity
of
in
+ Kf)-ATPase that
the
explained
by
the
of
manner, to
digesting
a Ca2+-dependent
cytoplasm
a Cazf-dependent
(Nat
a protein
be
the
destroys
the
ability
(Nat
+ Kt)-ATPase.
by
membrane
This 977
and
inhibition
of
human
red
increases Cazt.
extract either
protein
proteinaceous
cells,
the Evidence
in to
blood
other
affect
trypsin
in or
the factor
is
it
Vol. 111, No. 3, 1983
BIOCHEMICAL
Cazt-dependent,
because
of Ca*+.
are
not
There
calmodulin.
membranes second
The first
is that
be established
directly
depends
on the
the
protein (Nat
free
variety
presence
it
could
sensitivity
of the
(Nat
reltionship
between
the
the
be involved
generation
and the
point
it
to play the
t K+)-pump (Nat
in the
could
is
which It
is must now
extract
that
via
a mechanism
Ca*+-dependnent
acts
effect
of
by changes
Such an increase regulatory
increased muscle,
and the
inhibi-
Ca*+ sensitivity
be regulated
an important
in smooth
inotropic
low
range.
protein
t K+)-pump
of hypertension
The
due to a single
effect
the otherwise
in the physiological
if
to the
(14).
membrane
of this
where
instance,
is
factors.
increase
be anticipated For
is
significance
to the
protein
hemolysate,
Ca*+ inhibition
the
presence
(Na+ t K+)-ATPase,
stable
of other
this
calmodulin
of the
or whether
in the
that
to be heat
hemolysate
would
Ca*+
of cells.
thereby
the
+ K+)-ATPase
t K+)-ATPase
intracellular
purified
effect
increased
+ K+)-ATPase
of the
is known
the
that
(Nat
RESEARCH COMMUNICATION:
to suggest
adding
the
physiological
is
sensitivity
that
calmodulin
(Nat
The possible tory
is
in both
on the
the
of evidence
destroyed
whether
present
affects
on Ca*+ inhibition
boiling
because
component
only
two pieces
had no effect
signifiant
that
it
AND BIOPHYSICAL
it
Ca*+
could
alter
Na+/Ca*+
of cardiac
in
role
the
exchange
in a
the system
glycosides
of
(15)
and
and
(16).
ACKNOWLEDGEMENTS I thank This
work
and with part
Mrs.
Daniela
was supported funds
contributed
by a PMA Foundation
M. Polasek
for
by a Grant-in-Aid in
part
Research
excellent from
technical the American
by the Michigan Starter
Heart
assistance. Heart
Association
Association
Grant.
REFERENCES 1. 2. 3. 4.
Hoffman, J.F. (1962) Circulation. 26, 1201-1213. I.M. (19611 J. Physfol. (London). Dunham, E.T., and Glynn, 274-293. Skou. J.C. (1960) Biochim. Biophys. Acta. 42, 6-23. Whittam. R. and Blond, D.M. (1964) Biochem. J. 92, 147-158. 978
156,
and in
Vol. 111, No. 3, 1983
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B. (1977) Acta. Biol. Med. Ger. Gardos, G., Szasz, I., and Sarkadi, 36, 823-829. Yingst, D.R. (1982) Fed. Proc., Fed. Am. Sot. Exp. Biol. 41, 974. Muallem, S. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta. 597, 631-636. Dodge, J.T., Mitchell, C., Hanahan, D.T. (1963) Arch. Biochem. Biophys. 100, 119-150. Mauldin, D. and Roufogalis, B.D. (1980) Biochem. J. 187, 507-513. Brotherus, J.R., Moller, J.V., Jorgensen, P.L. (1981) Biochem. Biophys. Res. Commun. 100, 146-154. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, A.J. (1951) 3. Biol. Chem. 193. 265-275. Wolf, H.U. (1973) Experientia 29, 241-249. Martell, A.E. and Smith, R.M. (19741 Critical Stability Constants, Amino Acids, p. 204, Plenum Press, New York. Cheung, W.Y. (1971) 3. Biol. Chem. 246, 2859-2869. Biedert, S., Barry, W.H., and Smith, T.W. (1979) J. Gen. Phys. 74, 479-494. Blaustein, M.P. (1977) Am. J. Physiol. 232, C165-C173.
