Vol. 254. No. 8, issue of April 25, pp.

29282934, 1979
l’rinkdin U.S.A.

The Effects of Vanadate on the Plasma Membrane ATPase of

Neurospom crassa*
(Received for publication, July 31, 1978)

Barry J. Bowman and Carolyn W. Slayman

From the Departments of Human Genetics and Physiology, Yale University School of Medicine, New Haven, Connecticut
06510

The plasma membrane ATPase of Neurospom cmsa, the dynein ATPase from cilia and flagella (3,4). Two features
which is believed to function as an electrogenic proton of inhibition by vanadate are of particular interest: its site of
pump, is extremely sensitive to inhibition by vanadium action (a low affinity binding site at the inner surface of the
in the +5 oxidation state (vanadate). At pH 6.7 (the pH membrane, in the case of the (Na+, K’)-ATPase; 56) and its
optimum of the ATPase), inhibition is half-maximal specificity. Other ATPases tested, the (Ca”)-ATPase of sar-
between 0.45 and 1.0 PM vanadate, depending upon the coplasmic reticulum (l), actomyosin ATPase (l), and mito-
ionic composition of the reaction medium. Inhibition chondrial ATPase (1, 7), are unaffected by vanadate.
also depends upon pH, with the result that 1.6 PM van- Thus, we were interested to discover recently that the
adate causes an apparent shift of pH optimum to 6.0. plasma membrane ATPase of Neurospora is extremely sen-
EDTA prevents inhibition of the Neurospom ATPase

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

sitive to vanadate (7). Physiologically, this ATPase (8, 9) and
by vanadate. It does not act by chelating M%‘, since
other chelators (trens-1,2diaminocyclohexanetetra- a similar one in yeast (10) are analogous to the ATPase
acetic acid (CDTA), ethyleneglycol bis(P-aminoethyl complexes of mitochondria and bacteria. Their function is to
ether)N,N,N,N-tetraacetic acid (EGTA)) are ineffective transport protons outwards across the plasma membrane,
and since, over a broad range of concentrations, MgClz generating a large electrochemical H+ gradient (11-14); the
has little effect on the degree of inhibition by vanadate. H’ gradient, in turn, drives the co-transport of sugars, amino
Bather, EDTA appears to complex vanadate directly. acids, and inorganic ions (15-18). Biochemically, however, the
as has previously been reported in the chemical litera- fungal plasma membrane ATPases are more closely related
ture. to the (Na+, K’)-ATPase of animal cells. Both are integral
We have used vanadate to explore the reaction mech- membrane proteins, solubilized only with the aid of deter-
anism of the Neurospom ATPase in two ways. 1) When gents. In addition, Dufour and Goffeau (19) have recently
assayed with vanadate-free ATP, the ATPase shows a shown that the plasma membrane ATPase from Schizosac-
sigmoid dependence upon substrate concentration, charomyces pombe contains a single large subunit of M, =
consistent with the notion that the enzyme has two or 100,000 reminiscent of the large 95,000-dalton polypeptide of
more substrate binding sites acting cooperatively. Van- the (Na+, K+)-ATPase (20); preliminary studies in our labo-
adate stimulates ATPase activity at low substrate con- ratory point to a similar subunit composition for the Neuro-
centrations and inhibits at high concentrations, thus spora enzyme (21).
reducing the apparent cooperativity between sites. In their response to inhibitors, the fungal plasma membrane
2) We have previously reported (Bowman, B. J., and
ATPases show a distinct pattern. They are insensitive to
Slayman, C. W. (1977) J. BioL Chem. 252, 3357-3363)
oligomycin and to ouabain, but sensitive to DCCI and (as we
that Neumspom plasma membrane ATPase activity is
have recently found for the Neurospora enzyme) to vanadate
stimulated 20 to 62% by K+ or NH+. Under these con-
ditions, vanadate still inhibits more than 95% of the (7). The present report explores the mechanism by which
activity, and Dixon plots of the data are linear. The vanadate acts on the plasma membrane ATPase of Neuro-
results can best be accounted for in terms of a single spora. We find that the degree of inhibition depends upon the
plasma membrane ATPase which is subject to nonspe- ionic composition of the medium and the ATP concentration
cific salt effects, and are difficult to reconcile with the at which the enzyme is assayed, and that inhibition is reversed
idea that the K+-stimulated portion of ATPase activity by norepinephrine or by EDTA.
represents a discrete enzyme involved in K+ transport.
MATERIALS AND METHODS

