Bioscience Reports, Vol. 13, No. 3, 1993
REVIEW
Divalent Cation and Lipid-Protein
Interactions of Biomembranes
F. Y . Y a n g , ~'2 Y . G . H u a n g , ~ and Y . P. Tu ~
Received March 22 1993
Divalent cations play an important role in the functions of biomembranes. This review deals with
three topics: (1) MgZ+-mediated change in physical state of phospholipid induces conformation and
activity change of reconstituted mitochondrial H+-ATPase, (2) a proper transmembrane Ca2+
gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of
transmembrane Ca 2+ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca2+-ATPase
activity.
KEY WORDS: divalent cation; lipid-protein interaction; H+-ATPase; adenylate cyclase; Sarcoplasmic reticulum CaZ+-ATPase.
INTRODUCTION
Metallic cations, particularly calcium and magnesium, play an important role in
the function of biological m e m b r a n e s . It has been extensively found that divalent
cations can induce a series of changes in the physical state of lipid bilayers, such
as neutralization of the surface charge, increase of the surface pressure,
enhancement of lipid phase transition temperature, and decrease of the lipid
fluidity (1-4). In addition, the change in orientation of phospholipid headgroups,
induced by divalent cations has been reported (5); this will produce a local
electric field across the m e m b r a n e . H o w e v e r , there are up to now few available
data concerning the consequences that modifications in the physical state of lipids
by divalent cations may have on the function of m e m b r a n e proteins. O v e r the
years, the focus of our laboratory has been on the divalent cation-mediated
change in physical state of phospholipid modulates the function of m e m b r a n e
proteins. In the present article we first show that MgZ+-mediated alteration in
lipid fluidity induces an activity change of reconstituted mitochondrial H +ATPase. Second, we report that a proper t r a n s m e m b r a n e Ca 2+ gradient is
essential for the optimal fluidity of phospholipid bilayer, favouring the formation
~National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing
100101, China.
2To whom correspondence should be addressed.
143
11144-8463/93/06[X)-01435117.00/11
~) 1993 Plenum Publishing Corporation
144
Yang, Huang and Tu
of suitable conformation of adenylate cyclase with higher enzyme activity. Third,
results showing that transmembrane Ca 2+ gradient modulates reconstituted
sarcoplasmic reticulum Ca2+-ATPase (SR Ca2+-ATPase) activity will be
described.
I. Mg2+-MEDIATED C H A N G E IN THE PHYSICAL STATE OF
PHOSPHOLIPID MODULATES MITOCHONDRIAL H+-ATPase
ACTIVITY
Over the years, studies of the reconstitution of mitochondrial H§
with artifical phospholipid liposomes by the cholate dialysis method were carried
out in our laboratory. We found that 1 mM Mg 2§ in the dialysis medium could
greatly enhance the ATPase activity, 32pi-ATP exchange, ATP-driven membrane
potential (A~p) and ApH formation, as well as sensitivity to oligomycin or DCCD
(dicyclohexylcarbodiimide) of the reconstituted enzyme [6-8]. These results were
easily reproducible. The effect of Mg 2+ on the lipid fluidity and conformation of
the reconstituted H§
has been measured. On the basis of the results
obtained, we tentatively suggest that the effect of Mg 2§ may induce a physical
state of phospholipids that favours the formation of a suitable conformation of
the reconstituted H§
complex with higher enzyme activity (Fig. 1).
This assumption is further supported by the results of a series of biochemical
and biophysical experiments, which will be presented in the following sections.
A. Difference in Lipid Packing between MgZ+-containing and MgZ+-free
H+-ATPase-incorporating Proteoliposomes: L.(H+-ATPase)+M~+
and L.(H+-ATPase)_Mg2§
The study of fluidity of the proteoliposomes reconstituted in the presence
and absence of Mg 2+ using spin label 5-NS, [5-(N-oxyl)-4', 4'-dimethyloxazolidine stearic acid] has shown that the order parameters(S) calculated from
the ESR spectra of the Mg2§
proteoliposomes was higher than that of
the Mg2§
proteoliposomes. However, no significant difference was noted
Mg2*
M g z*
Fig. 1.
Mg2 §
M g 2§
g~§
M g 2§
Mg2 +
M~: "
A hypothetical scheme of the effect of Mg 2+ on the reconstituted H+-ATPase.
