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Phosphate dependent, ruthenium red insensitive Ca2+ uptake in mung bean mitochondria
Vol. 114, No. 3, 1983 August
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12, 1983
Pages 1176-1181
PHOSPHATE DEPENDENT, RUTHENIUM REDINSENSITIVE CA2+ UPTAKE IN MUNGBEANMITOCHONDRIA KARLE.O. WKERMAN'ANDANTHONYL. MOORER 'DEPT. OF MEDICALCHEMISTRY,UNIVERSITYOF HELSINKI, SILTAVORENPENGER lOA, SF-00270, HELSINKI, FINLAND n
'DEPT. OF BIOCHEMISTRY,UNIVERSITYOF SUSSEX, FALMER,BRICHTdNBN1 qQG,' U.K. Received May 4, 1983 Summary: Energy linked Ca2+ uptake into mungbean mitoch ndria has been studied. marsenazo III as a monitor2yf extramitochondrial Ca8+, we observe a respiration-linked uptake of Ca which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmo$!mgprotein/min. Acetate, sulphate and thiosulphate are unable to support Ca yptake. The results suggest that although plant mitochondria accumulate Ca in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.
Introduction:
Vertebrate
mitochondria isolated
from a wide variety 2+
species and tissues contain a very efficient
Ca 2+
reviews see l-5).
In these mitochondria Ca
transport
mechanism (for
uptake is linked to a
stoichiometric
increase in oxygen consumption, proton extrusion
depolarisation
of the membranepotential
plant mitochondria transport instance,
an energy-linked
electrophoretic
(1,2) and a
There is someevidence that mechanism(6-9).
For
uptake of large amounts of Ca2+ in the presence of of respiring
phosphate (7) have been reported. respiratory
(3-5).
Ca2+ by a similar
ohosphate (6) and a contraction
initial
of
mitochondria swollen in potassium
Indeed, it has been suggested that the
burst seen with corn mitochondria is associated with the
movementof a positively
charged calcium phosphate complex (7).
With respect to the role of mitochondria in the reRulation of cytosolic Ca2+, it has been suggested that in plant cells of free Ca*+ is mainly controlled (10).
Such a regulatory
by an active
the cytoplasmic concentration accumulation into mitochondria
role warrants the possession of a very efficient
0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in any form reserved.
1176
Vol. 114, No. 3, 1983
transport
Ca2+
system
system.
being
present
The aim of uptake
BIOCHEMICAL
To date in plant
the present
there
study
of cytosolic
Methods
is little
RESEARCH COMMUNICATIONS
evidence
in favour
of such
a
mitochondria. was,
in mung bean mitochondria
regulation
AND BIOPHYSICAL
to examine
therefore,
in an attempt
Ca 2+ in the plant
to determine
cell
Ca 2+
energy-linked its
role
in the
.
and Materials
Mung bean (Phaseolus aureus) mitochondria were grown and isolated as described previously (111. The basal experimental medium contained 0.3M mannitol and 1OmM Hepes pH 7.2 with KOH. Further additions as described in the figure 1eKends. Extramitochondrial Ca2+ was monitored with arsenazo III usinrJ the wavelength pair 665 nm - 685 nm in an Aminco DW2 spectrophotometer. The arsenazo III was purified as described by Scarpa (12) before use. For measurements of membrane potentials polyvinylchloride membranes sensitive to tetraphenylphosphonium (TPP+) were prepared as in (12) and were glued onto used F2112 Radiometer (Copenhagen) Ca2+ selective electrode tubes after removal of the Ca2+ selective membrane. The electrode was connected through an agar/KCl bridge to a KC1 reference electrode and the experimental protocol was essentially as in (14). FCCP was kindly donated by Dr. P.G. Heytler (DuPont, Wilmington, DE). A23157 was obtained from Calbiochem-Behring Corp. (La Jolla, CA), and oligomycin, antimycin A and tetrapheylphosphonium from Sigma Chemicals Co. (St. Louis, MO). Ruthenium red was purchased from BDH Chemicals Ltd. (Poole, England) and purified according to Luft (15) before use. All the other reagents were of the highest grade available. Results The endogenous bean mitochondria measured pulses since
the experimental back
of EGTA (2 ILM).