Growth of Cells and Cell Fractionation-Wild type strain RL2la

Vanadate has been found to be a potent inhibitor of the of Neurospora crassa was used in these experiments and grown for
(Na+, K’)-ATPase’ of animal cells (1, 2) and, more recently, 13 h in liquid minimal medium with vigorous aeration (7). Cells were
treated with snail digestive juice (Sigma P-glucuronidase) to weaken
* This work was supported by Research Grant GM-15761 from the their cell walls and disrupted by osmotic lysis (22). As previously
National Institute of General Medical Sciences and Grant PCM described (7), unbroken cells and large fragments were removed by
7725199 from the National Science Foundation. The costs of publi- centrifugation at 1,000 X g, and mitochondria, by two cycles of
cation of this article were defrayed in part by the payment of page centrifugation at 10,000 X g. Finally, a particulate fraction containing
charges. This article must therefore be hereby marked “advertise- the plasma membrane ATPase activity was obtained by centrifuga-
ment” in accordance with 18 USC. Section 1734 solely to indicate tion at 48,000 X g (7). The 48,000 X g pellet was suspended at 25 mg
this fact. of protein/ml in 10 mru Tris, 5 mM MgCL, pH 7.5, and stored at
’ The abbreviations used are: (Na’, K’)-ATPase, sodium and po-
tassium ion-dependent adenosine triphosphatase; DCCI, N,N’-dicy-
clohexylcarbodiimide, Pipes, piperazine-N,N’-bis(2-ethanesulfonic raacetic acid; CDTA, trans- 1,2diaminocyclohexanetetraacetic acid,
acid); EGTA, ethylene glycol bis(P-aminoethyl ether)N,N,N’,N’-tet- S, substrate; v, velocity in ymol/min/mg of protein.

2928
 Effects of Vanadate on the Plasma Membrane ATPase of N. crassa 2929
-70°C. This plasma membrane fraction was used for all ATPase inhibition of ATPase activity (60% at pH 6.7). In addition,
measurements. because the degree of inhibition varies with pH, vanadate at
ATPase Assays-In most experiments, ATPase activity was as- this concentration brings about an apparent shift of pH opti-
sayed at 30°C in 0.5 ml of the following reaction mixture: 5 mM
Na*ATP (Boehringer), 5 mu MgClz, 5 mu phosphoenolpyruvate, 2.5 mum from 6.7 (the true optimum observed with vanadate-free
d of Sigma pyruvate kinase (25 pg of protein, 11 mM (NH&SO+ final ATP) to 6.0 (the value observed previously with Sigma ATP;
concentration), 5 mM potassium azide (to inhibit residual mitochon- Ref. 8). The reason for the pH dependence of vanadate
drial ATPase; Ref. 7), and 10 mM Pipes, adjusted to pH 6.7 with Tris. inhibition is not clear. According to published data, at concen-
The reaction was started by the addition of enzyme and stopped after trations below 0.1 mu and at pH values between 4 and 8,
10 min by the addition of 0.1 ml of 50% (w/v) trichloroacetic acid. vanadate exists in solution predominantly as HzV04- (28).
The tubes were then centrifuged at 2000 x g for 5 min, and inorganic
phosphate was measured in a portion of the supernatant by the The Neurospora ATPase is approximately 7-fold less sensitive
method of Dryer et al. (23). High concentrations of vanadate (above to vanadate at pH 6.0 than at pH 6.7, however, with half-
20 PM) interfere with the phosphate assay, causing a decrease in maximal inhibition produced by 4.5 and 0.75 pM vanadate,
absorbance at 710 nm. Corrections were made by assaying phosphate respectively, (pH 6.7, data shown in the next section; pH 6.0,
standards in the presence of appropriate concentrations of vanadate. data not shown).
A second problem involving vanadate is that a slight delay in the Effect of Norepinephrine and EDTA-Norepinephrine and
onset of inhibition causes an error of about 8% in a IO-min assay. We
other catecholamines have been shown to prevent vanadate
have ignored this error in the experiments depicted in Figs. 1,2,4,5,
and 9. In the experiment of Fig. 10, where such an error could be inhibition of the (Na+, K+)-ATPase (1, 2), and Fig. 2 shows
s..d;z.t, we included a I-min preincubation in the presence of that the same effect is seen with the Neurospora ATPase.
Because high concentrations of norepinephrine are needed,
To assay ATPase activity at low substrate concentrations (see Figs. and because norepinephrine is known to form a complex with
6 to 8), 3.5 ml of reaction mixture was used. In this mixture, the vanadate (29), it is unlikely that this is a physiological effect
concentration of Tris/ATP was equal to the concentration of MgCL
of the hormone.
In addition, the mixture contained oligomycin (1 pg/ml, to inhibit