Divalent cations and biomembranes
145
between the rotational correlation times (r0) of proteoliposomes labeled with
12-NS or 16-NS. This may indicate that the fluidity of these two proteoliposome
preparations was different only near the polar faces, but not in the deeper region
of the phospholipid bilayer [6]. Similar results were obtained by using fluorescent
probes, namely a set of fatty acids with 9-anthroyloxy groups at the seventh,
twelfth and sixteenth positions [9]. Moreover, merocyanine 540 (MC 540), a
lipophilic and sensitive probe for measuring lipid packing, was also used to detect
the Mg2+-inducing change of physical state of phospholipid in the reconstituted
H+-ATPase system. Binding of the probe to the lipid bilayer can be measured by
the enhancement of fluorescence intensity in consequence of its entering the
hydrophobic environment of the membrane. Results showed that the fluorescence
intensity of the L.(H+-ATPase)_Mg~+ was 30% higher than that of L.(H +ATPase)+Mg2+ [10]. This may indicate that lipid molecules in the bilayer are
closely spaced and become more ordered in the presence than in the absence of
Mg 2+. Furthermore, an obvious difference could be detected in the efficiency of
energy transfer between 2-AP and MC 540 of Mg2+-containing and Mg2+-free
proteoliposomes. This difference would appear less significant, if the 2-AP was
substituted by 7-AS or 16-AP. This also suggests that the change in the lipid
packing detected by MC 540 occurs mainly in the region of the bilayer surface.
B. Effect o f Mg 2+ on the Conformation o f the Reconstituted H + - A T P a s e
The assumption that a Mg 2+ effect on the state of the phospholipid affects
the ATPase is supported by studies of the effect of Mg 2§ on the conformation of
the reconstituted H+-ATPase. In addition to Circular Dichroism (CD) studies [6],
the induction of a conformational change in the H+-ATPase, when reconstituted
in the presence of Mg 2+, can also be verified by a shift of the break in the
Arrhenius plot of the reconstituted enzyme complex from 22~ to 19~ and the
difference in the tryptophan intrinsic fluorescence spectra of L.(H §
ATPase)+MG2+ and L.(H+-ATPase)_Mg2+ [11, 12].
To further explore the difference in molecular arrangement of L-(H +ATPase)+Mg2+ and L-(H+-ATPase)_Mg2+, five maleimide spin probes were used.
Figure 2 shows the spectra of 3-maleimido-PROXYL-labeled H+-ATPase reconstituted in the presence or absence of Mg 2+ measured at different temperatures. The spectra are composed of at least two components, and their heights are
designated as S and W (strongly and weakly immobilized components). The ratio
W/S is used as a conformation index of membrane proteins. It could be seen that
the W/S calculated from ESR spectra in Fig. 2 is consistently lower for the
MgZ+-containing than for the MgZ+-free proteoliposomes [7]. As the strongly
immobilized component of the spectrum is thought to be due to spin labels bound
to deeply buried sulfhydryl groups, and the weakly immobilized component
originates from less deeply buried ones, it may be deduced from the ESR spectra
that more sulfhydryl groups in the H+-ATPase molecule are deeply buried in
Mg2+-containing proteoliposomes.
146
Yang, Huang and Tu
-Hg2+
B
+Hg2+
O~ S
5~
~
~
20~
30~
I OO
I,,
,~
I OG
Fig. 2. ESR spectra of 3-maleimido-PROXYL-labeled-H+-ATPase reconstituted in the
presence (B) or absence (A) of Mg2§ measured at different temperatures.
C. Mg 2+ Effect on the Reconstitution of H+-ATPase in Liposomes is
Dependent on the Nature of Phospholipids
The Mg 2§ effect of enhancing the reconstituted H+-ATPase activity might be
interpreted as resulting mainly from the following: (1) a Mg2+-mediated change in
the physical state of lipids, in turn ensuring conformation of H+-ATPase
possessing higher activity, and (2) direct interaction of Mg 2§ with the H §
ATPase. To discriminate between these two possibilities, porcine heart mitochondrial H§
was reconstituted in neutral (PC, PE) or acidic (PI, PG, PA,
PS, DPG) phospholipid (instead of soybean phospholipid) iiposomes separately.
The ATPase activity as well as sensitivity to oligomycin of the reconstituted
enzyme was measured and compared. Results obtained showed that 1 mM Mg 2+ in
the dialysis medium consistently, but unevenly, enhanced the ATPase activity and
its sensitivity to oligomycin in the acidic phospholipid proteoliposomes, but had
little or no effect in neutral phospholipid vesicles [7-9].