In the
presence
a fast
trace)
probably
causes
a slow
mitochondria The Ca2+ is inhibited
medium
the arsenazo
Most
experimental
causes
is
into
by titrating
the
Ca2+ concentration
extramitochondrial
of this
III Ca
medium contains
2+
only
decrease with
slowly
released
by respiratory
shown 1 .
ATP on the other
presence
0: phosphate
upon chain hand
(Fip;. lb 1.
10 !LY Ca
A further
rate
to the mitochondria
inhibitors
is unable A further 1177
deflection
an uptake
of 5 nmol/mp:
protein
The phosphate
of
addition
the
of phosphate into
the
per min
(Fig-la).
induced
uptake
such as CD- and antimycin to support
small
.
addition
that
anaerobiosis.
by adding
the mitochondria
2+
Ca 2+ (upwards
of Ca2+.
an average
with
mung
of 40 uM as
shown)
of succinate
in medium Ca 2+ indicating;
occurs
(not
about
in extramitochondrial
due to a binding
the order
associated
of 10 NM Ca2+ an addition
decrease
is of
signal is
upon suspending
A (not
Ca2+ uptake
even
in
of succinate
in
the
the
f I A
Vol. 114, No. 3, 1983
BIOCHEMICAL
AND ElOPHYSlCAL
RESEARCH COMMUNICATIONS
-02
a)
[CaZ 10 PM
SUCC pi 5-J
‘PM
2 PM
A 23107 1
TPP+
FCCP +
cl 2 YM I
01
a SUCC
02 Fig.1.
Ca2+ uptake by mung bean mitochondria during respiration The mitochondria were suspended at a concentration,of 0.5 mg protein/ml in the basal medium containing 10 pM Cad+ and 106 pM Arsenaso III (a,bl, 2 mM MgCl , 0.4 mM ADP and 4 mM succinate (c,d). Additiq:s: 4 mM succi6ate (succ); 0.4 mM KH PC (P 1, 2 mM Mg and 2 mM ATP (ATP), 0.4 $4 FCCP 0r~3.2 pM A23107 as indicated. Upwards deflection-increase in Ca2+ uptake. In (bl the initial jump upon succinate addition has been removed from the trace.
Fig.2.
Effect of A23187 on the membrane potential in mung bean mitochondria Conditions as in Fig.1 except with the inclusion of 0.4 mM were made of 1 KH.&+yd 10 uM Ca2+. Further additions 0.5 mg mitochondrial protein, 4 mM succinate (succ), 3.2 pM A23187 and 0.4 @l FCCP. An upward deflection corresponds to the formation of a membrane potential (positive extramitochondriallyl.
presence is
of ATP and phosphate
released
mammalian
upon addition
addition.
to exclude
membrane sensitive
there
of the divalent
extranitochondrial order
(3-5)
Ca 2+ is
Interestingly
restores
potential electrode.
the Ca2+ uptake.
of the uncoupler,
mitochondria
up upon addition
phosphate
i5sec
released This
Ca 2c
is
to the same level that
a real
uptake
a possible
uncouolinE
was measured A23157
fast
ionophore
suERests is
FCCP (FiE.lb).
a very
cation
release A23187
into by the
affect 1178
Similar
(Fip;.lc
decrease
the mitochondria ionophore, conditions the membrane
up
to
of the Ca 2+ taken
as observed
the observed
in identical
does not
The Ca2+ ta’xen
and d).
prior
to
in matrix.
In
the mitochondrial using
a TPP+
potential
(Fig.2)
BIOCHEMICAL
Vol. 114, No. 3, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2- min Fig.3.
