Electrogenic H+ translocation by the plasma membrane ATPase of Neurospora. Studies on plasma membrane vesicles and reconstituted enzyme.

Abstract

Fluorescent probes have been used to measure electrogenic proton pumping by the plasma membrane ATPase of Neurospora. In isolated plasma membrane vesicles, greater than 85% of which are inverted, ATP hydrolysis is accompanied by the formation of an inside acid pH gradient (delta pH) which can be detected by acridine orange fluorescence quenching and an inside positive membrane potential (delta psi) which can be detected by oxonol V fluorescence quenching. Maximal values of delta pH were generated in the presence of a permeant anion (SCN-, NO-3, or Cl-) and maximal delta psi, in the absence of such anions. Cation effects were much less pronounced and can probably be accounted for by non-specific salt effects on the rate of ATP hydrolysis. In addition, a rapid method is described for the reconstitution of the [H+]-ATPase, starting from isolated plasma membranes. When the membranes are solubilized with deoxycholate in the presence of asolectin and detergent is removed by passage through a Bio-Gel P-10 column, vesicles are reformed in which the Mr = 104,000 polypeptide of the ATPase constitutes 35% of the protein. Freeze-fracture electron microscopy of the vesicles has revealed intramembrane particles with a diameter of 116 A, equally distributed between the two fracture faces. Measurements with acridine orange and oxonol V indicate that the reconstituted ATPase retains its transport activity, generating both delta pH and delta psi during the hydrolysis of MgATP.

Full text

Page 1

THE JOURNAL
0 1984 by The American Society of Biological Chemists, Inc
OF BIOLOGICAL
CHEMISTRY
Vol. 259, No. 12, Issue of June 25, pp. 7884-7892,1984
Printed in U.S.A.
Electrogenic H+ Translocation by the Plasma Membrane ATPase
Neurospora
STUDIES ON PLASMA MEMBRANE VESICLES AND RECONSTITUTED ENZYME*
of
(Received for publication, July 11, 1983)
David S. PerlinS, Kunihiro Kasamo, Robert J. Brookers, and Carolyn W. Slayman
From the Departments of Human Genetics and Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
Fluorescent probes have been used to measure elec-
trogenic proton pumping by the plasma membrane
ATPase of Neurospora. In isolated plasma membrane
vesicles, >85% of which are inverted, ATP hydrolysis
is accompanied by the formation of an inside acid pH
gradient (ApH) which can be detected by acridine or-
ange fluorescence quenching and an inside positive
membrane potential (A*) which can be detected by
oxonol V fluorescence quenching. Maximal values of
ApH were generated in the
anion (SCN-, NO;, or C1-) and maximal A*, in the
absence of such anions. Cation effects were much less
pronounced and can probably be accounted for
specific salt effects on the rate
In addition, a rapid method is described for the re-
constitution of the [H+]-ATPase, starting from isolated
plasma membranes. When the membranes are solubi-
lized with deoxycholate in
and detergent is removed by passage through a Bio-
Gel P-10 column, vesicles are reformed in which the
M, = 104,000 polypeptide of the ATPase constitutes
35% of the protein. Freeze-fracture electron micros-
copy of the vesicles has revealed intramembrane par-
ticles with a diameter of 116 A, equally distributed
between the two fracture faces. Measurements with
acridine orange and oxonol V indicate that the recon-
stituted ATPase retains its transport activity, gener-
ating both ApH and A*
during the hydrolysis
MgATP.
presence of a permeant
by non-
of ATP hydrolysis.
the presence of asolectin
of
It is now well established that the fungal plasma membrane
contains a primary proton-translocating ATPase,
iological function is to generate an
gradient and thereby to supply energy to a variety of [H’I-