As a proton translocator, the reconstituted mitochondrial H+-ATPase can
pump protons from outside to the interior of the vesicles using the energy
released from hydrolysis of externally added ATPI Thus, a A/~.+ composed of
transmembrane potential (A~) and transmembrane pH difference (ApH) can be
generated across the membrane. This pumping activity of the H+-ATPase is a
more significant measure of its function. Here, by using the voltage-sensitive
probe oxonoI-VI [Bis(3-propyl-5-oxoisoxazol-4-yl) pentamethine oxonol] and the
pH-sensitive probe ACMA (9-amino-6-chloro-2-methoxy acridine), the ATPdriven A~, and ApH were measured and compared separately. Results showed
that for the H+-ATPase-incorporating PC + PE + PG proteoliposomes, l m M
Mg 2§ in the dialysis medium markedly increased the ATP-induced oxonol-VI
Divalent cations and biomembranes
147
absorbance change or the A C M A fluorescence quenching. However, this Mg 2+
effect could not be detected for the proteoliposomes reconstituted with only
neutral phospholipids (PC + PE).
D. MgZ+-Mediated Alteration in Physical State of Phospholipid Induces a
Conformational Change in Fo and F~
The mitochondrial H+-ATPase is comprised of two functional units, E, and
Fi. Fo is believed to contain a transmembrane channel through which protons
flow to F1, while F1 is a water-soluble peripheral membrane protein. F0 and F1 are
connected by oligomycin-sensitivity-conferring-protein (OSCP) and other factors.
If the Mg 2§ effect on the reconstitution of H+-ATPase is not a consequence
of direct interaction between Mg 2+ and the enzyme, some conformational change
in the F0 protein after Mg2+-induced alteration in the fluidity of the phospholipid
bilayer would be detected. So, after depletion of F~ by treatment with trypsin and
urea, the conformation of L.(H+-ATPase)+~g2+ and L-(H+-ATPase)_Mg2+ were
compared. Conformational studies were carried out by ESR spectroscopy using
3-maleimido-PROXYL as a probe. In a separate experiment, the Mg 2+ effect on
the conformation of purified soluble F~ was also investigated. The results clearly
showed that Mg 2+ can bring about a conformational change in the Fl-depleted
portion of the reconstituted H+-ATPase (Fig. 3A), whereas in purified F1ATPase no conformational change could be detected after treatment with Mg 2§
(Fig. 3B) (8-9, 13).
A similar conclusion can be drawn from the following experiments: (1) the
activity of H § translocation of Fo-containing proteoliposomes (L-F0), reconstituted in the presence of Mg 2+, was higher than that of Mg2§
vesicles. Here,
the H+-translocation of proteoliposomes was monitored by measuring the change
of fluorescence intensity using 9-amino acridine (9-AA) or the pH difference
.-.
L.(Fo)_Mg
2+
F1
L'(F0)+Mg 2+
10G
10G
Fig. 3. ESR spectra of 3-maleimido-PROXYL-labeled L'(Fo)_MgZ+ (Ft-depleted H+-ATPase,
reconstituted in the absence of Mg2+) and L-(Fo)+~g2+(A), or purified F 1 dialyzed in the presence
or absence of Mg2+ (B). Microwave power, 5 mW; modulation, 2.5 G; time constant, 0.5 second;
scan time, 8 minutes 18~
Yang, Huang and Tu
148
Table 1. Effect of Mg 2+ on ATP-induced fluorescence quenching of aurovertin-H+-ATPase proteoliposomes reconstituted with
PC, PC + D P G , or soybean phospholipids
A T P - i n d u c e d decrease in
aurovertin fluorescence(%)
Proteoliposomes
PC
PC/DPG = 3 : 1
Soybean phospholipids
- Mg 2+
+ Mg 2+
21.2
20.0
32.1
19.3
13.1
25.4
0.5 mg protein of the proteoliposomes was added to the cuvette
with 2ml of medium containing 10raM Tris-H2SO4 (pH 7.7),
0.5mM EDTA, 0.5 mM DTT, and 50mM sucrose at 300(2.
Aurovertin was added to a final concentration of 2 #M. Fifteen
microliters of 200mM ATP (pH 7.7) were injected into the
cuvene. The maximal ATP-induced decrease in aurovertin fluorescence (excitation at 370nm and emission at 470nm) was
expressed as a percentage decrease after the addition of ATP.
(ApH) by fast response-electrode method. (2) the difference of oligomycin
sensitivity of reconstituted H+-ATPase (L'FoF1) activity between Mg 2§
containing and Mg2+-free vesicles became more significant when the OSCP was
added during the reconstitution (unpublished results). It may be deduced that
OSCP was involved in the transmission of Mg 2§ effect from F0 to F1.
Furthermore, if the Mg 2+ effect is not direct, the conformational change in
F0 caused by Mg2+-mediated alteration in the physical state of phospholipid
would be transmitted to F~. It has been shown that aurovertin could be used as a
probe for the conformational changes of the/3 subunit (the catalytic site of F1 for
ATP synthesis) [14-15], which can be detected by the ATP-induced fluorescence
quenching of the enzyme-aurovertin complex [16-18].
