Functional and Regulatory Properties of H + Pumps
at the Tonoplast and Plasma Membranes of Zea mays Coleoptiles
A. Hager and W. Biber
Institut für Biologie I der Universität Tübingen, Auf der Morgenstelle 1, D-7400 Tübingen 1,
Bundesrepublik Deutschland
Z. Naturforsch. 39c, 927-937 (1984); received August 6 , 1984
ATPase, H+-Pumping, Ion Transport, Coleoptile, Zea mays
A microsomal membrane fraction (6000 x g supernatant of a cell homogenate), isolated from
coleoptiles of Zea mays, was separated by isopycnic sucrose density gradient centrifugation
in the presence of EDTA and without a prior pelleting step to avoid irreversible sticking of
different membrane species. The membrane fractions were characterized by assaying commonly
used marker enzymes, and the levels of activity investigated of ATP hydrolysis, ATP-dependent
H+ transport, and co- and countertransport o f ions, such as Cl- , fumarate2-, K+ and Li+. The
following results were obtained:
(1) ATP hydrolysis is performed by different enzymes associated with different membranes:
- vacuolar acid phosphatase (AP; inhibited by molybdate);
- Golgi phosphatase, revealing IDPase, pNPase and ATPase activity (not inhibited by
molybdate);
- ATPase activity of residual submitochondrial particles (sensitive to azide);
- a H+-translocating ATPase at tonoplast membranes (Km(ATP) = 0.29 mM; pH 7.5; stimulated by
uncouplers and completely inhibited by N O j);
- the H+-translocating ATPase of the plasmalemma (Km(ATP) = 0.39 mM at pH 6.5, inhibited
by vanadate, but not by NO^).
The latter activity is evident only after an osmotic shock, indicating that PL vesicles primarily
exist as inside-in-vesicles.
(2) ATP-fueled H+ pumps are localized at tonoplast (TO) and plasmalemma (PL) vesicles,
they differ to some extent in their properties:
(a) The PL H+ pump has a very narrow pH optimum and exhibits highest levels o f activity
at pH 6.5 with a pronounced increase of activity between pH 7.5 and 6.5 (properties,
obviously important in vivo for the regulation of active H+-extrusion by certain growth
substances, which affect the cytoplasmic pH (Hager and Moser, Planta 1984, in press);
in contrast, TO H+ pumps show a considerably wider pH optimum with highest levels
of activity around pH 7.5.
(b) In variance to the PL H+ pump the activity of TO pumps (Km(ATP) = 0.24 mM) is regulated
via the oxidation state of essential thiol groups. Their oxidation to the S -S -fo r m (e.g. by
blue light in the presence of a flavin) causes an inactivation, whereas a re-reduction by GSH
or cystein restores the activity [51].
(c) The ATP-fueled H+ transport into TO vesicles depends on an anion co-transport; most
effective is Cl- , but there is also a stimulation by organic ions, C4 and C 5 dicarboxylates,
such as malate, succinate, fumarate, 2-oxoglutarate and aspartate; NO J is inhibitory.
(d) H+-transport into sealed PL vesicles is also anion dependent. In this case, however,
N O j is as effective as Cl~.
(3) The TO membranes contain a H+/K + exchange mechanism responsible for a secondary
active K+ uptake into the vacuole. This mechanism could be the reason for a lower (ATP
dependent) acidification of TO vesicles in the presence o f K+ compared with Li+. - Similar
effects are observed with plasmamembrane vesicles, but in this case there is still the question
whether a H+/K +-exchange, a K+ channel, or both are acting.
Abbreviations: AP, acid phosphatase; BSA, bovine serum
albumin (fract. V, defatted); CCO, cytochrome c oxidase;
CCR, cytochrome c reductase; DTT, DL-dithiothreitol;
EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene
glycol-bis (/?-aminoethyl ether) N, N, N', N,'-tetraacetic
acid; ER, endoplasmatic reticulum; FC, fusicoccin; FMN,
flavin mononucleotide; GS, glucan synthetase; GSH,
glutathione (reduced form); HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC, high pressure
liquid chromatography; LAA, indole-3-acetic acid; IDP,
inosine 5'-triphosphate; NEM, N-ethylmaleimide; NR,
neutral red; pHMB, /»-hydroxymercuribenzoate; PL,
plasmalemma; pNP, /j-nitrophenyl phosphate; TO, tono­
plast; TRIS, tris (hydroxymethyl) aminomethane.
0341-0382/84/0900-0927
Reprint requests to Prof. Dr. A. Hager.
