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Reconstitution of proton pumping activity of a plasma membrane ATPase purified from radish
Plant Science Letters, 37 (1985) 189--193 Elsevier Scientific Publishers Ireland Ltd.
189
RECONSTITUTION OF P R O T O N PUMPING ACTIVITY OF A PLASMA MEMBRANE ATPase P U R I F I E D F R O M RADISH
MARIA CECILIA COCUCCI, MARIA IDA DE MICHELIS, MARIA CHIARA PUGLIARELLO and FRANCA RASI-CALDOGNO Cen tro di Studio del C.N.R. per la Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia "L. Gorini', UniversiM di Milano, via G. Celoria 26, 20133 Milan (Italy) (Received May 24th, 1984) (Revision received September 14th, 1984) (Accepted September 14th, 1984) Plasma membrane ATPase partially purified from radish seedlings (Raphanus sativum L.) (2.4--3.5 umol Pi min 1 mg I protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg : ATP. Mg : ATP-dependent proton pumping is prevented by N,N'-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase. Key words: proteoliposomes; plasma membrane ATPase; proton transport; H*-ATPase; vanadate; radish
Introduction
The existence of an electrogenic proton pumping ATPase at the plasma membrane of higher plants has been postulated on the basis of evidence coming from in vivo work [1,2]. Direct evidence that the plasma membrane ATPase drives an electrogenic transport of protons comes from the partial inhibition by vanadate of Mg : ATP-dependent electrogenic transport of protons measured in microsomal vesicles from radish, oat and t o b a c c o [3 --5]. Plasma membrane ATPases of yeasts and fungi have been purified and their capability to drive electrogenic transport of protons demonstrated both in native vesicles and in reconstituted proteoliposomes ([6] and references therein). Partial purification has been
achieved also for the plasma membrane ATPase of higher plants [7--9]. The partially purified plasma membrane ATPase from oat roots has been reconstituted in proteoliposomes and ATP-dependent electrogenic transport of protons has been shown to occur in such proteoliposomes [ 9]. However sensitivity to vanadate or other specific inhibitors of plasma membrane ATPase of this ATPdependent transport of protons has not been reported, thus weakening the conclusion that the observed proton transport is really driven by the plasma membrane ATPase. Here we show that plasma membrane ATPase partially purified from radish seeds [7] and reconstituted in proteoliposomes is able to drive transport of protons as shown by the sensitivity of ATP-dependent transport of protons in such proteoliposomes to vanadate. Materials and methods
Abbreviations: AO, acridine orange; DCCD, N,N',dicyclohexylcarbodiimide; FCCP, carbonyl cyanide p-trifluoro methoxyphenylhydrazone; HEPES, N-2 hydroxyethylpiperazine-N'-2-ethanesulphonicacid; MES, 2-(N-morpholyno)ethanesulphonic acid.
Plant material Radish seeds (R. sativum L. cv. Tondo Rosso Quarantino, Ingegnoli, Milan, Italy) were germinated for 24 h as described [ 10].