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

residual mitochondrial ATPase; Refs. 7 and 8) and 10 mM Pipes EDTA also prevents inhibition of the Neurospora ATPase
buffer, titrated to pH 6.7 with Tris. Hydrolysis of ATP was measured by vanadate, an effect first observed in the preliminary exper-
from M to 6% min. This procedure was adopted as a compromise
which would partially allow for the delay in the onset of vanadate
inhibition while keeping total ATP hydrolysis below 4%. Control I.3
experiments showed that under these conditions, a steady state rate t
of hydrolysis had been achieved by M min at low substrate concentra-
tions. At higher substrate concentrations, longer times were required
to achieve a true steady state rate in the presence of vanadate (Fig.
3), and the compromise procedure used in this experiment resulted in
a slight overestimation of rates (at 5 mu MgATP and 1.5 PM vanadate,
for example, by about 12%).
Tris/ATP was prepared from NazATP (Boehringer) by ion ex-
change chromatography on Dowex-50 resin.
Estimation of Protein-Protein was assayed by the method of
Lowry et al. (24) with bovine serum albumin as standard.
Reagents-/?-glucuronidase (type H-2), pyruvate kinase (type II),
phosphoenolpyruvate, oligomycin, norepinephrine, ethylenediamine-
tetraacetic acid (EDTA), ethylene glycol bis(/?-aminoethyl
ether)N,NJV’,N’-tetraacetic acid (EGTA), trans-1,2-diaminocyclo-
hexanetetraacetic acid (CDTA), piperazine-NJ’-bis(2-ethanesuIfonic
acid) (Pipes), tris(hydroxymethy1) aminomethane (Tris), pyrophos- I
01 ’ I 1 I 1 I I 1
phate, tripolyphosphate, and trimetaphosphate were purchased from 5 6 7 8 9
Sigma Chemical Corp. In all experiments, Naz ATP from Boehringer PH
Mannheim was used. Sodium vanadate (ortho) was obtained from FIG. 1. The effect of pH on ATPase activity in the presence and
Fisher Scientific Co. absence of 1.5 pM vanadate. The pH of the standard reaction medium
(see “Materials and Methods”) was varied from 5.2 to 8.4 by titration
RESULTS with Tris.
Dependence of ATPase Activity on the Source of ATP-As
has been observed for the (Na+, K+)-ATPase of animal cells
(1,2,25-27), the activity of the Neurospora plasma membrane
ATPase depends upon the source of ATP used in the assay.
In a preliminary experiment, we found ATPase activity to be
62% higher with Boehringer ATP than with Sigma ATP at
pH 6.0 and 153% higher at pH 6.7. At both pH values, addition
of EDTA (0.5 mM) to the reaction mixture increased the
activity seen with Sigma ATP to nearly the level seen with
Boehringer ATP. These results point to the presence of an
inhibitor in Sigma ATP; the reversal of inhibition by EDTA
will be discussed further in a later section.
Inhibition by Vanadate; Dependence on pH-Consistent
with the results of Cantley et al. (2) on the (Na+, K’)-ATPase
of animal cells, the inhibitor can be identified as vanadate, 0 IL
0.05 0.5 5.0 50.0
now known to be present in Sigma ATP purified from equine
muscle. Five miIlimolar Sigma ATP, the concentration used Na,VO, (PM)
in our standard assay, contains 0.3 to 2.4 pM vanadate (Sigma FIG. 2. Inhibition of ATPase activity by vanadate; prevention by
Technical Bulletin). Fig. 1 illustrates that 1.5 pM vanadate, norepinephrine (2.5 mu) and EDTA (1 mu). Activity was assayed as
when added to 5 mu Boehringer ATP, causes a significant described under “Materials and Methods.”
2930 Effects of Vanadate on the Plasma Membrane ATPase of N. crassa
iment described above. This result was explored further over
a range of vanadate concentrations (Fig. 2). In the absence of
EDTA, the ATPase was inhibited over the range of 0.05 to 5.0
p vanadate, with half-maximal inhibition at 0.75 pM. In the
presence of 1 mu EDTA, much higher concentrations of
vanadate (greater than 5 pM) were required to produce a
significant effect, with half-maximal inhibition at 25 PM.
In addition to preventing vanadate inhibition, EDTA can
also reverse inhibition, as shown in Fig. 3. In this experiment,
plasma membrane ATPase was added to the standard reaction
mixture and a linear rate of Pi production was measured for
the first 5 min. At that time, vanadate (1.5 pM) was added; 0 I 2 3
after a lag of about 1 mm, enzyme activity was reduced to 25% Chelator (mM)
of the initial rate. EDTA was then added to the inhibited FIG. 4. The effect of chelators on vanadate inhibition of ATPase
enzyme and a slow increase in activity was observed, such activity. Activity was assayed as described under “Materials and
that 8 min after EDTA addition, the reaction was proceeding Methods.” Control activity, indicated by the dashed line, was 1.75
at 80% of the initial rate. Fol/min/mg of protein. All other tubes contained 1.5 pM Na:sVOl
and the desired concentration (0 to 3 mM) of EDTA. EGTA. or
One possible interpretation of these results is that the
CDTA. Addition of 1 mM of any of the chelators, in the absence of
sensitivity of the ATPase to vanadate might depend critically vanadate, lowered the ATPase activity by about 5%.
upon the concentration of free Mg*+ (in addition to that
present as Mg. ATP); EDTA could then act by chelating the
excess Me. This explanation is made unlikely by the fact
that two structurally different chelators (EGTA and CDTA)