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928
A. Hager and W. Biber ■ Properties of H+ Pumps at the Tonoplast and Plasma Membranes
Introduction
D uring recent years a large body o f evidence has
accum ulated which indicates th a t electrogenic
proton pum ps localized at cell m e m b ran es are
responsible for m aintaining the m e m b ran e p o ten tial
[1, 2] and are the m ajor driving force for p rim a ry
and secondary active ion and solute tran sp o rts
through m em branes [3 -5 ]. In ad d itio n , H + p u m p s
are thought to be directly involved in inducin g
elongation growth [6, 7], It has been suggested th a t
growth substances, such as IAA or F C [8] stim u la te
the level o f H+-ATPase activity at cell m em b ran es
via up to now unknow n processes, th e re b y in ­
creasing proton extrusion and finally cell wall
loosening and cell elongation. In o rd e r to get m ore
inform ation about the characteristics and the m odes
o f regulation of H + pum ps we used m e m b ran es
from coleoptiles, a plant organ, w hich exhibits a
strong proton extrusion and elongation grow th.
Using isolated m icrosom al m em b ran e vesicles o f
low density from coleoptiles, H ager et al. [9] w ere
the first to dem onstrate the existance o f A T P
dependent H + pum ps by m eans o f a sp ectro p h o to m etric method. These H + A T Pase activities o f
isolated vesicles exhibited a high specificity for th e
substrate ATP. Surprisingly, the energy req u irin g
H + transport into the vesicles was strongly d ep en d en t
on a Cl- co-transport, not influenced by v an a d ate,
but completely inhibited in the presence o f N O j
and to a m inor degree by sulfate [10].
Subsequently, proton pum ps ex h ib itin g sim ila r
properties have been reported for (v acuolar) m e m ­
branes from different plant species and organs
(com roots: [11, 12]; oat roots [13, 14]; H evea latex
[15, 16]; Hevea vacuoles: [17]; corn coleoptiles: [18];
pum pkin hypocotyls: [19]; tulip petals: [20]; red b eet
storage tissue: [21]; Saccharomyces: [22]; tobacco
callus: [23]; pea stem: [24]; radish: [25]).
Furtherm ore, the inhibition by N O j o f th e
tonoplast located A T P-dependent H + tran sp o rt has
been recognized as an im p o rtan t featu re to d istin ­
guish between tonoplast and p lasm a lem m a H +
ATPases [17, 26. 27]; in ad d itio n v a n a d ate was
found to specifically inhibit the p la sm a lem m a
H + ATPase in vitro [28, 29] and in vivo [30]. A n o th er
characteristic o f vacuolar m em branes was suggested
to be a H+/C a 2+ antip o rter m echanism w hich is
thought to function as a secondary active C a 2+
pum p [31-33].
The aim o f the present study was to get m ore
inform ation about properties and functions o f
ATPases and proton pum ps belonging to d ifferen t
m em brane species o f the m icrosom al m em b ran e
fraction o f coleoptiles and to characterize th e coand countertransport o f ions accom panying the
active H+ transport. In addition, m echanism s w hich
possibly regulate the H + p u m p activities o f to n o ­
plast and plasm a m em branes are reported and im ­
plications for m etabolic processes, e.g. elongation
growth, discussed.
Material and Methods
Plant cultivation
T he hybrid corn variety “ In frafrü h ", category 4 b
(Nungesser KG, D arm stadt) was irrigated w ith
w ater for 5 h and germ inated on m oistened cellu ­
lose. The seedlings were cultivated over a p erio d o f
96 h at 26 °C , 90% hum idity and red light for 2 h
daily.
Preparation o f m icrosom al vesicles
D eleaved coleoptiles (norm ally 10 g) w ere plasmolized (10 min) by vacuum infiltration in m ed iu m I
(1 0 m l/g fr. w eight) for 10 m in, th ereafter ch o p p ed
with a razor blade in 3.0 ml m edium Il/g fresh
weight. The hom ogenate was pressed th ro u g h
4 layers o f surgical gauze and m ade up to 35 ml
w ith m edium II. T hen it was centrifuged for 20 m in
at 6000 x g (Beckman, JA 20). T he su p e rn a tan t
(m icrosom al m em brane fraction) was centrifuged
for another 30 m in at 143 000 x g (K ontron T G A 65;
45 Ti), and the pellet suspended in 1.2 ml m edium
Ill/g fr. wt with a hom ogenizer (Potter-E lvehjem ).
M edium I: 450 mM sucrose, 10 m M H E P E S /K O H ,
pH 7.5. M edium II: 300 m M sucrose; 75 m M H E P E S /
KOH. pH 7.5; 10 m M EGTA; 20 m M ascorbic acid;
2.5 mM DTT, 0.1% BSA (w: v). M edium III: 300 m M
sucrose; 10 mM H EPES/K O H pH 7.5; 1 mM M g S 0 4,
0.1% BSA. W hen the m em brane p rep aratio n s w ere
assayed im m ediately after isolation, the a d d itio n o f
ascorbic acid and BSA was not essential. If re ­
quired, the pH o f the m edia was adjusted to 7.5
w ith LiOH, CsO H or TRIS instead o f KOH.