Table 1 indicates that in PC reconstituted proteoliposomes, a slight
difference in ATP-induced fluorescence quenching of aurovertin-H+-ATPase
complex exists between the Mg2§
and the Mg2+-containing samples. This
difference became much more obvious, however, with PC + D P G or soybean
phospholipid vesicles. The results indicate that a change in the conformation of
/3 subunit in the F1 portion may be involved in the Mg2§
effect on the
reconstituted H+-ATPase complex.
Based on the above-mentioned results, it seems that Mg 2§ plays a role in
altering the lipid fluidity of the bilayers, which induces a change in conformation
of F0 buried in the lipid core. Such a change can be transmitted to F1, the
conformation of which is in turn changed, resulting in higher enzymatic activity.
In addition to mitochondrial H+-ATPase, similar Mg z+ effects have also
been observed in our laboratory on the reconstitution of cytochrome oxidase [19],
porcine kidney medulla Na, K-ATPase [20], chloroplast H§
and Ca 2+ATPase from rabbit sarcoplasmic reticulum into liposomes [21].
Divalent cations and biomembranes
149
II. EFFECT OF T R A N S M E M B R A N E Ca z+ G R A D I E N T ON FLUIDITY
A N D ACTIVITY OF A D E N Y L A T E CYCLASE-CONTAINING
PROTEOLIPOSOMES
The cytosolic free Ca 2+ in most cells is around 10-7--10 - 6 M , whereas the
extracellular Ca 2§ concentration is about 10 -3 M. This results in a 1000-10,000
fold transmembrane Ca 2§ gradient [22]. It is well known that the maintenance of
such concentration gradient is of vital importance in the cell function [23].
Generally, attention has been paid to the change in the activities of cytosolic
protein kinases in the consequence of increase in Ca 2§ concentration, while the
effect of the transmembrane Ca 2§ gradient and its change on the conformation
and activity of transmembrane proteins (e.g. Ca2+-ATPase, adenylate cyclase)
was more or less neglected. What is the role of transmembrane Ca 2§ gradient for
lipid-protein interaction of biomembranes? Is it essential for the maintenance of a
suitable physical state of lipid bilayer and hence an optimal conformation of
membrane enzymes with proper activity? First of all, the catalytic unit of
adenylate cyclase from bovine brain cortex was purified and reconstituted into
soybean phospholipid vesicles with (1000 fold) or without transmembrane Ca 2§
gradient. The enzyme activity, conformation and fluidity of four types of
proteoliposomes (the active center of enzyme facing outside) were compared [24].
A. Effect of Transmembrane Ca 2+ Gradient on Enzyme Activity of Adenylate
Cyclase (ACc)
The reconstituted activity of ACc in asolectin vesicles could be markedly
stimulated by forskolin (6 fold) or Mn e§ (5 fold), but no stimulation was observed
with guanidine nucleotide. This may indicate that the ACc used for incorporating
into asolectin vesicles was highly resolved from GTP regulatory protein. The
efficiency of the reconstitution of ACc ranged from 40-60% as estimated from
protein determination.
The proteoliposomes were prepared with sufficiently low Ca 2§ permeability.
Additon of ionophore A23187 would lead to a release of entrapped Ca 2§
outward, and hence a rapid increase of absorbance (675-680 nm) using arsenazo
III as Ca 2§ indicator. Following treatment with sodium cholate, no obvious
difference in enzymatic activity of the reconstituted proteoliposomes would be
detected. So, it could be deduced that ACc was inserted in a highly oriented
manner with most of the active sites facing outside (ACc.L), similar to inside-out
cell preparations.
Then, the enzymatic activities of four types of proteoliposomes with or
without Ca 2§ gradient were determined and compared. From Table 2, it could be
seen that the highest activity was observed in the case of A C e - L e a + - (lower
Ca 2§ outside) which is similar to physiological situation. If the transmembrane
Ca 2+ gradient was in the inverse direction ( A C c . L c a - + , higher Ca z+ outside), a
lower enzyme activity would appear. Proteoliposomes without transmembrane
Ca 2+ gradient ( A C c . L c a - - , A C c . L c a + + ) exhibited intermediate activities.