979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
29, 1983
Pages
EFFECT OF HEMOLYSATE ON CALCIUM INHIBITION (Na+ + K+)-ATPase Douglas Department
and Michael
Wayne State
'Detroit, Received
February
OF THE
OF HUMAN RED BLOOD CELLS
R. Yingst
of Physiology,
970-979
J. Marcovitz
University
Michigan
School
of Medicine,
48201
14, 1983
The sensitivity of the (Nat + K+)-ATPase in human red cell membranes to inhibition by Ca*+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca*+ causing 50% inhibition of the (Nat + Kt)-ATPase is shifted from greater j?a;hz; uM free Cazt in the absence of hemolysate to less than 10 uM free Ca hemolysate diluted 1:6U compared to --in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Cazt. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the to Ca*+ in a dose-dependent fashion, causing over 80% (Nat f Kt)-ATPase inhibition of the (Nat + Kt)-ATPase at 10 $I free Ca*+ at the highest concentration of the extract tested. The active factor in this membrane because it had no effect on the (Nat + Kt)-ATPase extract is Ca*+-dependent, in the absence of Ca*+. Trypsin digestion prior to the assay destroyed the ability of this rotein extract to increase the sensitivity of the (Nat + Kf)-ATPase to Ca 8t .
It (1)
has been known
and the
(Nat
for
some time
t K+)-ATPase
(Nat
t Kt)-ATPase
from
free
Ca*'
50% inhibition
causing
has been estimated to the
concentration
other
cells
membranes
of the
reported
that
(Nat
from
inhibits
the
(Nat
cells,
+ K')-pump
100 IJM (5)
which
50% inhibition
human red cells
+ K+)-pump
(Nat
of resealed
of
is
970
intracellular
reasonably (Nat
close
+ Kt)-ATPase
More
recently
human red cell
ghosts
Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N'-Z-ethanesulfonic acid; EGTA, ethyleneglycol-bis(B-aminoethyl ether)N,N'-tetraacetic EDTA, ethylenediaminetetraacetic acid. 0006291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
does the
in human red cells
of the (2).
+ K')-pump
as it
The concentration
(3,4).
of Ca*+ causing
unsealed
Ca*'
of human red blood
to be approximately
in white
the
(2)
that
it
acid;
was
and
BIOCHEMICAL
Vol. 111, No. 3, 1983
containing
Ca2+- sensitive
the
cellular
Ca*+ is
free
approximately (Nat
ten
t K+)-pump
sensitivity cells
may simply
afforded
by the
does
explain
not
pump in
resealed
explain
this
showed
the
measuring if
the
Cazt
the
in
improved
the
difference
ghosts
it
broken
membranes
(Nat
contains
arsenazo
ghosts
+ K+)-ATPase that
had been washed activity. a factor
the Ca2+
compared
to
intact free
reasoning,
between of broken
the
Ca2+
however, (Nat
+ K+)-
membranes.
To
the
resealed
ghosts
cytoplasm
diluted
20 fold
free
The present which
either
higher
Similar
intra-
Ca2+ (6),
of intracellular
III.
to Ca*+ contained
+ K+)-ATPase
hemolysate
resealed detection
(Nat
for
The apparent
may be significant
sensitivity
10 PM free
in Ca*+ sensitivity
and the
to monitor
estimated
t K+)-ATPase.
t Kf)-pump
RESEARCH COMMUNICATIONS
ITI
than
previously
use of entrapped
difference
(Nat
on the
(Nat
arsenazo
50% by less thdn
(Nat
reflect
a higher
whereas
lower
or the
of the
chromophore
inhibited
fold
AND BIOPHYSICAL
increases
of cytoplasm study the
prior
was designed inhibitory
which
to to test effect
of
t K+)-ATPase.