A. Hager and W. Biber ■ Properties of H+ Pumps at the Tonoplast and Plasma Membranes
Separation o f membrane fractions
bv density gradient centrifugation
In this case coleoptiles w ere chopped in 1 ml
m edium Il/g fresh weight, and m edium II (pH 7.8)
contained 1 mM EDTA.
Eleven ml o f the 6000 x ^-su p ern atan t, layered
onto a linear sucrose gradient (m edium IV: 15-4 5 %
sucrose (w/w), 7.5 mM HEPES, pH 7.0 or 7.5, 2 m M
EGTA, 1 mM D TT, 0.05% BSA) were centrifuged at
1 1 0 0 0 0 x 0 (Beckman, SW 27) for 15 h. T he g ra ­
dient was separated into 1.5 to 1.8 ml fractions
(Isco density gradient fractom eter 185), and the
density m onitored.
Demonstration o f proton accumulation
in microsomal vesicles
The experim ents were perform ed according to
H ager et al. [9], and H ager and H elm le [10]. In o rd er
to test fractions o f the sucrose gradient, 450 pi a li­
quots were m ixed w ith 50 pi o f m edium V (final
concentration: 2 mM M g S 0 4, 50 mM salt, 4 pm N R
and other com pounds tested). In variance to the
norm al m ethod H + transport was m easured in 2 m in
intervals after addition o f 2 mM A T P as AA o f
neutral red at 490 m inus 430 nm (m edium pH 7.5)
and 480 m inus 430 nm (m edium pH 7.0) respec­
tively using a Hewlett P ackard 8450 A spectrom eter.
929
Results
Isopycnic distribution o f m icrosom al m em branes
in a linear sucrose gradient
Fig. 1 shows the d istrib u tio n o f m icrosom al m e m ­
branes from m aize coleoptiles, Fig. 11 those from
oat coleoptiles, in a sucrose density gradient.
Organelles and particles o f the h o m ogenate w ith
higher density, such as cell wall fragm ents, nuclei,
m itochondria, etioplasts, w ere rem oved by ce n tri­
fugation (6 0 0 0 x 0 ). T he su p ern atan t, co n tain in g
“m icrosom al” m em branes, was layered onto a
density gradient w ithout any fu rth er cen trifu g atio n
step in order to avoid m em b ran e sticking; for the
same reason ED TA was present in the m ed iu m . T he
gradient was centrifuged for 15 h. In ad d itio n to
soluble proteins (strong ab so rp tio n at /-280nm) sm all
vacuoles occur at 18% sucrose. T hey b an d to g eth er
with the vacuolar enzym e AP (36) and /?- m , an
enzyme bound to tonoplast m em b ran es [37], T he
AP activity w ith pN P or A TP as su b strates was
com pletely inhibited by m o ly b d ate (0.1 m M ).
Assays o f enzymes
Enzymes were assayed as follows: N A D H -cytochrom e-c-reductase (EC 1.6.99.3), antim ycin A re­
sistant [34]; cytochrom e-c-oxidase (EC 1.9.3.1) [35];
acid phosphatase, 5 m M /?-nitrophenylphosphate as
substrate [36]; /?-m annosidase (EC 3.2.1.25), 10 m M
/?-nitrophenyl-/?-D-mannopyranoside as substrate [37];
glucan synthetase I and II (EC 2.4.1.12) and the
latent IDPase [38]. A TPase (EC 3.6.1.3) was assayed
by m easuring the decrease o f A TP via lum inescence
(LK B -W allace-Lum inom eter 1250) or by d e te rm in ­
ing the form ation o f P x (39) after 45 m in incu b atio n
at 35 °C (or after 25 m in incubation, if the K m was
determ ined); latent inosine d ip h o sp h a ta se (EC
3.6.1.6) was assayed according to [38], in the
presence or absence o f 0.15 m g/m l d igitonin,
followed by the m easurem ent o f P x as d escribed for
the ATPase-test. Protein was determ ined according
to [40] using BSA as a standard.
Fig. 1. Distribution o f microsomal membranes ( 6 K x g
supernatant) from cells of the maize coleoptiles in a linear,
isopycnic sucrose gradient. Characterization by protein
content (/428o) and various marker enzymes; AP, molybdate
sensitive (vacuolar enzyme); /?-M (tonoplast membrane);
CCR (ER); carotenoids (membranes of etioplasts); GS I
(Golgi apparatus); CCO (residual mitochondrial mem­
branes). ß-M = /?-mannosidase.