It is interesting to note that following the dissipation of transmembrane Ca 2+
150
Yang, Huang and Tu
Table 2. Effect of transmembrane Ca 2+ gradient (1000 fold) on the enzymatic activity of
ACc-containing proteoliposomes
Proteoliposomes
ACc-Lca+ -
ACe.Lea- -
ACc.Lca+ +
ACe.Lea- +
880
370
270
180
Enzyme activity
(pmol cAMP/min/mg protein)
The figures listed in this table are average of five experimental results + - ; - - ; + + and - + refer to
high Ca 2+ inside, low Ca2+ outside; low Ca 2+ both sides; high Ca 2+ both sides and low Ca 2+ inside,
high Ca 2+ outside, respectively.
g r a d i e n t by A23187, t h e d i f f e r e n c e in e n z y m e activity b e t w e e n A C e - L e a + - a n d
ACe.Lea-+
was d i m i n i s h e d ( T a b l e 3). It l e a d s to a d e c r e a s e in e n z y m a t i c
activity o f t h e f o r m e r (close to A C e . L e a - - ) ,
b u t i n c r e a s e in t h e l a t t e r case
Table 3. Change in adenylate cyclase activity
following the dissipation of transmembrane Ca 2+
gradient by A23187
Enzyme activity
(pmol cAMP/min/mg)
Vesicles
ACe-Lea+ ACc-Lca- +
- A23187
+ A23187 t
780
170
350
280
~Vesicles were pretreated with A23187 (10/~g/ml)
for 10 min at 0~ before assay.
(similar to that o f A C c - L c a + + ) . T h e s e results p r o v i d e a n o t h e r i n d i c a t i o n t h a t t h e
e n z y m a t i c activity o f t h e r e c o n s t i t u t e d a d e n y l a t e cyclase was m a r k e d l y a f f e c t e d b y
the p r e s e n c e o f a C a 2+ g r a d i e n t across m e m b r a n e a n d a p r o p e r t r a n s m e m b r a n e
C a 2+ g r a d i e n t is e s s e n t i a l for t h e h i g h e r e n z y m e activity.
B. Effect of Transmembrane Ca 2+ Gradient on the Conformation of
ACc-incorporating Proteoliposomes
In o r d e r to c o m p a r e the c o n f o r m a t i o n o f i n c o r p o r a t e d A C e in t h e s e
proteoliposomes, fluorescence spectroscopy and CD have been used.
T h e f l u o r e s c e n c e e m i s s i o n s p e c t r a o f A C e - i n c o r p o r a t i n g p r o t e o l i p o s o m e s as
c o m p a r e d with u n r e c o n s t i t u t e d A C e a r e s h o w n in Fig. 4. F l u o r e s c e n c e was
m e a s u r e d with an e x c i t a t i o n w a v e l e n g t h o f 285 n m , a n d d i f f e r e n c e in e m i s s i o n
intensity was r e c o r d e d at 338 nm. A s can be s e e n f r o m Fig. 4, r e c o n s t i t u t i o n o f
A C e in f o u r t y p e s o f a s o l e c t i o n vesicles with (1000 fold) o r w i t h o u t t r a n s m e m b r a n e C a 2§ g r a d i e n t w e r e a c c o m p a n i e d by a d e c r e a s e in i n t e n s i t y o f intrinsic
fluorescence with no m a r k e d shift in e m i s s i o n m a x i m u m at 338 nm. T h e intrinsic
p r o t e i n f l u o r e s c e n c e o f f o u r t y p e s o f p r o t e o l i p o s o m e d e c r e a s e d in t h e o r d e r :
ACe.Lea+- > ACe-Lea-- > ACe.Lea++ > ACe.Lea-+.
It m a y i n d i c a t e t h a t
t h e m i c r o e n v i r o n m e n t o f t r y p t o p h a n y l r e s i d u e s o f A C e in t h e s e t y p e s o f a s o l e c t i n
vesicles was different.
T h e C D s p e c t r a in t h e w a v e l e n g t h r a n g e f r o m 2 0 0 - 2 5 0 n m o f A C c - c o n t a i n i n g
Divalent cations and biomembranes
70
151
A
- 6o
"~5o
~4o
0
,
300
320
340
360
W a v e l e n g t h (nm)
I
380
~
400
Fig. 4. Intrinsic fluorescence spectra o f A C c and ACccontaining vesicles with o r w i t h o u t t r a n s m e m b r a n e Ca 2+
gradient. Curve A, ACe; curve B, A C c . L c a + - ; curve C,
A C c - L c a - - ; curve D, ACc.Lca+ + ; curve E, ACc-Lca+ +.
vesicles with (1000 fold) or without transmembrane C a 2+ gradient were shown in
Fig. 5. It is interesting to note that the order of decrease in the estimated o~-helix
contents of ACc in four types vesicles coincides exactly with that of increase in
the enzymatic activities of ACc.