dependent cotransport systems (1). Biochemically, the fun-
gal [H+]-ATPase belongs to the class
ATPases that also includes the [Na+,K’]-, [Ca2+]-, and
[H+,K’]-ATPases of animal cells. It has a polypeptide subunit
of M, = 100,000 which is deeply embedded within the lipid
bilayer of the membrane and requires detergents for solubili-
zation and purification (2, 3). During the reaction cycle, the
.
whose phys-
electrochemical proton
of ion-translocating
* This work was supported by National Institutes of Health Grant
GM-15761. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “aduertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
$ Present address, Department of Biochemistry, University of
Rochester Medical Center, 601 Elmwood Avenue,
NY 14642.
Present address, Department of Physiology, Harvard Medical
School, 25 Shattuck Street, Boston, MA 02115.
Rochester,
M, = 100,000 polypeptide forms an acylphosphate interme-
diate (4, 5); probably for this reason, ATPase activity is
extremely sensitive to inhibition by vanadate (6).
Much of our knowledge about the transport function
[H’I-ATPase has come from electrophysiological experiments
on whole cells (hyphae) of Neurospora. Because of their
relatively large diameter, the cells can be penetrated by mi-
cropipette electrodes; and it was this kind of direct measure-
ment that first demonstrated the ATP-dependent generation
of membrane potentials as high as -250 mV, inside negative
(7). Later experiments equated the electrogenic pump with
the [H+]-translocating ATPase (l), and computer techniques
have now been developed which allow the pump current (H’
efflux) to be extracted from the total membrane current (8,
9). By means of such experiments, it is possible to explore the
behavior of the ATPase in situ as the two components of the
electrochemical H+ gradient are varied separately: the mem-
brane potential, by passing current through a separate elec-
trode (8,9), and the pH gradient,
extracellular pH (10, 11).
Complementary information about the Neurospora [H+] -
ATPase has come from the study of isolated plasma mem-
brane vesicles, an approach pioneered by Scarborough and
his colleagues (12-14). Using vesicles prepared from the slime
mutant (12), which are assumed to be partially or completely
inverted (13, 14), these workers have shown that ATP hy-
drolysis leads to the formation of an inside acid pH gradient
(measured by the distribution of [‘4C]imidazole (14)) and an
inside postiive membrane potential (measured by the distri-
bution of [14C]SCN (13)). Their results provide strong support
for the notion of electrogenic proton translocation.
Nevertheless, a number of important questions remain con-
cerning the transport function of the fungal plasma membrane
ATPase. In studies on ATPases prepared
related yeast species, Saccharomyces cereuisiae and Schizosuc-
charomycespombe, Malpartida and Serrano (15) and Villalobo
(16) have raised the possibility that the fungal enzyme may
mediate an exchange of H’ for K’ rather than a purely
electrogenic movement of H+. A similar idea has long been
prevalent in the literature on the plasma membrane ATPase
of higher plants (17), while the plant tonoplast ATPase has
been postulated to couple anions to the translocation
protons (18).
In order to address these questions, we have examined the
effects of a variety of cations and anions on the ATP-depend-
ent formation of ApH and A* by the Neurospora plasma
membrane ATPase. To permit a ready comparison with elec-
trophysiological results obtained in uiuo, our experiments have
been carried out with vesicles isolated from the wild type
strain of Neurospora (19), and we have used the optical probes
acridine orange and oxonol V to provide a continuous record
of the
by varying intracellular and
from two closely
of
7884