930
A. Hager and W. Biber • Properties o f H+ Pumps at the Tonoplast and Plasma Membranes
Fig. 2. (a) Absorption spectrum of the yellowish membrane fraction from the sucrose gradient (Fig. 1 “carotenoids”);
(b) first derivative of the spectrum given in (a); (c) the extracted pigments in ethanol, and (d) separation of the
pigments by HPLC (2, 3 = neoxanthin; 4 = violaxanthin; 5= antheraxanthin; 7 = lutein + zeaxanthin; 13 = /7-carotene).
Between 20 and 30% sucrose several m e m b ran e
species were localized. A narrow b an d a ro u n d 24%
sucrose contained ER (m arker enzym e C C R ).
A round 28% sucrose, a very sm all am o u n t o f a
carotenoid-containing m em brane fraction was lo c al­
ized, possibly derived from etioplasts or ca ro ten o id containing envelopes o f plastides [41] w ith a re ­
ported density o f 1.113 [42, 43]. T h e ca ro ten o id s
were determ ined spectroscopically and by H P L C
analysis (Fig. 2). W hen using coleoptiles w hich w ere
grown in the light to develope plastides in th e
bundle sheets, chlorophyll-containing m e m b ran es
were recovered from the gradient at densities h ig h e r
than those of carotenoid-containing m em b ran es.
At about 32% sucrose G olgi m e m b ran es b an d e d
which showed G S I (Fig. 1) and phosphatase activity
expressed not only as ID Pase activity (Fig. 4) b u t
also as AP activity (with the su b strate p N P or
ATP). This phosphatase was, in contrast to th e
vacuolar AP, m olybdate insensitive. W hen A T P was
used as a substrate for the G olgi p h o sp h atase, th en
its hydrolysis was also insensitive to m o ly b d ate and
azide (Figs. 3 and 5). At 38% sucrose a resid u al
CC O activity from su b m itochondrial p articles was
present which coincided w ith p lasm alem m a m e m ­
brane markers. It should be noted, how ever, th a t th e
usually em ployed m ark er enzym e for p lasm alem m a,
GS II, was inactive d ue to the presence o f E D T A
and EG TA in the hom ogenization m ed iu m . T hus,
K+-stim ulated pH 6.5-ATPase was used as a m a rk e r
(not shown).
Distribution o f A T P hydrolyzing activities
in the linear density gradient
Fig. 3 dem onstrates the isopycnic d istrib u tio n o f
ATPase activities m easured at pH 7.5. Specific in ­
hibitors were used to discrim inate different ATPases.
931
A. Hager and W. Biber • Properties o f H+ Pumps at the Tonoplast and Plasma Membranes
Fraction N9
10
12
30
14
35
16
Fraction Nr.
40
Sucrose concentration (% )
Fig. 3. ATPase activity o f microsomal membranes on a
sucrose gradient (as in Fig. 1) and effect o f inhibitors;
molybdate inhibits ATPase activity o f AP, azide that of
residual mitochondrial ATPases. In the presence of molyb­
date and azide a residual hydrolyzing activity is observed
which is termed ATPasei: it consists of the NO J sensitive
H+ ATPase of the TO, the phosphatase o f the Golgi
apparatus (latent IDPase at about 30% sucrose) and the
PL H+ ATPase (around 39%); the latter shows very low
activity at pH 7.5 which was used in this experiment
(pH optimum: 6.5).
10
12
F ra ctio n NQ
14
16
Fig. 4. Distribution of the entire ATPase activity on micro­
somal membranes in the sucrose gradient at pH 6.5 and 7.5
and the IDPase activity with and without digitonin
(0.15 mg/ml); substrate concentrations: 1 m M ATP resp.
2.5 m M IDP.
At lower densities high levels o f A T P-hydrolysis
activity were recovered, a great p art o f w hich
ap peared to be due to the AP, localized w ithin or
set free from vacuoles. T his A T Pase activity was
com pletely inhibited by m olybdate.
In addition, the A TPase activity resulting from
subm itochondrial particles (38% sucrose) was
com pletely inhibited by azide. T he plasm alem m a
Fig. 5. Distribution of membrane-bound ATPasei activity
over the sucrose gradient:
part of the NOJ-sensitive
moiety is identical with the H+ ATPase of the TO, the
vanadate-sensitive part coincides with the PL H+ ATPase,
the vanadate and NOj-resistant ATPase with the Golgi
apparatus.
H + ATPase, occurring in th e sam e d ensity range,
was inactive at pH 7.5. A fter in h ib itio n o f A P by
m olybdate (50 |iM) and m ito ch o n d rial A T P ase by
azide (500 (im) or oligom ycin (20 ^m) a resid u al
ATPase activity could be observed w hich is n am ed
ATPase^ It obviously represents
(at p H 7.5) the
tonoplast H + A TPase and the G olgi p h o sp h atase.