C. Difference in Fluidity of Liposomes and ACc-containing Proteoliposomes
with or without Transmembrane Ca 2+ Gradient
Friedlander et al. [25] reported that lipid fluidity could influence the
adenylate cyclase activity. So, the lipid fluidity of four types of ACe-incorporating
0~
0
0
i
c)
tD~
0)
7cD
i
~zJ
200
I
210
I
I
I
220
230
240
W a v e l e n g t h (nm)
I
250
Fig. 5. CD spectra o f four types o f A C c - c o n t a i n i n g
p r o t e o l i p o s o m e s . Curve A, A C e . L e a + - ; curve B,
ACc-Lca--;
curve C,
ACc-Lca++;
curve D,
A C e . L e a - +.
152
Yang, Huang and Tu
Table 4. Difference in fluidity of ACc-incorporating proteoliposomes with or without transmembrane Ca2+ gradient
Proteoliposomes
ACc.Lca+ -
ACc.Lca- -
ACc-Lca+ +
ACc.Lca- +
DPH polarization
0.160 + 0.001
P < 0.02(6)
0.165 + 0.002
0.167+ 0.002
P < 0.02(6)
0.175 + 0.001
P < 0.02(6)
Values of fluorescence polarization are mean value + S.E. with the number of experiments in
parentheses. P<0.02 indicates that the difference is statistically significant compared with
ACc.Lca
proteoliposomes was measured using D P H (diphenylhexatriene) as fluorescent
probe. For comparison the degree of fluorescent polarization for D P H in
liposomes with or without Ca z+ gradient was also determined. Results showed
that the degree of fluorescent polarization for D P H in these liposomes decreased
in the order: L c a + + > L c a - + > L c a + - >
L c a - - . It is reasonable that Ca z+ is
able to decrease the lipid fluidity and two-side Ca 2+ effect is m o r e obvious than
one-side. In the case of four types of ACc-incorporating proteoliposomes the
fluidity decreased in another order: A C c . L c a + - > A C c - L c a - - > A C c - L c a + +
>ACc.Lca-+
(Table 4), which followed however, the same order as the
enzyme activity (Table 2).
It was known that higher Ca 2+ concentration ( > I / ~ M ) would inhibit
adenylate cyclase [26]. H o w e v e r , from our results, it seems that in addition to
direct effect, Ca2+-mediated change in lipid fluidity may also modulate the
reconstituted adenylate cyclase activity.
Based on the above-mentioned results, we tentatively suggest that a proper
transmembrane Ca z+ gradient may offer both in the outer and inner layer a
suitable fluidity of phospholipid, favouring the formation of an optimal conformation of the reconstituted adenylate cylcase with higher enzymatic activity.
III. TRANSMEMBRANE Ca 2+ GRADIENT MODULATES ACTIVITY
AND CONFORMATION OF RECONSTITUTED SARCOPLASMIC
RETICULUM Ca2+-ATPase (SR Ca2+-ATPase)
It is well known that in sarcoplasmic reticulum also, a 1000-10,000 fold
transmembrane Ca 2+ gradient exists, which is maintained by CaZ+-ATPase [27].
In order to explore the role of t r a n s m e m b r a n e Ca 2+ gradient in the modulation of
CaZ+-ATPase during contraction and relaxation cycle of muscle cells, the
enzyme-containing proteoliposomes with (1000 fold) or without t r a n s m e m b r a n e
Ca z+ gradient were reconstituted. Results showed that a proper t r a n s m e m b r a n e
Ca 2+ gradient is essential for the inhibition of the SR CaZ+-ATPase activity and
then the dissipation of the gradient will lead to an activation of C a z+ pumping.
And it was also revealed that phospholipids (especially PC) may be involved in
such modulation process.
A. Transmembrane C a 2§ Gradient and SR Ca2+-ATPase Activity
When skeletal SR (6 mg/ml) was treated with deoxycholate (3 m g / m l ) , 80%
of proteins could be solubilized. The detergent could be r e m o v e d f r o m solubilized
Divalent cations and biomembranes
153
Table 5. Effect of transmembrane Ca 2+ gradient on the enzyme
activity of Ca2+-ATPase-incorporatingproteoliposomes
Proteoliposome
(Cai:Cao)
A
B
C
D
E
(i00:100) (100:1) (1000:1) (100:1000) (1:1)
Enzyme activity (ttmol/mg-min)
-A23187
+A23187
4.20
4.80
1.28
--
0.29
4.50
0.20
3.10
3.85
--
samples by dialysis in different solutions ( 8 m M Hepes, 0 . 2 5 M sucrose,
0.4 M KCI, 1.5 mM Mg 2+, 1 mM E D T A and different concentrations of Ca 2+, p H
7.25) for 24hrs. Five types of vesicles with or without transmembrane Ca 2+
gradient were reformed with lower Ca 2+ permeability and ATP-dependent Ca z+
transport activity. The phospholipid content of the proteoliposomes was similar to
the native SR membrane, but in the vesicles most of protein was CaZ+-ATPase
(>95%).