Effect of anions and mersalyl on Ca*+ uptake by mung bean mitochondria Conditions as in Fig.1. c,d. Additions: 0.4 mM K-acetate (AC), 0.4 mM K-sulphate (sulph), 0.4 mt4 K-phosphate (PiI and 0.2 mt+fmersalyl (mers) as indicated.
even in
the presence
ionophore
is not
hie;hly
due to an uncoupling
effective
mitochondria
inhibitor (Ki*20nM,
phosphate-induced the
case with
arsenazo
III
response
Ca2-I- uptake
in
ability
to ta!te
control
ratio
sulphate
shows (Fie;.3a)
2+
fron
the anion nor
Ruthenium
mechanism
this
ion
affect
of ruthenium
a
the
of 5 111.1(Fip;.ld). considerably
The calculated
shown).
red,
of mammalian
significantly
even at a concentration
stored (not
The
reduces
rates
red and Mp *+ are
at +7’C
shown)
potential
of Mf:2+ and ADP. depicted
not
of the
for
the
the
respectively
4.7
per min.
or membrane
upon addition
Fig.3
does
to Ca *+ (not
are
up Ca
the effect
of the mitochondria.
10 ml’4 Mg2+ although
protein
1.Jhen mitochondria
experiments
ref.141
the presence
nmol/mg
that
of the Ca*+ uptake
Ca*+ uptake
same is
and 5.1
of 10 IIM Ca2+ , supaestinK
a few hours
althouRh
no chan,qe
is
observed.
dependency
they in
lose
their
the resoiratory
The uptake
is
restored
ADP and HP:2+ l;!ere included
Therefore
Pig.lc
thiosulphate
for
in
forwards. of Ca (not
2+
uptake.
shown) 1179
are
Neither able
acetate,
to support
Ca-?+
again the
BIOCHEMICAL
Vol. 114, No. 3, 1983
uptake.
AND BIOPHYSICAL
an inhibitor
The Ca*+ taken up is released if mersalyl,
phosphate translocator,
RESEARCH COMMUNICATIONS
of the
is added during the Ca*+ uptake phase (Fig.3b).
Discussion The results of the present study indicate up Ca2+ from the external
that mung bean mitochondria take
mediumin an energy-linked
manner. The uptake
mechanismis slow with a rate of uptake at the order of 5 nmol/mg protein per min in the presence of about
uM Ca2+ i.e.
10
magnitude lower than the vertebrate
a rate which is two orders of
counterpart
(for reviews see 2-4).
to ruthenium red and Mg2+
absolute requirement for phosphate, insensitivity as well as the inability
The
of other weak acid anions to promote uptake
suggests
that the mechanismof uptake is different
from the uniporter present in 2+ A slow phosphate dependent Ca uptake has been
mammalianmitochondria. observed in blowfly
flight
mechanismis sensitive the uniporter
muscle mitochondria
(17,181.
However, this
to ruthenium red (19,20) and is thus probably related
to
present in mammalianmitochondria. that the role of phosphate in Ca2+ uptake by mung bean
It appears likely
mitochondria is simply to form complexes with Ca*+ in the matrix space (as evidenced by electron dense precipitates
(2111, although a Ca*+/phosphate
symport mechanismcannot be excluded at present (see 7). symporter as suggested in (7), however, appears unlikely inhibition
of Ca*+ uptake by mersalyl,
an inhibitor
A Ca*+/phosphate in view of the
of the H+/phosphate
symporter, unless of course the supposed Ca*+/phosphate symporter is inhibited unlikely
by mersalyl.