Page 2

Proton Transport by the Neurospora [H+]-ATPase
7885
of ApH and A+. In addition, as an extension of the work with
plasma membrane vesicles, we have developed
stitution procedure which yields proteoliposomes enriched in
the ATPase and capable of rapid rates of ATP-dependent
proton translocation.
a simple recon-
MATERIALS AND METHODS
Isolation of Plasma Membranes-Wild type strain RL2la of Neu-
rospora crassa was grown for 12 to 14 h at 25 “C in Vogel’s minimal
medium with 2% sucrose (20). Plasma membranes were prepared
according to the method of Bowman et al. (19) in which cells are
treated with snail-gut enzyme to weaken their cell walls, disrupted by
sequential homogenization and sonication, and fractionated by dif-
ferential centrifugation. The resulting plasma membranes were sus-
pended in 1 m M EGTAl-Tris, pH 7.2, and stored at -70 “C. When
assayed for ATPase activity under optimal conditions (pH 6.7 (21))
they had specific activities of 4 to 6 pmol/min/mg of protein. Freeze-
fracture electron microscopy revealed the presence of closed mem-
brane vesicles, as will be described in a later section.
Fluorescence Quenching-ApH and A* were assayed by fluores-
cence quenching of acridine orange (22) and oxonol V (23), respec-
tively. In experiments designed to establish optimal conditions, the
pH dependence of ATP-induced oxonol quenching was similar to the
pH dependence of ATP hydrolysis with a broad optimum between
pH 6 and 7. For acridine orange, however, the optimum for both the
initial rate and the extent of ATP-induced quenching was at pH 7.5.
Two factors probably contribute to this discrepancy: the fact that the
buffer capacity of isolated plasma membranes, like that of intact cells
of Neurospora (11), drops sharply as the pH is increased from 5.5 to
7.5’; and the fact that, at higher external pH, fewer protons need to
be transported to generate a given pH gradient. The best compromise
between the optimal conditions for the two probes was found empir-
ically to be pH 7.2, which was adopted for all further measurements.
Thus, for the assay of ApH under standard conditions, vesicles
(50-100 yg of protein) were incubated in a stirred fluorescence cuvette
containing 2.0 ml of reaction medium (100 m M KCl, 20 m M HEPES-
KOH buffer, pH 7.2, and 2 PM acridine orange) at 22 “C. The reaction
was initiated by the injection of 1.5 to 5.0 m M MgS04 and Na2ATP
through a light-tight delivery port in the top of the cuvette. Relative
fluorescence was monitored with an Aminco-Bowman spectrofluoro-
meter, using excitation and emission wavelengths of 429 and 600 nm,
respectively. The assay of membrane potential was carried out in the
same way, except that the standard reaction medium lacked KC1 and
contained 1 to 3 p~ oxonol V and 100-200 yg of membrane protein.
Excitation and emission wavelengths were 580 and 640 nm, respec-
tivley.
Control experiments showed that, as expected from the known
substrate requirements of the plasma membrane ATPase, the fluo-
rescence quenching of acridine orange and oxonol V was specific for
ATP; no quenching was obtained with other nucleoside triphosphates
(GTP, ITP, CTP, UTP) added at 5 m M in the presence of 5 m M
MgSO4 (24). The ATP-induced quenching of both probes was abol-
ished by FCCP (2 yg/ml), which would be expected to collapse ApH
and A*, and by vanadate (100 p ~ ) ,
membrane ATPase (6). It was not affected by KN, (1 mM), an
inhibitor of mitochondrial ATPase (25).
Reconstitution of the [H+]-ATPase-A
sion was prepared containing (in 2.0 ml) 3 mg of membrane protein,
10 m M MES-KOH or HEPES-KOH, pH 7.2, 150 m M KC1, 1 m M
a potent inhibitor of the plasma
plasma membrane suspen-
Na’EDTA, and 10 mg of acetone-washed asolectin (26). To this cloudy
suspension, purified deoxycholate (27) was added to give a final
concentration of 0.6%. Upon vigorous homogenization, the suspen-
sion became clear. It was then rapidly applied to a Bio-Gel P-10
column (20 X 1.5 cm), which was eluted with buffer containing 10
’ The abbreviations used are: EGTA, ethylene glycol bis(p-amino-
ethyl ether)-N,N,N’,N‘-tetraacetic acid; FCCP, carbonyl cyanide p-
trifluoromethoxyphenylhydrazone; HEPES, N-2-hydroxyethylpiper-
azine-N’-2-ethanesulfonic acid MES, 2-(N-morpholino)ethane-
sulfonic acid; oxonol V, bis[3-phenyl-S-oxoisoxazol-4yl]pentamethine