MATERIALS
AND METHODS
Membranes used in the experiments shown in Figures 1, 2, and 3 were prepared according to Muallem and Karlish (7). Two to four day old aircontaminated bank blood was washed 3 times in 10 volumes of cold 172 mM Tris-HCl (pti 7.6 at 22'C) by centrifuging for 1 minute at 12,000g at 4°C in a Sorvall RC-56 with an SS-34 rotor (Newtown, CT). The buffy coat was removed by aspiration after each wash. Cells were centrifuged at 12,DOOg for 5 minutes, the supernatant was removed, and one volume of packed. cells were hemolysed in 10 volumes of distilled water at 0°C and stirred for five minutes. This solution was centrifuged for 10 minutes at 45,OOOg, and the supernatant which constitutes the hemolysate was aspirated and frozen at -20°C for later experiments. The ghosts were washed and centrifuged three times in 20 volumes of ice cold 1 mM MgC12 and 2 mM Hepes-Tris (pH 7.4 at 22°C) at 45,000g for 5 minutes and then suspended in 25 volumes of a solution containing 2 mM Hepes-Tris and 5 mM EGTA-Tris (pti 7.4 at 22°C). This solution was frozen for 30 min in liquid nitrogen, thawed and incubated for 30 min at 37"C, then refrozen and reincubated, a procedure designed to remove proteins such as calmodulin which may bind to the membranes in a Ca-dependent fashion (7). The ghosts were then centrifuged, washed four times in the solution consisting of 1 mM MgC12 and 2 mM Hepes-Tris and frozen at -20°C for future experiments. Membranes used in Figures 4 and 5 were prepared by the Dodge procedure Two to four day old air-contaminated bank blood was washed 3 times in (8). equal volumes of 310 ideal mOsm sodium phosphate buffer, pH 7.4, by centrifuging for 1 min at 12,OOOg at 4°C. The buffy coat was removed by aspiration after each wash. After the last wash the cells were centrifuged for 5 minutes at 12,000g and all of the supernatant removed. Approximately 20 ml 971
Vol. 111, No. 3, 1963
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of these packed cells were combined with 20 ml of the 310 mOsm buffer, mixed and then rapidly poured into 560 ml of ice cold 20 mOsm sodium phosphate buffer, pH 7.4. This solution was stirred on ice for 5 minutes and centrifuged 40 minutes at 40,OOOg at 4°C. The supernatant was decanted, the button on the bottom of the tube aspirated off, and the membranes washed two more tiIWS in 30 volumes of the 20 mOsm buffer. The resultant ghosts were combined into 4 centrifuge tubes and washed 2 more times in the 20 mOsm buffer centrifuging at 40,OOOg for 30 minutes. In order to remove proteins that bind to the membrane in a Ca-dependent manner, the membranes were suspended in 5 volumes of 0.1 mM EDTA, 1.0 mM Tris, pH 8.0 and incubated at 37°C for 30 minutes following the procedures of Mauldin and Roufogalis (9). The mixture was cooled for 5 minutes and centrifuged for 20 minutes at 40,OOOg. The supernatant was saved (EDTA extract see below) and the membranes resuspended to the original volume with 5.0 mM Tris:HCl, 15 mM NaCl, pH 7.2 and frozen at -20°C. Before an assay the ghosts were thawed and washed 3 times in 30 volumes of 5 mM Tris:HCl, 15 mM NaCl, pH 7.2, centrifuging at 30,OOOg for 5 minutes. The membranes were then suspended in the same buffer and added to the assay at the appropriate time. The supernatant obtained after the first centrifugation following the EDTA extraction was concentrated 6 fold on an Amicon PM 30 membrane under nitrogen pressure at 25 psi. It was then diluted 10 fold with 5 mM Tris:HCl, 15 mM NaCl, pH 7.2 and reconcentrated under the same conditions. This procedure was repeated twice more to assure that all the phosphate and EDTA was washed through the PM 30 membrane and out of the sample. The final protein concentration of the extract was approximately 1 mg/ml. Inorganic phosphate was analyzed by the method and protein according to Lowry (11). Free Ca2+ and the total concentration of EDTA, Ca2+, and Mg2+ (12) appropriate associations constants from Martell and 37°C and 0.17 M. All chemicals were reagent grade. muscle prepared to be low in Ca2+ and "vanadium free"
of Brotherus et al. (10) Mg2+ were calculated from using constants for the Smith (13) corrected for ATP was from equine (Sigma, St. Louis, MO).