Similarly,
a slow uniport mechanismof Ca2+ uptake is
since in this case one would
in the presence of respiration
predict
a slow uptake towards equilibrium
alone (in the absence of phosphate). The release
of Ca2+ by A23187, however, indicates
that a Ca*+ gradient is formed across
the membrane. It may therefore
be concluded that although plant mitochondria do accumulate
Ca2+ in . an energy dependent fashion, uniport
and
furthermore
it
is not via a simple electrophoretic
the low rates of transport 1180
reported cast somedoubt on
vol. 114, No. 3, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the postulated role (10) of plant mitochondria in the rerrulation
of cytosolic
Ca2+ in the plant cell. Acknowledgements This work was supported in part by grants from the SERCand ARC (to A.L.M.) and the S&rid Juselius Foundation (K.E.O.A.). Wewish to thank Ms. Kaija Niva for technical assistance. References 1. Lehninp;er, A.L., Carafoli, E. and Rossi, C.S. (1967) Adv.Enxymol. 29, 259-319. 2. Bygrave, F.L. (147' ) Curr.Top.Bioenerq. 5, 250-318. 3. Saris, N-E.L. and i kerman, K.E.O.. (198OT Curr.Top.BioenerE. 2, 103-178. 4. Nicholls, 3.G. and fkerman , K.E.O. (1982) Biochim.Biophys.Acta. -'683 57-45. 5. Ackerman,K.E.O. and Nicholls, D.G. (1952) Rev.Physiol.Biochem. Pharmacol. in press. 6. Chen, C-H. and Lehninger, A.L. (lQ73) Arch.Biochem.Biophys. 157, 183-196. 7. Day, D.A. and Hanson, J.B. (1978) Biochim.Biophys.Acta. 502, 289-297.
8. Russell, M.J. and Wilson, S.B. (1478) In "Plant Mitochondria" (G. Ducet and C. Lance, eds.) pp 175-l=. Elsevier/North Holland Biomedical Press. Amsterdam/NewYork. 9. Hanson, J.B. and Day, D.A. (1980) In "The Biochemistry of Plants" (N.E. Tolbert, ed.) Vol.1, pp 315-ES. Academic Press, London. 10. Dieter, P. and Marme, D. (1980) Planta 150, l-9. 11. Moore, A.L. and Proudlove, M.O. (1983) In "Isolation of Membranes and Organelles from Plant Cells" (J.L. El1 and A.L. Moore, eds.) pp. 153-184. Academic Press, London. 12. Scarpa, A. (1979) Methods Enzymol. Vol.LVl, 301-338. 13. Kamo, M., Muratsuga, M., Hon,qoh, R. and Kobatake, Y. (1979) J.Membr. Biol. E, 105-121. 14. Lotscher, H-R., Winterhalter, K.H., Carafoli, E. and Richter, C. (lo80). J.Biol.Chen;lGj 93~~X3;",',".,,, 15. Luft, J.H. -) 347-368. 16. Reed, K.C. and By.orave, F.J,. (lo741 Bi0chem.J. 140, 143-155. 17. Carafoli, E., Hansford, R.G., Sacktor, B. and Lehninger, A.L. (1971) J.Biol.Chea. 275, 964-972. 18. Smith, R.L. andBygrave, F.J,. (1978) Bi0chem.J. 170, 81-85. lo. Carafoli, E. and Sacktor, B. (1972) Biochem.Biophys.Res.Comnun. 2, 1498-1503. 20. Wohlrab, H. (1974) Biochemistry 13, 4014-4017. 21. Peverly, J.H., Miller, R.J., Malone, C. and Koeppe, D.E. (1974) Plant Physiol. 54, 408-411.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
12, 1983
Pages 1176-1181
PHOSPHATE DEPENDENT, RUTHENIUM REDINSENSITIVE CA2+ UPTAKE IN MUNGBEANMITOCHONDRIA KARLE.O. WKERMAN'ANDANTHONYL. MOORER 'DEPT. OF MEDICALCHEMISTRY,UNIVERSITYOF HELSINKI, SILTAVORENPENGER lOA, SF-00270, HELSINKI, FINLAND n
'DEPT. OF BIOCHEMISTRY,UNIVERSITYOF SUSSEX, FALMER,BRICHTdNBN1 qQG,' U.K. Received May 4, 1983 Summary: Energy linked Ca2+ uptake into mungbean mitoch ndria has been studied. marsenazo III as a monitor2yf extramitochondrial Ca8+, we observe a respiration-linked uptake of Ca which requires phosphate and is insensitive to ruthenium red. The rate of uptake is of the order of 5 nmo$!mgprotein/min. Acetate, sulphate and thiosulphate are unable to support Ca yptake. The results suggest that although plant mitochondria accumulate Ca in an energy dependent fashion, it is not via a simple electrophoretic uniport mechanism.