The latter is visible in Fig. 3 in th e region b etw een
25% and 32% sucrose, after in h ib itio n o f th e to n o ­
plast H+ ATPase by N O j, and coincides w ith th e
IDPase activity show n in Fig. 4 w hich is n o rm ally
used as a m arker for G olgi m em branes. A t pH 7
both the H + A TPases o f to n o p last an d o f the
plasm alem m a are active; th erefo re th e A T Pasej
hydrolizing activity can be referred to 3 d iffe ren t
enzymes (Fig. 5): the H + A T Pase o f to n o p la st
vesicles (K m (ATP) = 0.28 m M at pH 7.5), w hich can
be inhibited by N O j; th e G olgi p h o sp h atase, w hich
correlates w ith G S I (Fig. 1) and ID P ase activity
(Fig. 4) and the A TPase activity in th e dense region
of the gradient, resulting from the v an a d a te sen­
sitive H+-ATPase o f the p lasm alem m a (K m (ATP) =
0.39 m M at pH 6.5).
The H +-ATPase activities o f TO and PL w ere
stim ulated by uncouplers (Fig. 6), in d ic atin g th a t
they are electrogenic p u m p s and localized in m e m ­
branes o f sealed vesicles.
In this context it is interesting to know th a t m ost
of PL vesicles are originally form ed inside-in.
Therefore the binding site for A T P at th e H +-
932
A. Hager and W. Biber • Properties o f H+ Pumps at the Tonoplast and Plasma Membranes
0
2
U
6
8
Fraction Nr.
10
12
14
16
Sucrose concentration ( % )
Fig. 6 . ATPase! activity of microsomal membrane vesicles
in the sucrose gradient. The ATP-hydrolysis associated
with a H+ transport into vesicles is stimulated by the
uncoupler nigericin (1 (iM ). Stimulation is only observed in
the low-density fractions containing the NOJ-sensitive
TO H+ ATPase (20-30% sucrose) and in the dense frac­
tions due to the NOj-resistant PL H+ ATPase (30-43% ),
however not in the Golgi fraction.
0
2
U
6
8
Fraction Nr.
10
12
14
16
A T P dependent transport o f H + across TO a n d P L
W ithin the sucrose density g rad ien t m e m b ra n e
vesicles occur, which exhibit an A T P d e p e n d e n t
intravesicular acidification as show n by d ifferen ce
spectroscopy with N R [9, 10]. At pH 7.5 this activity
isonly found in m em brane fractions w ith low density
(isolated from maize: Fig. 8 - 1 0 , and from oat:
Fig. 11). Sometimes, two peaks o f activity w ere
found (Fig. 9 and 10). O ne peak was a ro u n d 18%
the other at 28% sucrose. T he am o u n t o f th e vesicle
moiety changed alternatively in d iffe ren t e x p e ri­
ments. The vesicles w ith the low est d en sity w ere
obviously small vacuoles. Both these vesicle p o p u la ­
tions exhibited identical properties w ith resp ect to
H + pum ping and ion transport: th e C l- d e p e n d e n t
intravesicular acidification [10] was sm aller in th e
presence of K+ com pared to Li+ (Fig. 8). T h is effect
is assumed to be caused by a H +/K + a n tip o rte r
m echanism at the TO vesicles lead in g to an influx
o f K+ and an efflux o f H +. A sim ilar m ech an ism has
already been postulated for the PL [23]. In a d d itio n ,
the two TO vesicle m oieties revealed th e sam e
anion dependency o f A T P-fueled acid ificatio n :
besides Cl", organic anions, such as fu m a ra te or
m alate were also used (Fig. 9).
Fraction Nr.
Fig. 7. ATPase) activity of microsomal membranes in a
sucrose gradient. N o ATPase activity can be shown in the
high-density regions of the gradient (PL), since a dilution
step (osmotic shock) during the ATPase test has been
avoided. An indication that PL vesicles are in their
original inside-in conformation.
ATPase is not accessible, leading to very low levels
o f ATP hydrolizing activity (Fig. 7). O nly afte r an
osmotic shock, i.e. osm otic lysis w hich usually
occurs during the ad d itio n A TP, an A T P h y d ro l­
izing activity can be dem onstrated.
Fig. 8 . Density gradient distribution of microsomal mem­
brane vesicles exhibiting an ATP-fueled, Cl_-dependent
acidification (TO vesicle, pH 7.5) Acidification (AA of
NR) was measured 8 min after addition o f ATP (2 m M )
and 4 min after the further addition of gramicidin
(10 |ig/ml). The term "unspecific acidification” describes a
decrease of the medium pH, which is caused by the
phosphatase activity (IDPase) of Golgi membranes and
which is not reversible by uncouplers. The lower rate of
H+ accumulation by the vesicles in the presence o f K+ and
compared to Li+ is a consequence of a H+/K + anti porter
mechanism at the TO membranes.
A Hagerand W. Biber • Properties o f H+ Pumps at the Tonoplast and Plasma Membranes
933
Fra ctio n Nr.