RESULTS The effect the
presence
Figure
1.
ATPase,
activity
inhibits
absence (Ca2+ the
and absence The membranes
the
which
of Ca2+ on the ATPase
(Nat
of ouabain
types
the
In the
of Ca2+ stimulates 1).
contain
activity
Mg2+ - ATPase.
(Fig.
from
the
Ca*+,
increases
ATPase,
and increases
in the
(2)
the
human red blood of ATPase
measured
(Nat
in
presence presence
+ Mg2+)-ATPase
inhibitory
is
activity:
effect
shown
(1)
mixture
the the
in the and (3)
(Nat
+ K+)10,
of Ca2+ on the
(Ca*+
(Nat
minus
concentration
containing
of Ca2+ on the
in
of ouabain
of Ca2+ and ouabain increasing
in
Mg2+-
activity
of ouabain;
and inhibits
to assay
effect
cells
+ K+)-ATPase,
of hemolysate
stimulating
membranes
of Ca2+ and presence
in the
hemolysate the
of human red cell
absence
activity
absence
(Cazt
Adding
UM free
three
t K+)-ATPase;
minus
t Mg2+)-ATPase,
ATPase
of hemolysate
measured
the
activity
25 and 50 t Mg*+)-
+ K+)-ATPase
Vol.
111,
No.
BIOCHEMICAL
1983
3,
Ouob co*+ Herno,
Fig.
(Fig.
1.
Kf)-ATPase
0 IJM free
or
the
component
of
free
the
of the
- + 2525 - -
-t 00 ++
- + 5050 - -
(Nat
and
-+ 1010 ++
-+ 2525 ++
-+ 5050 tt
(PM)
hemolysate
on
is
maximal
of
the
hemolysate
to
10
The
increased
by
(Nat free
the
inhibition similar
shift
in caused
to
that
effect
only
the
1 is
shown
and
is
Cazt-inhibition
+ K+)-ATPase Ca2+
no
on
either
the
(Na+
At
0 uM
1).
+ K+)-ATPase
inhibition
is
(Fig,
has
Figure
evidenced
Ca2+
hemolysate
in
2).
$I
the
presented
is
experiment
- + 10 10 - -
-ATPase
data
hemolysate
this
Mg 2i-ATPose
Ca2+
(Fig.
in
0
Ca
Mg*+
hemolysate
increase
(No'+ K+)-ATPose (Co*++Mg*')-ATPose
- + 0 0 - -
At
effect
Ca2+
m m
RESEARCH COMMUNICATIONS
The effect of Ca2+ and hemolysate on the (Nat + K+)-ATPase, (Ca2+ + Mg2+)-ATPase, and Mg2+-ATPase in human red cell membranes. The assay was conducted by first preincubating the membranes for 10 minutes at 37°C in the assay mixture. The reaction was initiated by the addition of ATP to a final concentration of 1 I#!. Fifteen minutes later the reaction was stopped and the concentration of inorganic phosphate analyzed. The final assay volume after the addition of the ATP was 0.6 ml and contained 204 ua membrane orotein. 18 nF1 NaCl, 30 ai KCl, 112 mM choline Cl, 20 at! Hepes-Tris, 5 n@lEDTA, 0.2% bovine serum albumin, pH 7.3, and where a propriate, 5.5 mM Mg (0 free Ca2+), 1.84 mM Ca2+ and 3.68 mM f4g$+ (10 uM free Ca2+, 500 LIM fr e Mg2+), 2.99 p Ca2+ and 2.56 r&l Mg2+ (25 PM free Ca2+, 500 PM M#+), 3.79 mM Ca +, 1.79 mM Mg2+ (50 UM free Ca2+, 500 UM free Mg +I, 0.5 mM ouabain, and 0.1 ml hemolysate, representing a 60 fold dilution of the original cellular contents. The rate of ATP hydrolysis was calculated as phosphate liberated per mg membrane protein per minute after subtracting off less than 1.3% of the ATP found hydrolyzed in the absence of membranes. Mean values with standard deviations are given for triplicate samples. These results from one experiment are representative of 4 others.