The A T P hydrolysis activity of the four types of CaZ+-ATPase-containing
proteoliposomes with or without transmembrane Ca 2+ gradient was determined
(Table 5). From Table 5, it can be seen that the highest enzyme activity was
observed in the case of proteoliposome A (100/~M Ca 2+ on both side, without
transmembrane Ca 2+ gradient), which is similar to the physiological situation
when Ca 2+ is released from SR. If there existed a transmembrane Ca 2+ gradient,
no matter what the direction was, a lower enzyme activity would appear.
Comparing the enzyme activities of proteoliposome A, B and C, it is evident that
the higher transmembrane Ca 2+ gradient was, the lower the enzyme activity
appeared, especially for C (with 1000 fold transmembrane C a z+ gradient), only
7% of enzyme activity of A being observed [28]. These results may indicate that
under the physiological situation, when Ca 2+ was pumped into SR, the
Ca2+-ATPase activity would be gradually inhibited in consequence of increase in
transmembrane Ca 2+ gradient.
Furthermore, as shown in Fig. 6, all of the three types of proteolipos0mes
could accumulate Ca z+ (without oxalate inside) and the proteoliposome A
( C a i : C a o = 100:100) has the highest activity of Ca 2+ uptake. If there existed
transmembrane Ca 2+ gradient, no matter what the direction was, a lower Ca 2+
uptake activity would appear.
It can also be seen in Table 5 that following the dissipation of transmembrane Ca 2+ gradient by Ca 2+ ionophore A23187, enzyme activity of
proteoliposome C and D increases and hence the difference of enzyme activity in
above-mentioned CaZ+-ATPase-containing proteoliposomes is markedly diminished. This result provided another indication that the enzyme activity of
reconstituted Ca2+-ATPase was markedly inhibited by the presence of the
transmembrane Ca 2+ gradient.
B. Comparison of Conformation of SR CaZ+-ATPase-incorporating
Proteoliposomes with and without Transmembrane C a 2+ Gradient
In order to compare the conformation of Ca2+-ATPase incorporated in these
proteoliposomes, fluorescence quenching technique has been used, According to
154
Yang, Huang and Tu
800
z~
"d
600
m
v
400
m
a
a
200
a
3
5
Time
A
7
( rain )
o
I00:100
+
1000:1
I00:1000
Fig. 6. Effect of transmembrane Ca 2+ gradient on Ca 2+uptake of Ca2§
proteoliposomes.
1.80
A
O
o
A
1.60
o B
C
D
u
Q
1.40
O
a
1.20
O
O
ea
ee
1.00 0.00
!
0.20
0.40
Acrylamide
0.60
(
M
0.80
1.00
)
Fig. 7. Quenching of PM-Ca2+-ATPase by acrylamide.
(Ca~: Cao = 100: 100); B, (100: 1); C, (1000 : 1); D (100: 1000).
A,
Divalent cations and biomembranes
155
the results of Andesen and Hammes [29], SR Ca2+-ATPase has 18-19 SH
groups, some of which are exposed on the surface of CaZ+-ATPase and can be
labeled by NPM [N-(1-pyrenyl)maleimide]. If the concentration of NPM is lower
(NPM: CaZ+-ATPase, 1 : 1), only the SH group near catalytic site can be labeled.
From fluorescence quenching of PM.Ca2+-ATPase with acrylamide, we can
determine the change of micro-environment of this SH group (Fig. 7). Our results
showed that quenching is highest in the proteoliposome A (without transmembrane Ca 2+ gradient). The ksv (the Stern-Volmer constant) were 1.00 M -1,
0.79 M -1, 0.71 M -1 and 0.61 M -~ for proteoliposome A, B, C and D respectively.
It is evident that the conformation of CaZ+-ATPase in above mentioned
proteoliposomes varies with transmembrane Ca 2+ gradient.
C. Difference in Fluidity between SR CaZ+-ATPase-containing
Proteoliposomes with and without Transmembrane Ca2+ Gradient
It has been suggested that the rate-limiting step in the reaction of the
SR.Ca2+-ATPase could be dependent on the fluidity of the surrounding lipid
bilayer [30]. In order to study the molecular mechanism of the role of
transmembrane Ca 2+ gradient in the modulation of Ca2+-ATPase, the physical
state of the membrane lipids in the three types proteoliposomes was investigated
by using fluorescence probe DPH. Results in Table 6 show that the fluidity
decreases in the same order as the enzyme activities.