10
12
Fraction N9
14
10
12
14
16
Su crose concentration ( % )
Fig. 9. Density gradient distribution of membrane vesicles
showing an ATP-dependent intravesicular acidification
(reversible by uncouplers, e.g. 0.5 (am nigericin) in the
presence of 50 m M Cl- or 25 m M fumarate2-. Reaction
time after addition of 2 m M ATP with KC1 resp. K2
fumarate was 8 min resp. 10 min. The co-transport of Cl~
or fumarate2- with H+ occurs at the same membrane
species (TO). In most preparations the proton pump at
17% sucrose was not distinct (Fig. 10) or not detectable
(Fig. 8 ) (see text).
Fra ctio n Nr.
10
12
30
14
16
35
40
Sucrose concentration (% )
Fig. 10. Distribution of membrane vesicles in a density
gradient exhibiting an ATP-fueled acidification (14 min
after adding ATP; pH 7.0). The acidification is Cl~-dependent and completely inhibited by N O j in the case of
TO vesicles. The H+ transport into PL vesicles cannot be
inhibited by N O j and is smaller in the presence of K+
compared to Li+.
The plasm alem m a H +-A TPases could be d e m o n ­
strated together w ith th a t o f the TO at pH 7.0
(though its optim al activity is at pH 6.5). A fter
inhibition o f the TO p u m p by N O j the PL H +pum ping activity becam e distinguishable. In respect
Fig. 11. Isopycnic distribution of microsomal membrane
vesicles from cells of coleoptiles of Avena sativa over a
linear sucrose gradient. Characterization (a) by protein
(A2iio) and marker enzymes, (b) by the ATP hydrolyzing
activity (at pH 7.0), and the NOj-sensitive part of ATPi
activity (at pH 7.5) which corresponds to ATP-dependent
(NOj-sensitive) intravesicular acidification (^ ^ 4 9 0 - 430 )-
to the ATP hydrolysis activity o f PL and TO H +
pumps, the am ount o f acid ificatio n in PL vesicles is
low com pared with th at in TO vesicles. T his could
im plicate that PL vesicles are m ore leaky to w ard H +
[44] or that part o f the PL form s m e m b ran e sheets
during the assay.
Sim ilar to conditions at th e TO , PL vesicles show
a lower acidification in the presence o f K + com pared
with Li+ (Fig. 10). T his could result from a K +
channel, already p ostulated for the PL [53] o r from
a H +/K + co u ntertransport (Fig. 10). Interestingly,
the N O j-insensitive p ro to n p u m p in g activity (PL )
could only partially be in h ib ited by v an ad ate
(Fig. 12; also shown by [21]) w hereas th ere was
nearly a com plete in h ib itio n o f its co n sid erab ly
higher ATPase activity (see D iscussion). Fig. 12
shows th at the A TP d ep en d en t ac id ificatio n o f
sealed PL vesicles like th a t o f TO vesicles is also
anion-dependent but th a t the an io n tran sp o rte r is
very unspecific; acid ificatio n also occurs in the
presence o f N O 3 .
934
A. Hager and W. Biber • Properties o f H+ Pumps at the Tonoplast and Plasma Membranes
3 ]
B B
Different sensitivity o f P L and TO H + A TP ases
toward p H and sulfliydryl reagents
= K - S a lt
- Li - Salt
□
none
50 mM N 0 3"
25 mM a - 0 6 2' 2 5 mM S O j'
50 mM NO,"
5 0 mM C I'
*0,2 mM VOj-
Fig. 12. Dependency of ATP-fueled acidification of sealed
PL membranes (41% sucrose fraction of a density gradient
as in Fig. 1 or 10) on anions and cations. In contrast to the
ATP hydrolyzing activity (Fig. 5) the H+-pumping activity
is only partially inhibited by vanadate. Reaction time after
addition of ATP was 14 min.
Fig. 13 dem onstrates the very narrow p H o p ti­
m um o f PL H +-ATPases, ex hibiting highest levels
of activity at pH 6.5 and a significant increase o f
activity between pH 7.5 and 6.5. It has b een su g ­
gested [52] that in vivo a reg u latio n o f H + p u m p
activity occurs by small changes o f the cy to p lasm ic
pH (see Discussion). In Fig. 14 d ata on th e sen si­
tivities o f TO and PL associated H + p um ps to w ard
SH reagents is given. Only the TO H + p u m p w as in ­
hibited by SH-blocking com pounds such as pH M B
(Fig. 15) or N EM (Fig. 14, 16). Its in h ib itio n by
pHMB could be restored by D T T (or G S H and
Cys; not shown). F urtherm ore, th e TO H + p u m p
F r a c t io n
10
12
35
N9
K
16
40
S u c ro s e conc
(% )
Fig. 14. Different sensitivities of ATP-hydrolyzing enzymes
to SH-blockers. NEM (0.1 m M ) inhibits the TO ATPase
only, pHMB (50 ^m) the TO ATPase and, partially, the
PLATTase; the Golgi-Phosphatase was not sensitive to
these reagents.
p-Hydroxymercun-
8.0
u 8.5
pH
Fig. 13. pH-dependency of PL (vanadate-sensitive) and
TO (nitrate-sensitive) H+ ATPase. For determination o f
TO ATPase the 25% sucrose band, for that o f the PL
ATPase, the 41% sucrose band was used. The difference
of P, release in the presence or absence o f 25 m M K N 0 3
(TO) or 0.5 m M K3 V 0 4 are plotted.