1).
The
AND BIOPHYSICAL
in
amount
from
50
the
presence by
the
found
973
in
not
Figure
2.
affected
by
produced of UM free of
hemolysate in
ouabain-sensitive
9 other
Ca2+
by
the
necessary
for
in
absence
Ca*+ hemolysate at
the
the (Fig.
10
separate
UM free
50%
2). Ca2+
experiments
of The in
t
Vol. 111, No. 3, 1983
BlOCHEMlCAL
0
Hemol. Fig.
using
2.
AND BIOPHYSICAL
0
10 IO
25 25
50 50
- +
- t
- t
- t
RESEARCH COMMUNICATIONS
(pM)
The effect of Ca2+ and hemolysate on the (Na+ + K+)-ATPase. The data are from Figure 1 and were calculated for a given set of conditions by taking the difference between randomly matched values plus and minus ouabain, and then calculating the mean and standard deviations of these differences. The standard deviations are larger for the conditions where both CaZ+ and hemolysate are present because the values are differences between two larger numbe s due to concomitant stfmulati n of the (Ca2+ + Mg2')-ATPase by Caht and hemolysate (Fig. 11. Cakt and the hemolysate interacted in a significant fashion (P < 0.051 to alter the activity of the (Nat + K+)dTPase, as shown by a two-way analysis of variance.
similar
concentrations
of hemolysate
under
comparable
conditions
(data
not shown. Boiling ability
to
(Fig. the
the
hemolysate
increase
the
3) and only (Ca*+
Mg2+)-ATPase of the
(Nat
In the of the
(Nat
as did
+ K+)-ATPase
(data
not
(data
its
not the
was carried
it
to the membranes
of Ca2+ on the ability
shown).
in place
hemolysate,
above experiments + K+)-ATPase
effect
reduced
calmodulin the
to adding
inhibitory
slightly
+ Mg2+)-ATPase
0.2 PM of purified
prior
of hemolysate
but
did
not
the
activity
experiments, simulated
increase
its
(Na+ t K+)-ATPase
to stimulate In other
destroyed
the
of
adding the
(Ca2+ +
Ca2+ inhibition
shown). effect
of the
out using 974
hemolysate red cell
on Ca2+ inhibition membranes
prepared
by
Vol.
111,
No.
6IOCHEMlCAL
3, 1983
AND BIOPHYSICAL
0 0 -tB
Hemol. Fig.
3.
a procedure were
prepared that
increase
the
that would Ca2+ deplete
the
lo lo 10 -tB
(PM)
The effect of boiled hemolysate on the (Nat + K+)-ATPase at 0 and 10 $4 free Cc?+. The experiment was carried out as described in the legend to Figure 1 except that in this case the preincubation was 15 minutes and 120 mM NaCl, 20 mM choline, and 232 ug membrane protein were substituted for the value of those constituents listed in Figure 1. The concentrations of both membranes and hemolysate are similar to that shown previously, but are from different preparations. The hemolysate sample marked "B" is from the same stock as that marked "t", but was boiled for 5 minutes prior to the assay, filtered on Whatman #2 paper, and then added in the same volume as "t". The ouabain-sensitive differences for each condition are between six values with ouabain randomly matched with six values without ouabain as described in Figure 2.
which
proteins
0
RESEARCH COMMUNICATIONS
depletes
in
this
might
removal
increase inhibition. membranes
in
bind
sensitivity of
the
manner also
Ca2+
membranes
such
of
an
attempt
the
membrane
of
the
(Na+
of
To
test
if
of
the
protein
these
calmodulin
to
to
components
probability
bound
prior
in
the an
procedures which
increases
975
other
The
(Nat effect
which
It
manner was
anticipated
+ Kt)-ATPase
assay
of
the
remove Ca*+
membranes
cytoplasmic
a Ca*+-dependent
+ K+)-ATPase. to
observing
remove
(7).
inhibition
hemolysate
and
on
calmodulin
also of
the
Vol. 111, No. 3, 1983
Fig.