Table 6. Effectof transmembrane Caz+ gradient on membrane fluidity
and activityof Ca2+-ATPase-incorporatingproteoliposomes
Proteoliposomes
(Cai:Cao)
DPH polarization(P)
Enzyme activityt
A
(100: 100)
B
(1000: 1)
C
(100:1000)
0.157 + 0.002 0.175+ 0.002 0.182+ 0.003
4.50
0.30
0.20
1/~mol/min.mgprotein.
The above-mentioned results may indicate that during the contractionrelaxation cycle of muscle cells, in consequence of release of stored Ca 2+ through
the channel, the transmembrane Ca 2+ gradient will decrease, resulting in an
increase of membrane fluidity, followed by activation of Ca2+-ATPase which will
take up Ca 2+ back into SR and reestablish the transmembrane Ca 2+ gradient.
This will lead to a decrease in lipid fluidity, and hence a conformation change of'
Ca2+-ATPase, resulting in inhibition of its activity. So, it seems probable that in
addition to the direct effect of Ca 2+ on the Ca2+-ATPase [31-32], phospholipids
may also be involved in the modulation of Ca2+-ATPase by transmembrane Ca 2+
gradient. This is further supported by the following results.
D. Phosphatidylcholine Plays an Important Role in the Modulation of SR
CaZ+-ATPase by Transmembrane Ca2§ Gradient
We suggested above that phospholipids may be involved in the transmembrane Ca a+ gradient-mediated modulation of SR Ca2+-ATPase. Such
156
Yang, Huang and Tu
postulation was tested by measuring the activity of the reconstituted enzyme in
different phospholipid vesicles with or without transmembrane Ca 2+ gradient. It
has been reported [33] that higher Ca 2§ uptake could be observed when the SR
Ca2+-ATPase was reconstituted in the presence of phosphatidylethanolamine(PE). It is also known that phosphatidylcholine(PC) is the main phospholipid (65%) of SR membrane but its role is not yet understood. In order to
explore the lipid requirement for the modulation of Ca2+-ATPase by the
transmembrane Ca 2§ gradient, the purified Ca2+-ATPase was reconstituted with
different phospholipid mixtures: PC-PE (M:M, 1:1), PS-PE (1:1) and P G - P G
(1 : 1) in the presence or absence of transmembrane Ca 2§ gradient. Here, the final
lipid to protein ratio was 30 : 1 (W: W) and all the proteoliposomes prepared with
lower Ca 2+ permeability.
The results clearly showed that a singificant inhibition of ATP hydrolysis and
Ca 2§ uptake by the transmembrane Ca 2§ gradient (Cai>Cao, similar to the
physiological situation) could be observed in the PC-PE vesicles incorporating
Ca2+-ATPase, while in the case of PS-PE or P G - P E proteoliposomes little or no
inhibition was detected. So, this probably reflects that PC may be involved in the
inhibition of reconstituted SR Ca2+-ATPase activity by the transmembrane Ca 2§
gradient (Cai>Cao). Furthermore, it is interesting to note that PC mainly
distributes in the inner leaflet of the native SR membrane. Presumably, the
asymmetry of PC distribution is a major determinant in the modulation of SR
Ca2+-ATPase by the transmembrane Ca 2§ gradient.
IV. PERSPECTIVES
Mg2+ and Ca 2+ are abundant cations within cells. Based on the abovementioned studies showing that a MgZ+-mediated change in lipid physical state
modulates activity of mitochondrial H+-ATPase and that transmembrane Ca 2+
gradient affects the conformation and activity of reconstituted adenylate cyclase
and SR CaZ+-ATPase, we suggest that the structure and function of many
membrane proteins may be modulated by divalent cations in a similar way. So, it
will be interesting to extend these investigations to other membrane proteins as
well as to study in more detail the mechanism of modulation by divalent cations
on membrane proteins mediated through lipid-protein interaction.
ACKNOWLEDGEMENTS
The work of our group quoted in this article was supported by Academia
Sinica and the National Natural Science Foundation of China.
The authors are also grateful to their colleagues Zhang, X. F., Guo, B. Q.,
Cheng, Q. S., Peng, H., Zhou, L. X., Che, Y. W., Zhang, Y. Z., Liu, Z. M.,
and Tong, J. C. who have contributed with enthusiasm to the studies presented
here and to Miss X. J. Luo and W. M. Zhong in the preparation of this article.
Divalent cations and biomembranes
157
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