oxonol; acridine orange, 3-bis-dimethyl aminoacridinium chloride;
SITS, 4-acetamido-4’-isothiocyano stilbene-2,2’-disulfonic acid
TMA, tetramethylammonium; CCCP, carbonyl cyanide p-
chlorophenylhydrazone; PIPES, 1,4-piperazinediethanesulfonic acid.
’ D. S. Perlin, unpublished experiments.
-
m M MES-KOH, pH 7.2, 100 m M KCl, and 0.1 m M NazEDTA at a
flow rate of 1.0 ml/min. The cloudy void volume fractions were pooled,
diluted 40-fold with HEPES/KCl/EDTA buffer, and centrifuged at
100,000 x g for 1 h. The pellet was suspended in 10 m M MES-Tris,
pH 7.2, 100 m M KC1 for assays of ApH and in 50 m M MES-KOH,
pH 7.2, for assays of A*.
Electron Microscopy-Electron microscopy of whole cells, plasma
membrane vesicles, and reconstituted ATPase was carried out follow-
ing freeze-fracture in order to visualize intramembranous particles.
Cells were fixed in 2.5% glutaraldehyde buffered with 0.1 M cacodyl-
ate, pH 5.8. Plasma membrane vesicles and reconstituted enzyme
were suspended in 1.0 m M EGTA (adjusted to pH 7.5 with Tris), 20%
glycerol, and centrifuged at 100,000 x g for 1 h. Specimens were
quickly frozen in Freon 22, cooled with liquid nitrogen, and fractured
at -130 “C using a Balzers BAF freeze-fracture apparatus. The spec-
imens were then shadowed with platinum and replicated with carbon,
the thickness of the films being controlled by a Balzers QSG 201
quartz crystal thin film monitor. Replicas were washed overnight in
sodium hypochlorite, picked up on 100-mesh Formvar-coated grids,
and examined in a Zeiss EM 10B microscope.
Other Methods-ATPase activity was assayed at 22 “C as described
by Bowman and Slayman (21). Protein was determined by the method
of Lowry et al. (28) with bovine serum albumin as standard. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis was performed on
8-16s gradient gels (3); after staining with Coomassie blue, the gels
were scanned with a Beckman model DU-8 densitometer.
Reagents-Acridine orange, HEPES, MES, orthovanadate, FCCP,
L-a-phosphatidylcholine, L-a-phosphatidylethanolamine, L-a-phos-
phatidylinositol, L-a-phosphatidylserine, and L-a-phosphatidic acid
were purchased from Sigma; oxonol V from Molecular Probes, Inc.;
asolectin from Avanti Polar Lipids; SITS from Pierce Chemical Co.;
and [3H]deoxycholate from New England Nuclear.
RESULTS
Sidedness of P h m a Membrane Vesicles-In order to ex-
amine the ultrastructure of the material to be used for trans-
port studies, freeze-fracture electron microscopy was carried
out on whole cells of wild type Neurospora and on plasma
membranes isolated by the method of Bowman et al. (19). In
the whole cells, the plasma membrane appeared highly asym-
metric, as has been observed for many types of biological
membranes (29). Tangential fracturing revealed a concave P
face, studded with intramembrane particles, and a convex E
face, relatively free of particles (Fig. 1, A-C). The average
particle frequency was 2672 f 74/pm2 on the P face and 348
f 29 per pm2 on the E face (Table I).
Isolated plasma membranes appeared
mostly empty but in a few cases contained smaller vesicles or
other membranous inclusions (Fig. 1D).
ameters ranged from 0.05 to 0.53 pm, with 90% of the values
falling between 0.08 and 0.30 pm; the spread of values presum-
ably reflects the fact that the vesicles were fractured at various
levels relative to their equatorial plane. Once again, the mem-
branes appeared highly asymmetric, with
cies of the P face and E face not significantly different from
those of the intact plasma membrane (Table I). This asym-
metry provided a means to determine the sidedness of the
vesicles: 1257 of 1450 vesicles, or 87%, were inverted relative
to the intact cell.
Measurement of ApH with Acridine Orange and A* with
Oxonol V-On
the basis of previous studies (13, 14), it is to
be expected that inverted plasma membrane vesicles should
hydrolyze ATP at the outer surface, pumping H’ inward and
generating a pH gradient (inside acid) and a membrane po-
tential (inside positive). Control experiments, summarized
under “Materials and Methods,” showed that ApH can be
followed by fluorescence quenching of the weak base acridine
orange (22, 30) and A+ by fluorescence quenching of oxonol
V (23, 31). With the two probes in
objective was to follow the time course with which ApH and
as vesicles which were
The measured di-
the particle frequen-
hand, an immediate