(Na'
4.
resultant
Methods). ATPase
at
increased up
to
extract
had
Dodge protein the
effect
0 and
10
uM free
percent
in
membranes
mixture
Then
the 80%
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
The effect of proteins from the membranes of Dodge ghosts on the (Na+ + Kf)-ATPase at 0 and 10 uM free Ca*+. Each panel shows one of four similar experiments with each point representing the mean and standard deviation of three values. The protein extract contains proteins that were removed from Dodge ghosts incubated at 37'C in 0.1 mM EDTA, 1.0 mM Tris:HCl, pH 8.0 and subsequently separated from the membranes and concentrated on an Amicon PM 30 membrane, as described in the Materials and Methods. The assay ghosts are those from which the proteins were extracted. Note, the protein extract has no effect on the (Na+ + K+)-ATPase measured at D $I free Ca*+, whereas it inhibits the (Na+ + K+)-ATPase in a dose dependent manner when 10 uM free Ca is present in the assay. This suggests that the extract increased Ca-induced inhibition of the (Na+ + K+)-ATPase. The assay was cdrried out under the conditions specified in the legend to Figure 1, except that in this case the incubation time was 40 minutes and 20 mM NaCl, 10 mM KCl, 89 mM choline Cl, 20 mM Pipes:Tris, and 120 to 160 &ml membrane protein were substituted for the value of those constituents listed in Fig. 1. In all four experiments the extract and the Ca*+ interacted in a si nificant fashion (P < 0.05) to alter the rate of the (Na 9 + Kf)-ATPase, as demonstrated in a two-way analysis of variance.
+ Kt)-ATPase,
the
BIOCHEMICAL
of
concentrated
and
sensitivity
of
indicating
that
on
washed the the
concentrated
the
extract (Fig.
inhibition
effect
at
(Nat
on
(Nat an
The
uM free
(Fig.
in
an
Amicon
PM 30
measured
on
4).
(see
+ K+)extract
Cazt
from
less
than
20
percent
absence
of
Ca2+
the
In
inhibition
976
filter
the
membrane
from
and
that
XM 100
distinct
(Na+
(9)
show
(Fig.
to
the
EDTA
data
+ Kt)-ATPase
Amicon
is
extracted on
was
10
+ Kt)-ATPase
component
first
4).
manner the
were
was
Ca2+
a dose-dependent no
(8)
the 4). had by
the
Similar no
Ca*+
non-calmodulin
effect (data
extracts on not
the shown),
activator
BIOCHEMICAL
111, No. 3, 1983
Vol.
cQ+
of
the If
f
the
the
Mg*+)-ATPase
to
which
concentrated
Ca2+
is
stays
extract
its
membranes,
ATPase
-
The rate of (Na+ + K+)-ATPase activity at 0 and 10 $i free Ca*+ in the presence and absence of the membrane extract, and in the presence of extract that had been digested with trypsin. The protein was digested by adding 1 Pg of trypsin/25 pg protein and incubating at 37°C for 12 hours.The (Nat+ K+)-ATPase was carried out as described in Figure 1 under the conditions of Figure 4, except that the membranes had a final concentration of 211 Kg/ml. Each value is the mean of the difference between 3 samples without The bars ouabain minus 3 randomly matched values with ouabain. This experiment are the standard deviations of these differences. was repeated four other times with similar results.
(Ca*+
(9). to
5.
RESEARCH COMMUNICATIONS
0 m
Extraa Trypein Fig.
AND BIOPHYSICAL
ability
is to
destroyed
on top digested
alter
(Fig.
of
the
an Amicon
with
XM 100
trypsin
sensitivity
prior of
the
membrane to
(Na+
adding t
Kf)-
5). DISCUSSION
The than
above
calmodulin
binds
to
the
sensitivity favor boiling Ca*+
results that
is
membrane of
of
could
the
present in
is
the
hemolysate
sensitivity
of
in
+ Kf)-ATPase that
the
explained
by
the
of
manner, to
digesting
a Ca2+-dependent
cytoplasm
a Cazf-dependent
(Nat
a protein
be
the
destroys
the
ability
(Nat
+ Kt)-ATPase.