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

are unable to prevent inhibition of the ATPase by vanadate
(Fig. 4). Thus, in the presence of 1.5 pM vanadate (which gave
83% inhibition of enzyme activity), addition of up to 3 mM
EGTA or CDTA had very little effect. By contrast, EDTA
restored activity to nearly the control level; restoration was
half-maximal at 0.5 IILM EDTA.
Effect of A¶$+-Further evidence that EDTA does not act
by lowering the free Mg2+ concentration comes from an ex-
periment in which [MgC12] was varied in the presence and 0 5 IO 15
absence of vanadate (1.5 pM) and EDTA (1 ITIM). This exper- Mg Cl, (mM)
iment also provides information about the complexity of the
FIG. 5. The effect of Mg*+ on ATPase activity. Activity was as-
interactions of vanadate, MgATP, and free Mg” with the sayed as described under “Materials and Methods,” except that MgCk
ATPase. was varied from 0 to 15 mru, and vanadate (1.5 PM) or EDTA (1 mM)
In the absence of vanadate and EDTA (0, Fig. 5), as (or both) were added as indicated. The ATP concentration was kept
[MgCIZ] was raised from 0 to 15 mM (with ATP fixed at 5 mM), constant at 5 mM.
ATPase activity increased to a maximum at approximately 5
mu MgClz (Mg:ATP = l:l), and then declined gradually to approximately 66% of the maximal rate at 15 mM MgClz (10
mM free Mg*‘). Thus, the Neurospora ATPase is inhibited by
excess free Mg*+, but not drastically so.
When 1.5 pM vanadate was added (0, Fig. 5), its effect
depended upon the MgC12 concentration in an unexpected
way. At low [MgClr], there was no inhibition of enzyme
activity by vanadate (and, in fact, an indication of a slight
stimulation, which will be discussed further in the next sec-
tion). As [MgCh] was raised, vanadate became progressively
more inhibitory, until at 5 mM MgClz it gave the 73% inhibition
seen in earlier experiments (Figs. 1 to 4). Above 5 mM MgC12,
the vanadate curve declined in parallel with the control curve,
and inhibition remained roughly constant at 73 to 78%. Thus,
the action of vanadate does not appear to depend critically on
the presence of MgClz in excess of the ATP concentration.
In the remaining two curves in Fig. 5, MgC12 was also raised
from 0 to 15 mM, this time in the presence of 1 mu EDTA,
with and without 1.5 PM vanadate. The addition of EDTA
alone (V, Fig. 5) shifted the control curve to the right by an
y
0 I I I I amount corresponding to the removal of 1 mM Mg*+, but did
0 5 10 15 20 not otherwise change the shape of the curve. When EDTA
Time (mid
was added together with vanadate (A, Fig. 5), its ability to
FIG. 3. Time course of inhibition by vanadate and reversal of protect against vanadate inhibition depended upon the MgCl2
inhibition by EDTA. The reaction was initiated by adding enzyme to concentration. Below 5 mM MgC12, vanadate produced no
25 ml of reaction mixture as described under “Materials and Meth-
ods.” At M-min intervals, 0.5~ml aliquots were withdrawn and assayed significant inhibition (compared with the curve obtained in
for inorganic phosphate. The rate of the reaction during the fist 5 the presence of EDTA alone), and thus protection was essen-
min was 2.65 mol/min/mg of protein. At the first arrow, 1.5 pM tially complete. This result is consistent with the notion that,
N&V04 was added; at the second arrow, 1.0 mru EDTA was added. at low MgC12 concentrations, essentially all of the free vana-
 Effects of Vanadate on the Plasma Membrane A TPase of N. crassa 2931

date as well as nearly 1 mM of the Mg2+ were complexed by TABLE I

the EDTA. Above 5 mM MgCh, however, the protective effect Effect of other oxyanions on ATPase activity
of EDTA progressively diminished. Presumably in this range, The reaction mixture contained 5 mM MgClz, 5 mM NazATP, 25
excess free Mg2+ began to compete with vanadate for the mM NH&l, 5 mM KN,, and 10 mu Pipes adjusted to pH 6.7 with
EDTA, the concentration of free vanadate in the reaction Tris. For the experiments in which PO4 and As01 were tested, [v-
mixture increased, and inhibition was seen again. 32P]ATP (40,008 cpm/0.5 ml) was added and the production of radio-
active Pi was measured (8). Control activities ranged from 1.10 to 1.53
The complexity of the reaction mixture means that the pol/min/mg. Phosphatase activity with the three polyphosphate
concentrations of all of the relevant species are impossible for compounds (0.08 to 0.27 pmol/min/mg) was determined in a parallel
us to calculate quantitatively. EDTA. vanadate complexes experiment by omitting ATP from the reaction mixture and the final
have been described in the chemical literature, however, with ATPase activity was corrected accordingly. Numbers in parentheses
association constants in the range of lo6 to 1O’5*6 (30). Fur- represent stimulation of activity.
thermore, we have observed that the addition of 1 mM EDTA Concentration tested % Inhibition
to reaction buffer containing 150 pM vanadate (pH 6.7) leads n&f %
to a pronounced change in the UV absorption spectrum of PO4 0.01 0
vanadate, with an increase in absorbance at 260 nm; addition 0.1 1
1.0 1
of excess of MgClz (5 mM) causes a return to the control
10.0
spectrum. The spectral results support the notion that under As04 0.01 (47,
the conditions of these experiments EDTA can bind vanadate 0.1 7
reversibly, and that Mg2+ competes with vanadate for the 1.0 7
EDTA. 10.0 19
Elyect of Substrate Concentration-One puzzling aspept of Pyrophosphate 0.01 9
the results in Fig. 5 was the slight stimulation of ATPase 0.1 4
1.0 4
activity by vanadate at low MgClz concentrations. To explore