Introduction:
Vertebrate
mitochondria isolated
from a wide variety 2+
species and tissues contain a very efficient
Ca 2+
reviews see l-5).
In these mitochondria Ca
transport
mechanism (for
uptake is linked to a
stoichiometric
increase in oxygen consumption, proton extrusion
depolarisation
of the membranepotential
plant mitochondria transport instance,
an energy-linked
electrophoretic
(1,2) and a
There is someevidence that mechanism(6-9).
For
uptake of large amounts of Ca2+ in the presence of of respiring
phosphate (7) have been reported. respiratory
(3-5).
Ca2+ by a similar
ohosphate (6) and a contraction
initial
of
mitochondria swollen in potassium
Indeed, it has been suggested that the
burst seen with corn mitochondria is associated with the
movementof a positively
charged calcium phosphate complex (7).
With respect to the role of mitochondria in the reRulation of cytosolic Ca2+, it has been suggested that in plant cells of free Ca*+ is mainly controlled (10).
Such a regulatory
by an active
the cytoplasmic concentration accumulation into mitochondria
role warrants the possession of a very efficient
0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. AN rights of reproduction in any form reserved.
1176
Vol. 114, No. 3, 1983
transport
Ca2+
system
system.
being
present
The aim of uptake
BIOCHEMICAL
To date in plant
the present
there
study
of cytosolic
Methods
is little
RESEARCH COMMUNICATIONS
evidence
in favour
of such
a
mitochondria. was,
in mung bean mitochondria
regulation
AND BIOPHYSICAL
to examine
therefore,
in an attempt
Ca 2+ in the plant
to determine
cell
Ca 2+
energy-linked its
role
in the
.
and Materials
Mung bean (Phaseolus aureus) mitochondria were grown and isolated as described previously (111. The basal experimental medium contained 0.3M mannitol and 1OmM Hepes pH 7.2 with KOH. Further additions as described in the figure 1eKends. Extramitochondrial Ca2+ was monitored with arsenazo III usinrJ the wavelength pair 665 nm - 685 nm in an Aminco DW2 spectrophotometer. The arsenazo III was purified as described by Scarpa (12) before use. For measurements of membrane potentials polyvinylchloride membranes sensitive to tetraphenylphosphonium (TPP+) were prepared as in (12) and were glued onto used F2112 Radiometer (Copenhagen) Ca2+ selective electrode tubes after removal of the Ca2+ selective membrane. The electrode was connected through an agar/KCl bridge to a KC1 reference electrode and the experimental protocol was essentially as in (14). FCCP was kindly donated by Dr. P.G. Heytler (DuPont, Wilmington, DE). A23157 was obtained from Calbiochem-Behring Corp. (La Jolla, CA), and oligomycin, antimycin A and tetrapheylphosphonium from Sigma Chemicals Co. (St. Louis, MO). Ruthenium red was purchased from BDH Chemicals Ltd. (Poole, England) and purified according to Luft (15) before use. All the other reagents were of the highest grade available. Results The endogenous bean mitochondria measured pulses since
the experimental back
of EGTA (2 ILM).
In the
presence
a fast
trace)
probably
causes
a slow
mitochondria The Ca2+ is inhibited
medium
the arsenazo
Most
experimental
causes
is
into
by titrating
the
Ca2+ concentration
extramitochondrial
of this
III Ca
medium contains
2+
only
decrease with
slowly
released
by respiratory
shown 1 .
ATP on the other
presence
0: phosphate
upon chain hand
(Fip;. lb 1.