Fig. 15. Inhibition of ATP-dependent acidification o f TOvesicles by pHMB and its reversion by DTT.
0^— —
AT P (+50 mM C D
±pHMB
---XI
A. Hager and W. Biber • Properties of H+ Pumps at the Tonoplast and Plasma Membranes
Fig. 16. Inhibition of ATP-dependent acidification of TO
vesicles by various concentrations of NEM.
Fig. 17. Blue light dependent inhibition of the ATPdependent acidification of TO-vesicles in the presence of
FMN (20 hm). ® After illumination (447 nm; 5.85 mW cm-2;
5 min) the degree of intravesicular acidification is reduced.
Q Illumination in tl\e presence o f GSH prevents the
inhibition by light. O The inhibitory effect of light on
H+-pumping can be restored by GSH (5 m M ) added im­
mediately after illumination [51]. Instead of HEPES
10 m M phosphate buffer was used in this experiment.
was inactivated by light and in the presence o f a
flavin, obviously due to an o xidation o f essential
SH-groups o f the p u m p to disulfides [51]. The
activity was restored by G SH (Fig. 17).
Discussion
From a linear sucrose gradient two fractions o f
microsomal m em brane vesicles from corn cole­
optiles were recovered w hich exhibited A TP d ep e n ­
935
dent H + pum ping activity (Fig. 10). T h e activ ity in
the low density region could be referred to sm all
vacuoles and TO vesicles w hereas th a t in th e high
density region coincided with PL m em brane m arkers.
TO vesicles were spread over a large density region
also covering the very narrow b an d o f E R m e m ­
branes. Sometimes, in the low est density region,
TO vesicles form ed a peak se p arated from th e o th er
TO vesicles m oiety (Figs. 9, 10). T his p eak consisted
o f vacuoles w hich are believed to be n ativ e u n ­
dam aged organelles, still co ntaining AP. S im ilar
results have been reported by [45]. O ne can assum e
that TO vesicles should be h eav ier th a n vacuoles
due to there form ation d u rin g h o m o g en izatio n ,
which is perform ed in a sucrose co n tain in g m e d iu m
and is accom panied by an exchange o f v acu o lar
content (AP is lost and sucrose is tak en up). H o w ­
ever, it is also possible th at a d ifferen t cleavage
of m em brane constituents could have led to tw o
TO m em brane m oieties o f d ifferen t density.
The properties o f the tw o TO d eriv ed vesicle
population are identical w ith respect to th e p ro p ­
erties of H + ATPases and ion tran sp o rt. T h e H +
pum ps o f TO m em branes have a high su b strate
specificity; A TP was show n to be the only n u cleo ­
side triphosphate [10, 12, 13, 19]. T his specificity
seems to be less expressed w hen A T P hydrolysis
instead of H+ p u m p in g is m easu red , o b viously d ue
to some other contam inating p hosphatases.
A TO associated H + p u m p in g activity occurs only
in the presence o f certain anions, C l- b eing m ost
effective [10, 11]. F o r the tran sp o rt o f C l- th ro u g h
the m em brane a channel or ca rrier has b een sug­
gested (Fig. 18) sim ilar to th a t found in an im al
m em brane systems [10]. T he u p tak e o f C l- into the
TO vesicles as a consequence o f the active H + tra n s­
port has also been show n by a filtratio n te ch n iq u e
using 36C1_ [46, 47]. In ad d itio n to C l- , o rg an ic ions
like C4 and C 5 dicarboxylates (Fig. 9), such as
malate, fum arate, succinate, a-o x o g lu tarate, oxaloacetate etc., can be co tran sp o rted w ith pro to n s
across the TO [48]. T h eir u p tak e has b een show n
using labelled com pounds, such as [14C ]succinate
[47], In the presence o f citrate no A T P d ep e n d en t
intravesicular acidification could be observed,
which is in contrast to results o b tain ed w ith TO
vesicles from Nitella [49] or luteoids from H evea [50].