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

10.0 9
this phenomenon further, the enzyme was assayed as a func- Tripolyphosphate (linear) 0.01 14
tion of Mg.ATP concentration at 0, 1.5, and 15 pM vanadate 0.1 16
(Fig. 6). 1.0 0
The control curve illustrates that, at its pH optimum (6.7) 10.0 82
and in the absence of vanadate, the ATPase displays a sigmoid Trimetaphosphate (cyclic) 0.01 16
0.1 15
dependence upon Mg . ATP concentration (Fig. 6a). A double
1.0 10
reciprocal plot of the same data is concave upward (Fig. 6b) 10.0
but can be linearized by plotting l/u versus l/S2 (not shown), K&r04 0.001 A
consistent with the notion that the ATPase possesses two or 0.01 (1)
more binding sites with positive cooperativity between the 0.1 6
sites (31). A Hill plot of the data (Fig. 7) was linear over the 1.0 a
NazMoOd 0.001 2
range 0.3 to 2 mM MgATP, and gave values of n = 1.9 and
0.01 1
[f3lo.5 = 1.8 mM. 0.1 2
The addition of vanadate to the reaction mixture caused a 1.0 1
Na2W04 0.001 4
0.01 4
0.1 2
1.2
1.0 25
G
E

.E 0.0
\
5 3.00
E
= 0.4
>

I.00
0
0 2 4 6
S (mM)
v
V mm+ 0.30

II
0.3 0.6 1.2 2.4
s
Fm. 7. Hill plot of substrate-velocity data, obtained as described
FIG. 6. Effect Mg. ATP concentration (S) on ATPase activity.
of in the legend to Fig. 6. S is substrate concentration (mru), v is ATPase
Au assays were done by the procedure described under “Materials activity @mol/min/mg), and V,,,,, is the maximal ATPase activity
and Methods” for low substrate concentrations. obtained from the intercept of a Lineweaver-Burk plot.
 2932 Effects of Vanadate on the Plasma Membrane ATPase of N. crassa
I.6

jol , , , ) , , , ,
0 50 (c-3 Is0 200
NH,CI (mM)

0
0 2 4 6

Mg ATP (mM)
FIG. 8. Effect of substrate concentration on ATPase activity in the
presence of 25 mu NH&l, KCl, or NaCl. All assays were done with
the procedure described under “Materials and Methods” for low

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

substrate concentration.