Page 3

7886
Proton Transport
by the Neurospora [H+J-ATPase
FIG. 1. Electron micrographs of freeze-fracture replicas of whole
and reconstituted vesicles. A, whole cells; bar, 0.5 pm, X 26,000. B and C, whole cells, P face and E face of
plasma membrane, respectively; bar, 0.1 pm, X 125,000. D, isolated plasma membrane vesicles;
40,000. E, reconstituted vesicles; bar 0.5 pm, X 40,000. In all cases, direction of shadowing is from bottom to top.
cells, plasma membrane vesicles,
bar, 0.5 pm, X
A* are generated and to analyze the factors that dictate their MgATP was added to each cuvette. There was a rapid decrease
relative sizes. In the experiment of Fig. 2, two identical
suspensions of membrane vesicles were made, one containing neous formation of a membrane potential. By contrast, acri-
acridine orange and the other, oxonol V. At the first arrow, dine orange fluorescence decreased more slowly, requiring 45
in oxonol fluorescence, corresponding to the nearly instanta-

Page 4

Proton Transport
by the Neurospora [H+]-ATPase
7887
TABLE I
Frequency of intramembrane particles
Cells and vesicles were subjected to freeze-fracturing and examined
by electron microscopy, as described under “Materials and Methods.”
For calculation of particle frequencies, areas were determined using
a sauare erid.
~~
Whole cell
Plasma membrane
vesicles
Reconstituted
vesicles
Frequency
particleslFm2
2672 f 74
348 f 30
2874 f 321
338 t 60
445 f 24
441 t 39
P face
E face
P face
E face
Concave face
Convex face
Mg ATP
I
H
IO sac
FIG. 2. Time course for ATP-induced fluorescence quench-
ing of acridine orange and oxonol V. Parallel assays were made
as described under “Materials and Methods” in the presence of 50
mM HEPES-KOH, pH 7.2, and 2.5 y~ acridine orange or 2.5 y~
oxonol V. Additions were 1.5 mM MgATP and 25 mM KNOB. The
acridine orange trace has been corrected for an instantaneous upward
shift in the base-line upon the addition of MgATP, which occurs in
the absence of vesicles and represents an interaction of ATP with the
probe. In the oxonol V trace, the small amount
quenching that remains after the addition of KNO, is insensitive to
FCCP and was shown in separate experiments to result
interaction between probe, ATP, and lipid vesicles.
of fluorescence
from an
s to approach a steady state value. Quantitative measurements
on three separate preparations of membrane vesicles gave an
average tIl2 of 1.9 f 0.1 s for the oxonol response (probably
limited by the mixing time in the cuvette) and 13.3 * 3.3 s
for the acridine orange response. Control experiments showed
that acridine orange could redistribute across the
membrane quite rapidly (55 s) in response to an artificial pH
jump, so the time course illustrated in Fig. 2 presumably
reflected the rate of ApH generation under the conditions of
that experiment.
It seemed reasonable to believe that, for vesicles suspended
in HEPES buffer electrogenic proton pumping might be lim-
ited by the relative impermeability of the buffer anion. Indeed,
when 25 mM KN03 was added (at the second arrow), there
was an immediate collapse of A*, accompanied by an increase
in ApH to a new steady state value (Fig. 2). Similar results
were obtained with 25 mM NaN03, KSCN, or KCl.
Effects of Anions on ApH, A*, and ATP Hydrolysis-The
experiment of Fig. 2 drew attention to the role of anions in
determining the relative magnitudes of ApH and A* and
vesicle
made it of interest to survey a variety of anions over a wider
range of concentrations. Fig. 3 illustrates the results of such
a study. In general, there was direct correlation between the
ability of a given anion to diminish the membrane potential
(oxonol V fluorescence quenching, middle) and its ability to
enhance pH gradient formation (acridine orange
quenching, top). Thus, as the concentration
was increased from 1 to 100 mM, the steady state level of A s
decreased dramatically, while over the same range of concen-
trations the steady state ApH increased. C1- followed the
same pattern but required higher concentrations. These re-
sults are consistent with the notion that
a lesser extent) C1- serve as permeant anions; when present
at a sufficiently high concentration in the external medium,
they can enter the vesicles to balance the
being pumped by the electrogenic ATPase.
SO$- and MES- were much less effective. Rather, as the
concentration of these two salts was raised, both A s and ApH
tended to increase somewhat (except at SO:- concentrations
fluorescence
of SCN- or NOJ
SCN-, NOT, and (to
charge of the protons
Y
0
1
I
I
0
IO I O
IO0
ANIOU CONC. (mM)
FIG. 3. Effect of anions on ApH (measured by acridine or-
ange fluorescence quenching), A* (measured by oxonol V
fluorescence quenching), and ATP
added as the K’ salt to a reaction medium containing 10 mM HEPES-
KOH, pH 7.2. All assays were performed as described under “Mate-
rials and Methods,” except that
4 mM MgATP was used for the assay
of ATP hydrolysis.
hydrolysis. Anions were