0304-4211/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
190
Solubilization and purification o f A TPase Plama membrane ATPase was solubilized and purified as previously described [7], except that the homogenization of seedlings was carried out in the presence of 1 mM phenylmethylsulfonyl fluoride and 5 mM ATP. The last step of the purification procedure (solubilization with lysolecithin) was omitted. The specific activity of the ATPase used in this work ranged from 2.4 to 3.5 ~mol Pi min -~ mg -1 protein, when the activity was assayed in the presence of 100 ~g m1-1 lysolecithin in the standard conditions described [71. Incorporation o f the purified A TPase in liposomes The cholate-dialysis m e t h o d of Kagawa and Racker was used [ 11]. Phospholipids (100 mg) (II S type, Sigma} homogenized in 3 ml of 50 mM NaC1, 50 mM KC1, 6 mM MgC12, 3 mM K -N- 2 -hydroxyethylpiperazine-N'- 2 -ethane sulphonic acid (HEPES), 0.035%o ~-mercaptoethanol (pH 6.4) (buffer A) were sonicated to clarity under nitrogen stream, then 0.8 ml of 10% K-cholate (pH 7.5) were added. A b o u t 0.2 ml of enzyme preparation (approx. 1.8 umol Pi min -1, when assayed in the presence of lysolecithin) containing 0.5--0.75 mg protein were brought to 0.5 ml with buffer A and mixed with 0.5 ml of the phospholipids-cholate solution. The mixture was sonicated, in ice under nitrogen stream, with a microtip for 2 min and dialysed against 500 ml of buffer A for 23 h at 4°C with three buffer changes. A TPase assay The ATPase activity was assayed in buffer A in the presence of 0.02% phospholipids, with 4--6 pg protein in a final volume of 0.5 ml. The reaction was started dy addition of 3 mM ATP and was run for 15 min at 30°C. The released Pi was assayed as previously described [ 7]. A TP-dependent changes o f A O absorbance Proteoliposomes (15 pl) were incubated in I ml of buffer A i n the presence of 1 or 2 ~M
acridine orange (AO) and 3 mM ATP in 1.75 mM Na--2- (N- morpholino ) ethanesulphonic acid (MES) (pH 6.6).. Changes in the ionic composition of the incubation medium always resulted in a lower degree of ATP-dependent proton pumping activity, probably due to a relatively high permeability of proteoliposomes to ions, including protons. Assays were performed at room temperature (22--25°C). AO absorbance was measured at 492 nm with 550 nm as the reference wavelength by a Sigma ZWS II dual wavelength spectrop h o t o m e t e r equipped with a Philips PM8222 recorder. The instantaneous change of A A 4 9 2 - - s s 0 n m due to the addition of ATP or of proteoliposomes has been corrected in all the traces. All the experiments were performed at least three times with three replicates; traces are from one representative experiment. Protein determination Protein content was determined by the Coomassie blue dye-binding procedure [12] with bovine serum albumin as a standard. Chemicals Sodium orthovanadate and A~) were purchased from BDH. AO was recrystallized according to Ref. 13. All other chemicals were purchased from Sigma. Cholate was recrystallized according to Ref. 11. Results
A vanadate-sensitive ATPase from radish seeds has been purified to a spec. act. of 13 pmol Pi min-1 mg -1 protein by Cocucci and Marr~ [7]. The enzyme has a pH o p t i m u m at pH 6.6, is insensitive to oligomycin and azide, highly specific for Mg:ATP, phospholipid-dependent (with a marked preference for lysophosphatidylcholine), activated by monovalent cations, insensitive to NO~ and other anions, inhibited (in addition to vanadate) by Ca 2÷ and DCCD. Biochemical characteristics of this ATPase closely resemble those reported for plasma membrane ATPases purified from yeasts, fungi and higher plants [6,8,9], thus
191