The influx o f K+ into vacuoles and TO vesicles
obviously takes place via a K + specific H +/K + a n ti­
porter m echanism at the T O m em b ran es (alread y
936
A. Hager and W. Biber • Properties of H+ Pumps at the Tonoplast and Plasma Membranes
H+ -pump
( pH optimum 7.5)
regulated by redox state (-SH)
NOT-sensitive
C I', NOJ
m ala te
(dicarboxylates)
H+K+
n H +-
C a 2"
cytop lasm
Fig. 18. Schematic presentation of the primary and
secondary active ion transport mechanisms at the TO
and PL, together with the properties suggested to be
important for a regulation of H+-pumping activity. The
H+/Ca2+ antiporter mechanism is described in [31].
proposed by [23] for the PL); as a consequence the
intravesicular acidification is sm aller in the presence
o f K+ com pared to L i+ or C s+ (Fig. 8). T h e K + u p ­
take into the vacuoles is a secondary energized
mechanism, leading to an A T P d e p e n d en t accu­
m ulation o f KC1 and K2-m alate (Fig. 18).
The activity o f TO H + pum ps reveal a bro ad
pH optim um w ith a m axim um aro u n d pH 7.5
(Fig. 13). At such a pH PL p roton p u m p s are no
longer active (Figs. 8 - 10). C onsequently, w ith in the
entire unseparated m icrosom al m em b ran e fraction,
at pH 7.5 only the TO H + pum ps are active and
can be studied w ithout fu rth er p u rifica tio n [10].
A regulation o f TO H + pum ps possibly proceeds
via the redox state o f specific SH -groups at the
enzyme. W hen these sulfhydryls w ere blocked by
pHMB (Fig. 15) or N EM (Fig. 16) an in h ib itio n o f
the H+ pum ps resulted. In the case o f pH M B this
inactivation was abolished by S H -reagents, such as
DTT, GSH or Cys (Fig. 15). In ad d itio n , the
reactive SH-groups o f the H + pum ps w ere oxidized
to disulfides by light in the presence o f flavins
(Fig. 17). The resulting inactivation can com pletely
be restored by the above m entioned S H -reducing
reagents [51]. This light-dependent m echanism o f
regulation o f TO H + pum ping (Fig. 18) could be an
initial step in a series o f blue light d ep e n d en t
processes in plants, though no experim ental evi­
dence exists till now.
Unlike TO H + pum ps the PL pum ps are insen ­
sitive to N O j (Fig. 11). T his p roperty can be used
to assay PL H+ A TPases in the p resence o f TO
pumps. As the results show, the isolated PL vesicles
are obviously rather perm eable to p rotons, an as­
sum ption already m ade by Perlin and Spansw ick
[44], Com pared to the A TP hydrolyzing activity th e
m easurable A T P-dependent in trav esicu lar a c id ific a ­
tion is small. The hydrolyzing activity b ecam e,
however, only m easurable w hen th e vesicles w ere
shocked by a dilution step. O bviously, PL vesicles
are originally form ed during h o m o g en izatio n
mainly inside-in, and thus the h y d ro ly tic site is
inaccessible for ATP (Fig. 7). As a co n seq u en ce o f
the osmotic shock they partially are in v erted , b u t
part o f them also seems to form m e m b ran e sheets.
Consequently, H + p u m p activity can be d e m o n ­
strated only with sealed inside-out vesicles (Fig. 10).
In contrast to the A T P-hydrolyzing activity o f
PL m embranes (Fig. 5) the H + p u m p in g activ ity is
scarcely inhibited by vanadate (Fig. 12), an o b se rv a­
tion also reported by [21]. It is possible th a t b in d in g
o f vanadate and ATP occurs at d iffe ren t and
opposing sites o f the enzym e and th a t in sealed
vesicles, which only exhibit H + p u m p in g activity,
the vanadate binding site is inside.
Rem arkable is the very narrow pH o p tim u m o f
PL H + ATPases exhibiting highest levels o f activ ity
at pH 6.5 and with a pronounced increase betw een
pH 7.5 and 6.5 (Fig. 13). In vivo this fea tu re seem s
to be im portant for a regulation o f PL H + p u m p s
and the H+ extrusion into the cell w all c o m p a rt­
ment; in this respect evidence has b een given [52]
that all conditions and com pounds lead in g to a
decrease of pH in the cytoplasm cause an en h an ced
rate of H+ extrusion and elongation grow th. L ip o ­
philic esters of acetic acid, w hich are h y d ro lized
w ithin the cytoplasm, have been show n to be m o st
effective.
Fig. 18 sum m arizes som e p ro p erties o f th e in ­
vestigated A T P-dependent PL and T O H + p u m p s
and the suggested co- and co u n tertran sp o rt m e c h a n ­
isms at these m em branes.
Acknowledgements
Critical reading o f the m an u scrip t by Prof.
R. Hampp, as well as experienced technical as­
sistance by Mrs. Ch. Piesch and K. Putz, is g ra te ­
fully acknowledged.
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