by
membrane
This 977
and
inhibition
of
human
red
increases Cazt.
extract either
protein
proteinaceous
cells,
the Evidence
in to
blood
other
affect
trypsin
in or
the factor
is
it
Vol. 111, No. 3, 1983
BIOCHEMICAL
Cazt-dependent,
because
of Ca*+.
are
not
There
calmodulin.
membranes second
The first
is that
be established
directly
depends
on the
the
protein (Nat
free
variety
presence
it
could
sensitivity
of the
(Nat
reltionship
between
the
the
be involved
generation
and the
point
it
to play the
t K+)-pump (Nat
in the
could
is
which It
is must now
extract
that
via
a mechanism
Ca*+-dependnent
acts
effect
of
by changes
Such an increase regulatory
increased muscle,
and the
inhibi-
Ca*+ sensitivity
be regulated
an important
in smooth
inotropic
low
range.
protein
t K+)-pump
of hypertension
The
due to a single
effect
the otherwise
in the physiological
if
to the
(14).
membrane
of this
where
instance,
is
factors.
increase
be anticipated For
is
significance
to the
protein
hemolysate,
Ca*+ inhibition
the
presence
(Na+ t K+)-ATPase,
stable
of other
this
calmodulin
of the
or whether
in the
that
to be heat
hemolysate
would
Ca*+
of cells.
thereby
the
+ K+)-ATPase
t K+)-ATPase
intracellular
purified
effect
increased
+ K+)-ATPase
of the
is known
the
that
(Nat
RESEARCH COMMUNICATION:
to suggest
adding
the
physiological
is
sensitivity
that
calmodulin
(Nat
The possible tory
is
in both
on the
the
of evidence
destroyed
whether
present
affects
on Ca*+ inhibition
boiling
because
component
only
two pieces
had no effect
signifiant
that
it
AND BIOPHYSICAL
it
Ca*+
could
alter
Na+/Ca*+
of cardiac
in
role
the
exchange
in a
the system
glycosides
of
(15)
and
and
(16).
ACKNOWLEDGEMENTS I thank This
work
and with part
Mrs.
Daniela
was supported funds
contributed
by a PMA Foundation
M. Polasek
for
by a Grant-in-Aid in
part
Research
excellent from
technical the American
by the Michigan Starter
Heart
assistance. Heart
Association
Association
Grant.
REFERENCES 1. 2. 3. 4.
Hoffman, J.F. (1962) Circulation. 26, 1201-1213. I.M. (19611 J. Physfol. (London). Dunham, E.T., and Glynn, 274-293. Skou. J.C. (1960) Biochim. Biophys. Acta. 42, 6-23. Whittam. R. and Blond, D.M. (1964) Biochem. J. 92, 147-158. 978
156,
and in
Vol. 111, No. 3, 1983
5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
B. (1977) Acta. Biol. Med. Ger. Gardos, G., Szasz, I., and Sarkadi, 36, 823-829. Yingst, D.R. (1982) Fed. Proc., Fed. Am. Sot. Exp. Biol. 41, 974. Muallem, S. and Karlish, S.J.D. (1980) Biochim. Biophys. Acta. 597, 631-636. Dodge, J.T., Mitchell, C., Hanahan, D.T. (1963) Arch. Biochem. Biophys. 100, 119-150. Mauldin, D. and Roufogalis, B.D. (1980) Biochem. J. 187, 507-513. Brotherus, J.R., Moller, J.V., Jorgensen, P.L. (1981) Biochem. Biophys. Res. Commun. 100, 146-154. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, A.J. (1951) 3. Biol. Chem. 193. 265-275. Wolf, H.U. (1973) Experientia 29, 241-249. Martell, A.E. and Smith, R.M. (19741 Critical Stability Constants, Amino Acids, p. 204, Plenum Press, New York. Cheung, W.Y. (1971) 3. Biol. Chem. 246, 2859-2869. Biedert, S., Barry, W.H., and Smith, T.W. (1979) J. Gen. Phys. 74, 479-494. Blaustein, M.P. (1977) Am. J. Physiol. 232, C165-C173.
979