10 !LY Ca
A further
rate
to the mitochondria
inhibitors
is unable A further 1177
deflection
an uptake
of 5 nmol/mp:
protein
The phosphate
of
addition
the
of phosphate into
the
per min
(Fig-la).
induced
uptake
such as CD- and antimycin to support
small
.
addition
that
anaerobiosis.
by adding
the mitochondria
2+
Ca 2+ (upwards
of Ca2+.
an average
with
mung
of 40 uM as
shown)
of succinate
in medium Ca 2+ indicating;
occurs
(not
about
in extramitochondrial
due to a binding
the order
associated
of 10 NM Ca2+ an addition
decrease
is of
signal is
upon suspending
A (not
Ca2+ uptake
even
in
of succinate
in
the
the
f I A
Vol. 114, No. 3, 1983
BIOCHEMICAL
AND ElOPHYSlCAL
RESEARCH COMMUNICATIONS
-02
a)
[CaZ 10 PM
SUCC pi 5-J
‘PM
2 PM
A 23107 1
TPP+
FCCP +
cl 2 YM I
01
a SUCC
02 Fig.1.
Ca2+ uptake by mung bean mitochondria during respiration The mitochondria were suspended at a concentration,of 0.5 mg protein/ml in the basal medium containing 10 pM Cad+ and 106 pM Arsenaso III (a,bl, 2 mM MgCl , 0.4 mM ADP and 4 mM succinate (c,d). Additiq:s: 4 mM succi6ate (succ); 0.4 mM KH PC (P 1, 2 mM Mg and 2 mM ATP (ATP), 0.4 $4 FCCP 0r~3.2 pM A23107 as indicated. Upwards deflection-increase in Ca2+ uptake. In (bl the initial jump upon succinate addition has been removed from the trace.
Fig.2.
Effect of A23187 on the membrane potential in mung bean mitochondria Conditions as in Fig.1 except with the inclusion of 0.4 mM were made of 1 KH.&+yd 10 uM Ca2+. Further additions 0.5 mg mitochondrial protein, 4 mM succinate (succ), 3.2 pM A23187 and 0.4 @l FCCP. An upward deflection corresponds to the formation of a membrane potential (positive extramitochondriallyl.
presence is
of ATP and phosphate
released
mammalian
upon addition
addition.
to exclude
membrane sensitive
there
of the divalent
extranitochondrial order
(3-5)
Ca 2+ is
Interestingly
restores
potential electrode.
the Ca2+ uptake.
of the uncoupler,
mitochondria
up upon addition
phosphate
i5sec
released This
Ca 2c
is
to the same level that
a real
uptake
a possible
uncouolinE
was measured A23157
fast
ionophore
suERests is
FCCP (FiE.lb).
a very
cation
release A23187
into by the
affect 1178
Similar
(Fip;.lc
decrease
the mitochondria ionophore, conditions the membrane
up
to
of the Ca 2+ taken
as observed
the observed
in identical
does not
The Ca2+ ta’xen
and d).
prior
to
in matrix.
In
the mitochondrial using
a TPP+
potential
(Fig.2)
BIOCHEMICAL
Vol. 114, No. 3, 1983
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2- min Fig.3.
Effect of anions and mersalyl on Ca*+ uptake by mung bean mitochondria Conditions as in Fig.1. c,d. Additions: 0.4 mM K-acetate (AC), 0.4 mM K-sulphate (sulph), 0.4 mt4 K-phosphate (PiI and 0.2 mt+fmersalyl (mers) as indicated.
even in
the presence
ionophore
is not
hie;hly
due to an uncoupling
effective
mitochondria
inhibitor (Ki*20nM,
phosphate-induced the
case with
arsenazo
III
response
Ca2-I- uptake
in
ability
to ta!te
control
ratio
sulphate
shows (Fie;.3a)
2+
fron
the anion nor
Ruthenium
mechanism
this
ion
affect
of ruthenium
a
the
of 5 111.1(Fip;.ld). considerably
The calculated
shown).
red,
of mammalian
significantly
even at a concentration
stored (not
The
reduces
rates
red and Mp *+ are
at +7’C
shown)
potential
of Mf:2+ and ADP. depicted
not
of the
for
the
the
respectively
4.7
per min.
or membrane
upon addition
Fig.3
does
to Ca *+ (not
are
up Ca
the effect
of the mitochondria.