striking shift in the kinetic behavior of the ATPase. The u

versus S plot became more hyperbolic (Fig. 6~); the double
reciprocal plot, more nearly linear (Fig. 6b). The plots in the
presence of 1.5 PM vanadate are equivalent to our previously
01 I I * I I 1 1 1
published data on the concentration dependence of the Neu- 0 so loo 150 200
rospora ATPase, obtained with Sigma ATP (8); they are also No Cl (mM)
consistent with the results of Fig. 5, in the sense that 1.5 pM
FIG. 9. Effectmonovalent cations on ATPase activity, in the
of
vanadate inhibited ATPase activity by 60% at 5 mu MgATP presence and absence of 1.5 pi vanadate. The reaction mixture
but stimulated it at MgATP concentrations below 1 mu. A contained 5 mu M&L, 5 mu Tris/ATP, and 10 rn~ Pipes adjusted
comparison of the double reciprocal plots with 1.5 and 15 PM to pH 6.7 with Trk.
vanadate (Fig. 6b) reveals that the linear portions share a
common intercept on the l/S axis, and thus that vanadate
affects the maximal velocity of the enzyme but is not compet-
itive with the substrate.
Effect of Other Oxyanions-A number of compounds which
share some properties with vanadate were tested for their
ability to inhibit the ATPase (Table I). Although it has been
suggested (2, 5, 6) that vanadate may act as an analog of
phosphate, neither phosphate nor arsenate (at concentrations
up to 10 mu) significantly affected the Neurospora enzyme,
nor did they alter inhibition by vanadate (data not shown).
Several polyphosphates were also tested because the ability
of vanadate to form stable polymers (28) could conceivably
allow it to mimic a portion of the ATP molecule. Of the three
Na,VO, (/A)
compounds tried, only linear tripolyphosphate, at 10 mu,
significantly inhibited the enzyme. Pyrophosphate and cyclic FIG. 10. Inhibition of ATPase activity by vanadate in the presence
of KC1 (25 mu) or NRC1 (25 mu). The reaction mixture contained
trimetaphosphate were ineffective. 5 mM MgCh, 5 mM Tris/ATP, oligomycin (1 pg/ml), and 10 mu Pipes,
Three compounds which are neighbors of vanadate in the adjusted to pH 6.7 with Triq hydrolysis of ATP was measured from
periodic table, chromate, molybdate, and tungstate, were also 1.0 to 10.0 min. Control activity was 1.31 pmol/min/mg in the presence
tested. None of these compounds significantly inhibited the of NH&l, 1.09 Fol/min/mg in the presence of KCl, and 0.88 gmol/
ATPase. All of these results, taken together, indicate that the min/mg with no added salt. The inset shows a Dixon plot (32) of the
action of vanadate upon the ATPase is highly specific. same data.
Effect of Monovalent Cations-Although there is no evi-
dence that the Neurospora plasma membrane ATPase is and was inhibited by Na+ (43%). None of these cations signif-
directly linked to potassium or sodium transport, we had icantly changed the KI,Z for MgATP, and combinations of the
previously observed a moderate stimulation of ATPase activ- ions had no synergistic effect (data not shown).
ity by cations (8). In view of reports that cations can affect To test the possibility that cations may alter the sensitivity
the sensitivity of the (Na+, K’)-ATPase to vanadate, it was of the enzyme to vanadate, a series of assays was done with
important to test the effect of cations on the Neurospora increasing concentrations of NH’, K’, or Na+, each in the
ATPase assayed with vanadate-free ATP. Fig. 8 shows that presence or absence of vanadate (Fig. 9). When 1.5 pMvana-
ATPase activity was stimulated by NHdf (30%) and K’ (12%), date was present, NH4’ and K+ no longer stimulated but
 Effects of Vanadate on the Plasma Membrane ATPase of N. crassa 2933
instead were slightly inhibitory. For example, the addition of drial ATPases; 33-35). An alternative possibility, and one that
25 mu NH4’ to a sample with vanadate resulted in a 23% loss has been advanced to explain similar monovalent cation ef-
of activity (Fig. 9). Na+, instead of inhibiting, had no effect on fects on the plasma membrane ATPase of higher plants (36-
ATPase activity when assayed with vanadate (1.5 PM). Thus, 38), is that the overall ATPase activity results from at least
although monovalent cations have a much smaller effect on two enzymes: a basal level of [Mg*+]-ATPase, and a K’-
the Neurosporu ATPase than on the (Na+, Kf)-ATPase of activated ATPase which functions in K’ transport.
animal cells (1, 2, 5, 6, 25-27), the interaction between vana- Two lines of evidence from the present study argue strongly
date and cations is qualitatively similar in the two cases: against this view for the Neurospora ATPase. In the fast
ATPase activity assayed in the presence of K’ or NH4’ is place, neither the sigmoid shape of the v versus S curve nor
more sensitive to vanadate than is ATPase activity assayed in the K1,2 is altered by K’ or NH4’, even though the amount of
the presence of Na+. activity does vary (Fig. 8). In the second place, inhibition of
To explore this interaction further, the activity of the Neu- ATPase activity by vanadate follows a simple monotonic
rospora ATPase was measured as a function of vanadate curve, interpretable in terms of a single K,, whether or not K+
concentration in the presence of K+ (25 mM), NHd+ (25 mM), or NH4’ is present (Fig. 10). Both of these results are most
and Na’ (25 mu). The data with KC and NH4+ are illustrated easily accounted for in terms of a single ATPase whose activity
in Fig. 10, the Na+ data (not shown) were superimposable is modulated slightly by monovalent cations.
with the control curve obtained in the absence of added alkali Reversal of Inhibition by EDTA-One finding of special
metal cations. All four inhibition curves were linear when interest is that the inhibition of the Neurospora plasma
plotted by the method of Dixon and Webb (32) with half- membrane ATPase by vanadate can be prevented (and re-
maximal inhibition produced by 1.0 pM vanadate in the ab- versed) by low concentrations of EDTA. Because neither
sence of added cation, 1.1 w with Na+, 0.64 pM with K’, and EGTA nor CDTA is effective, and because small changes in
0.45 PM with NH,‘. the concentration of free Mg’+ have no significant influence
on enzyme activity, we conclude that EDTA does not act by