Page 5

7888
Proton Transport
by the Neurospora [H+]-ATPase
above 50 mM, where AJI fell abruptly). The most plausible
interpretation of these results is that SO:- and MES- are
relatively impermeant anions and that the
in AJI and ApH reflected a moderate stimulation of the
ATPase as the K' concentration or ionic strength of the
medium was increased.
This interpretation was supported by direct measurements
of ATPase activity under the same ionic conditions (Fig. 3,
bottom). As expected for an electrogenic ATPase, the per-
meant anions SCN-, N0.5, and C1- were most effective in
stimulating enzyme activity, and they did so over the same
range of concentrations (1 to 50 mM) that were effective in
collapsing A s and stimulating ApH. (Above 50 m M there was
a slight inhibition of ATPase activity by NO; and C1- and a
strong inhibition by SCN-.) However, even the two relatively
impermeant anions produced some stimulation of ATP hy-
drolysis: SO:-, in the range from 5 to 50 mM, and MES-, in
the range from 10 to 80 mM. This stimulation, presumably a
result of increasing K+ concentration or ionic strength (6, 21),
can account at least qualitatively for the effects of SO:- and
MES- on AJI and ApH.
The experiment of Fig. 3 documented the relative permea-
bility of the Neurospora plasma membrane to the anions
tested but did not provide any direct information about the
mechanism of anion permeability. To ask whether a specific
SITS-sensitive anion exchange system might be involved, of
the kind that has been well described for animal cells, plasma
membrane vesicles were preincubated for 2 min with SITS (0
to 500 pM) in the presence of 100 m M el-, NO:, or SCN-,
and acridine orange fluorescence quenching was used to follow
ATP-dependent ApH. Below 40 p ~ ,
the rate of formation on the magnitude of ApH. At higher
concentrations, SITS progressivley inhibited ApH formation,
but it did so equally in the presence of all three anions (el-,
NO;, SCN-). This result suggested that SITS might be inhib-
iting the ATPase itself, rather than altering the anion perme-
ability of the membrane. Indeed, a direct assay of ATP
hydrolysis revealed 73-80% inhibition by 500 p~ SITS, re-
gardless of the anion that was present. Thus, there is no
evidence for a specific SITS-sensitive anion exchange system
in the Neurospora plasma membrane.
Effects of Cations on ApH, A*, and ATP Hydrolysis-The
experiments of Fig. 3 in which anion effects were analyzed
were all performed with K+ salts. In order to look for possible
cation effects on ApH, A*, and ATPase activity, an analogous
study was performed with six different chloride salts: K+, Rb',
Na+, TMA+, choline+, and Tris + (Fig. 4). All six salts gave
qualitatively similar patterns, involving a decrease in AJI
( ~ ~ d d ~ ) , increase in ApH (top), and a stimulation of ATPase
activity (bottom) as the salt concentration was raised from 1
to 100 mM. Thus, the dominant factor was the behavior of
C1- as a permeant anion. Nevertheless, there were consistent
quantitative differences among the various cations, with the
order of effectiveness being Rb',K+ > choline+ > TMA" >
Tris',Na+. These are related to differences that have been
reported previously (24) and will be treated further under
"Discussion."
Reconstitution of the [I€+]-ATPase-Once
translocation had been characterized in plasma membrane
vesicles, the next goal was to reconstitute the [H'j-ATPase
into liposomes so that its transport properties could be studied
in a better defined environment. Recently, the purified [H+]-
ATPase of S. pombe has been reconstituted by two procedures,
cholate dialysis (32) and freeze-thawing (33). Both methods
yielded proteoliposomes capable of ATP-dependent proton
translocation, and the enhancement of proton translocation
observed increases
SITS had no effect on
electrogenic H'
20 b/
I f 5 t
I
75 t
I
I
IO
100
CATION CONC (mM)
FIG. 4. Effect of cations on ApH, A*, and ATP hydrolysis.
Each cation was added as the C1- salt to a reaction medium containing
10 mM HEPES, adjusted to pH 7.2 with the same cationic base. ATP
was added as the same cationic salt, prepared by ion exchange
chromato~aphy using Dowex G-50. The total cation concentrat~on
present in the reaction mixture is given on the abscissa. All assays
were performed as described under "Materials and Methods."
by uncouplers (FCCP, CCCP) or by valinomycin plus K+
provided evidence that the process was electrogenic (16, 32,
33). Nevertheless, there were some limitations to the pro-
cedures described; in particular, the maximally stimulated
ATPase activity of the reconstituted vesicles was quite low
(3.7 to 6.6 pmol/min/mg of protein (16, 32, 33)), suggesting
that there might have been significant inactivation during
enzyme purification and/or reconstitution.
After attempts to reconstitute the purified ~eurospora
ATPase, in which similar problems were encountered, we
devised an alternative procedure that uses plasma membrane
vesicles as starting material and yields vesicles with high
ATPase activity and high rates of proton transport. The
starting point for this work was the discovery by Bowman et