10 ml’4 Mg2+ although
protein
1.Jhen mitochondria
experiments
ref.141
the presence
nmol/mg
that
of the Ca*+ uptake
Ca*+ uptake
same is
and 5.1
of 10 IIM Ca2+ , supaestinK
a few hours
althouRh
no chan,qe
is
observed.
dependency
they in
lose
their
the resoiratory
The uptake
is
restored
ADP and HP:2+ l;!ere included
Therefore
Pig.lc
thiosulphate
for
in
forwards. of Ca (not
2+
uptake.
shown) 1179
are
Neither able
acetate,
to support
Ca-?+
again the
BIOCHEMICAL
Vol. 114, No. 3, 1983
uptake.
AND BIOPHYSICAL
an inhibitor
The Ca*+ taken up is released if mersalyl,
phosphate translocator,
RESEARCH COMMUNICATIONS
of the
is added during the Ca*+ uptake phase (Fig.3b).
Discussion The results of the present study indicate up Ca2+ from the external
that mung bean mitochondria take
mediumin an energy-linked
manner. The uptake
mechanismis slow with a rate of uptake at the order of 5 nmol/mg protein per min in the presence of about
uM Ca2+ i.e.
10
magnitude lower than the vertebrate
a rate which is two orders of
counterpart
(for reviews see 2-4).
to ruthenium red and Mg2+
absolute requirement for phosphate, insensitivity as well as the inability
The
of other weak acid anions to promote uptake
suggests
that the mechanismof uptake is different
from the uniporter present in 2+ A slow phosphate dependent Ca uptake has been
mammalianmitochondria. observed in blowfly
flight
mechanismis sensitive the uniporter
muscle mitochondria
(17,181.
However, this
to ruthenium red (19,20) and is thus probably related
to
present in mammalianmitochondria. that the role of phosphate in Ca2+ uptake by mung bean
It appears likely
mitochondria is simply to form complexes with Ca*+ in the matrix space (as evidenced by electron dense precipitates
(2111, although a Ca*+/phosphate
symport mechanismcannot be excluded at present (see 7). symporter as suggested in (7), however, appears unlikely inhibition
of Ca*+ uptake by mersalyl,
an inhibitor
A Ca*+/phosphate in view of the
of the H+/phosphate
symporter, unless of course the supposed Ca*+/phosphate symporter is inhibited unlikely
by mersalyl.
Similarly,
a slow uniport mechanismof Ca2+ uptake is
since in this case one would
in the presence of respiration
predict
a slow uptake towards equilibrium
alone (in the absence of phosphate). The release
of Ca2+ by A23187, however, indicates
that a Ca*+ gradient is formed across
the membrane. It may therefore
be concluded that although plant mitochondria do accumulate
Ca2+ in . an energy dependent fashion, uniport
and
furthermore
it
is not via a simple electrophoretic
the low rates of transport 1180
reported cast somedoubt on
vol. 114, No. 3, 1983
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the postulated role (10) of plant mitochondria in the rerrulation
of cytosolic
Ca2+ in the plant cell. Acknowledgements This work was supported in part by grants from the SERCand ARC (to A.L.M.) and the S&rid Juselius Foundation (K.E.O.A.). Wewish to thank Ms. Kaija Niva for technical assistance. References 1. Lehninp;er, A.L., Carafoli, E. and Rossi, C.S. (1967) Adv.Enxymol. 29, 259-319. 2. Bygrave, F.L. (147' ) Curr.Top.Bioenerq. 5, 250-318. 3. Saris, N-E.L. and i kerman, K.E.O.. (198OT Curr.Top.BioenerE. 2, 103-178. 4. Nicholls, 3.G. and fkerman , K.E.O. (1982) Biochim.Biophys.Acta. -'683 57-45. 5. Ackerman,K.E.O. and Nicholls, D.G. (1952) Rev.Physiol.Biochem. Pharmacol. in press. 6. Chen, C-H. and Lehninger, A.L. (lQ73) Arch.Biochem.Biophys. 157, 183-196. 7. Day, D.A. and Hanson, J.B. (1978) Biochim.Biophys.Acta. 502, 289-297.
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