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

DISCUSSION chelating Mg2+. Rather, it seems likely that EDTA complexes
vanadium directly. In turn, excess free Mg’+ can compete with
Inhibition by Vanadute-In the light of the apparent struc- vanadate for EDTA. Such competition could well explain the
tural similarity between the proton-translocating ATPase of failure of Cantley et al. (2) to observe an EDTA effect on
Neurospora and the Na+- and K+-translocating ATPase of vanadate inhibition of the (Na+, K+)-ATPase, since their
animal cells (21), the fact that both enzymes are sensitive to standard reaction medium contained 28 mM MgCh.
vanadate is of special interest. Depending upon the ionic Overall, the EDTA effects seen in the present experiments
composition of the reaction buffer, inhibition of the Neuro- have important implications for the procedures used to pre-
spora ATPase is half-maximal between 0.45 and 1.0 ,uM van- pare and assay vanadate-sensitive ATPases. Since many cells
adate. At physiological Mg” concentrations, 0.25 pM vanadate and tissues contain vanadium (39), the presence or absence of
gives half-maximal inhibition of the (Na+, K+)-ATPase; at EDTA during the preparation of an ATPase may determine
higher Mg2’ concentrations, the (Na+, K’)-ATPase becomes how much vanadate remains bound to the enzyme. Further-
even more sensitive (1, 5). more, ifvanadate is bound to an ATPase, or if it is accidentally
It seems likely that experiments with vanadate will provide added during the assay (for example, via Sigma ATP), the
useful new information about the reaction mechanisms of presence or absence of EDTA in the assay medium may
both enzymes. The recent 4aV binding studies of Cantley et al. determine the activity that is measured. In the light of these
(6), for example, have shown that vanadate acts at a low possibilities, previously reported effects of EDTA on ATPases
affinity ATP-binding site of the (Na+, K+)-ATPase, and have (for example, Ref. 40-43), as well as reported shifts in pH
lent support to an alternating site model for that enzyme. In optimum with ATP of varying purity (44), may need to be
the case of Neurospora, the present experiments with vana- reinvestigated.
date-free ATP have revealed a sigmoid dependence of enzyme
activity upon ATP concentration, which can be fitted nearly Acknowledgments-We thank Mr. Kenneth Allen for expert tech-
quantitatively by assuming that two ATP binding sites must nical assistance, Drs. John 0. Edwards and N. A. Walker for helpful
be filled in order for the reaction to proceed (or alternatively, discussions, and Dr. L. C. Cantley for making available a preprint of
that the enzyme possesses an even larger number of ATP his unpublished work.
binding sites with a lesser degree of cooperativity). Vanadate
REFERENCES
causes a shift in concentration dependence from a sigmoid
1. Josephson, L., and Cantley, L. C., Jr. (1977) Biochemistry 16,
curve to a hyperbolic curve and, thus, clearly diminishes the
4572-4578
cooperativity between the sites. The precise way in which this 2. Cantley, L. C., Jr., Josephson, L., Warner, R., Yanagisawa, M.,
effect is brought about will have to await further studies with Lechene, C., and Guidotti, G. (1977) J. Biol. Chem. 252, 7421-
purified enzyme; in particular, measurements of 48V binding 7423
(analogous to those of Cantley et al. (6)) should prove valua- 3. Nagata, Y., and Flavin, M. (1978) Biochim. Biophys. Acta 523,
ble. 228-235
4. Gibbons, R., Cosson, M. P., Evans, J. A., Gibbons, B. H., Houck,
I.
Effects of Monovalent Cations-A second important piece
B., Martinson, K. H., Sale, W. S., and Tang, W.-J. Y. (1978)
of information regarding the reaction mechanism of the Neu- Proc. Natl. Acad. Sci. U. S. A. 75,2220-2224
rospora ATPase concerns the role of monovalent cations. We 5. Cantley, L. C., Jr., Resh, M. D., and Guidotti, G. (1978) Nature
reported earlier (8) that, although there is considerable ATP- 272,552-554
ase activity in the absence of alkali metal cations, activity is 6. Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J.
stimulated moderately by K’ or NHd+. Because of the physi- Biol. Chem. 253,7361-7368
7. Bowman, B. J., Maimer, S. E., Allen, K. E., and Slayman, C. W.
ological evidence that the Neurospora plasma membrane
(1978) Biochim. Biophys. Acta 512, 13-28
ATPase functions in proton translocation (ll-14), we pro- 8. Bowman, B. J., and Slayman, C. W. (1977) J. Biol. Chem. 252,
posed that these minor ionic effects are unrelated to transport 3357-3363
but, instead, represent the kinds of salt effects seen with many 9. Scarborough, G. A. (1977) Arch. Biochem. Biophys. 180,384-393
enzymes (for example, bacterial, chloroplast, and mitochon- 10. Delhez, J., Dufour, J.-P., Thines, D., and Goffeau, A. (1977) Eur.
2934 Effects of Vanadate on the Plasma Membrane ATPase of N. crassa
J. Biochem. 79.319-328 Commun. 77,1024-1029
11. Slayman, C. L. (1970) Am. 2001. 10, 377-392 28. Pope, M. T., and Dale, B. W. (1969) Q. Rev. Chem. Sot. Land. 22,
12. Slayman, C. W., and Slayman, C. L. (1975) in Molecular Aspects 527-548
of Membrane Phenomena (Kaback, H. R., Neurath, H., Radda, 29. Cantlev, L. C., Ferguson, J. H., and Kustin, K. (1978) J. Am.
G. K., Schwyzer, R., and Wiley, W. R., eds) pp. 233-248, Chem. Sot. 100,5210-5212
Springer-Verlag, Berlin 30. Przvborowski. L.. Schwarzenbach. G.. and Zimmermann. Th.
13. Slayman, C. L., Long, W. S., and Lu, C. Y.-H. (1973) J. Membr. (1965) Helv. Chim. Acta 48, 1556-1565
Biol. 14,305-338 31. Segel, I. (1975) Enzyme Kinetics, John Wiley and Sons, New
14. Scarborough, G. A. (1976) Proc. Nutl. Acad. Sci. U. S. A. 73, York
1485-1488 32. Dixon, M., and Webb, E. C. (1964) Enzymes, Academic Press,
15. Seaston, A., Inkson, C., and Eddy, A. A. (1973) Biochem. J. 134, New York
1031-1043 33. Adolfsen, R., and Moudrianakis, E. N. (1973) Biochemistry 12,
16. Cockburn, M., Earnshaw, P., and Eddy, A. A. (1975) Biochem. J. 2926-2933
146,705-712 34. Ahlers, J., and Gunther, T. (1975) Arch. Biochem. Biophys. 171,
17. Brocklehurst, R., Gardner, D., and Eddy, A. A. (1977) Biochem. 163-169
J. 162.591-599 35. Mainzer, S. E., and Slayman, C. W. (1978) J. Bacterial. 133,5&G
18. Slayman, C. L., and Slayman, C. W. (1974) Proc. Natl. Acad. Sci. 592
U. S. A. 71, 1935-1939 36. Hodges, T. K., Leonard, R. T., Bracker, C. E., and Keenan, T. W.
19. Dufour, J.-P., and Goffeau, A. (1978) J. Biol. Chem. 253, 7026- (1972) Proc. N&l. Acad. Sci. U. S. A. 69, 3307-3311
7032 37. Leonard, R. T., and Hotchkiss, C. W. (1976) Plant Physiol. 58,
20. Hokin, L. E., Dahl, J. L., Deupree, J. D., Dixon, J. F., Hackney, 331-335
J. F., and Perdue, J. F. (1973) J. Biol. Chem. 248, 2593-2605 38. Leigh, R. A., and Wyn Jones, R. G. (1975) J. Exp. Bot. 26, 508-
21. Bowman, B. J., Blasco, F., and Slayman, C. W. (1978) in Frontiers 520
of Biological Energetics (Dutton, P. L., Leigh, J., and Scarpa, 39. Underwood, E. J. (1962) Trace Elements in Human and Animal
A., eds) pp. 525-533, Academic Press, New York Nutrition, p. 354, Academic Press, London
22. Lambowitz, A. M., Smith, E. W., and Slayman, C. W. (1972) J. 40. Klodos, I., and Skou, J. C. (1975) Biochim. Biophys. Acta 391,

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

Biol. Chem. 247,4850-4858 474-485
23. Dryer, R. L., Tammes, A. R., and Rough, R. I. (1957) J. Biol. 41. Klodos, I., and Skou, J. C. (1977) Biochim. Biophys. Acta 481,
Chem. 225, 177-183 667-679
24. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. 42. Fagan, J. B., and Racker, E. (1977) Biochemistry 16, 152-158
(1951) J. Biol. Chem. 193,265-275 43. Wallick, E. T., Alien, J. C., and Schwartz, A. (1973) Arch. Bio-
25. Beauge, L. A., and Glynn, I. M. (1977) Nature 268, 355-356 them. Biophys. 158, 149-153
26. Beauge, L. A., and Glynn, I. M. (1978) Nature 272, 551-552 44. Katz, A. I., and Michell, A. R. (1976) J. Physiol. (Land.) 263,
27. Hudgins, P. M., and Bond, G. H. (1977) Biochem. Biophys. Res. 21OP
The effects of vanadate on the plasma membrane ATPase of Neurospora crassa.
B J Bowman and C W Slayman
J. Biol. Chem. 1979, 254:2928-2934.

Access the most updated version of this article at

http://www.jbc.org/content/254/8/2928.citation

Alerts:
• When this article is cited
• When a correction for this article is posted

Click here to choose from all of JBC's e-mail alerts

This article cites 0 references, 0 of which can be accessed free at

Downloaded from http://www.jbc.org/ by guest on March 26, 2019

http://www.jbc.org/content/254/8/2928.citation.full.html#ref-list-1