indicating that this enzyme is a plasma membrane ATPase. In the experiments described in this paper the plasma membrane ATPase from radish was purified as described by Cocucci and Marr~ [7], b u t the last step of purification (solubilization with lysolecithin) was omitted. The characteristics of the ATPase activity at our stage of purification are reported in Table I. The specific activity is lower than that reported for the lysolecithin-solubilized enzyme (approx. 13 pmol Pi min-1 mg -1 protein [ 7 ] ), nevertheless contamination by mitochondrial and tonoplast ATPases appears negligible: in fact the ATPase activity is insensitive to oligomycin and to NO3. The ATPase activity is severely inhibited by vanadate and DCCD. The concentration of DCCD required for maximal inhibition of the ATPase activity is somewhat higher than that reported for the lysolecithin-solubilized enzyme [7], b u t this difference might well depend on the high concentration of phospholipids in our assay conditions. The capability to drive transport o f protons of the ATPase reconstituted in proteoliposomes by the cholate-dialysis technique was
checked. Intraliposomal acidification was monitored by measuring absorbance changes of AO, a dye which has been extensively used [14] to monitor inside acid transmembrane ApH (a decrease of AA492- ss0 nm reflects an acidification of the intravesticular space). Figure 1 (line a) shows that addition of ATP to reconstituted proteoliposomes determines a net pumping of protons into the liposomes, which reaches a steady state after 3 to 4 min. This process is substantially unaffected by oligomycin (line b). ATP-dependent pumping of protons is immediately reversed by the addition of the ionophores carbonyl cyanide p-trifluoro methoxyphenylhydrazone (FCCP) or gramicidin or of (NH4)2SO4. Moreover no ATP-dependent
ATP
a
~
c ~
Table I. E f f e c t of inhibitors on ATPase activity. ATPase activity was assayed as described in Materials and methods. E t h a n o l ( 0 . 2 % ) w a s present in all assays. Specific activity in the presence o f 100 ug m1-1 lysolecithin was 2.97 u m o l Pi rain i mg-~ protein. No pnitrophenylphosphate hydrolzying activity was detectable. Data represent the mean of t w o experim e n t s run with three replicates. Addition
None 30 ~M vanadate 100 uM vanadate 2 ug m1-1 o l i g o m y c i n 30 uM DCCD 100 uM D C C D 300 uM D C C D 20 mM K NO3
FCCP
ATPase activity
d~
u tool Pi min - ~ mg- ~ protein
Relative activity
2.28 0.82 0.30 2.30 0.99 0.66 0.24 2.21
100 36 13 100 43 29 10 97
ATP
~,
T
(NH4)2S04
Fig. 1. A T P - d e p e n d e n t decrease-of AO absorbance. P r o t e o l i p o s o m e suspension (15 ul) (approx. 10 ug protein) was incubated in 1 ml of buffer A containing 1 uM AO. Line a, c o n t r o l ; line b, plus 2 ug m1-1 oligom y c i n ; line c, plus 0.2 uM valinomycin; line d, plus 10 uM FCCP. All samples c o n t a i n e d 0.2% ethanol. R e a c t i o n was started by addition o f 10 ul of 0:3 M ATP in 175 mM Na--MES (pH 6.7). A d d i t i o n s of 1 ul of 5 mM FCCP, 4 mM gramicidin or 3 M (NH4)2SO 4 were made at the indicated points.
192
a
c/f I
0.001 /tA 492-550 I
I 1 min
Fig. 2. Effect of DCCD on ATP-dependent decrease of AO absorbance. Experimental conditions as in Fig. 1, except that buffer A contained 30 pM (line b) or 100 pM (line c) DCCD.
t
I 2 min
To.o02
T
p r o t o n p u m p i n g is d e t e c t a b l e if p r o t e o l i p o s o m e s are p r e t r e a t e d with FCCP (line d). A d d i t i o n o f v a l i n o m y c i n to the assay m e d i u m (Fig. 1, line c) o n l y slightly increases the rate of ATP-dependent proton transport, t h u s suggesting t h a t in o u r e x p e r i m e n t a l c o n d i t i o n s (50 m M NaC1 plus 50 m M KCI) t h e p e r m e a b i l i t y to chloride or to Na÷ a n d K ÷ is large e n o u g h to collapse t h e A~I, built u p b y the electrogenic transport of protons. A T P - d e p e n d e n t t r a n s p o r t o f p r o t o n s is s t r o n g l y i n h i b i t e d b y 30 ~M D C C D (Fig. 2, line b) a n d b l o c k e d b y 100 p M D C C D (Fig. 2, line c). Figure 3 s h o w s t h a t v a n a d a t e drastically reduces ATP-dependent transport of protons w h e n s u p p l i e d at 30 p M (line b) a n d m o r e so at 100 p M c o n c e n t r a t i o n (line c). H o w e v e r in t h e p r e s e n c e o f 100 p M v a n a d a t e a n d o f A T P a d r i f t in A O a b s o r b a n c e i n d e p e n d e n t o f p r o t e o l i p o s o m e s o c c u r s which r e n d e r s it v e r y difficult to measure ATP-dependent transport o f p r o t o n s in this c o n d i t i o n .
492-550
t
Fig. 3. Effect of vanadate on ATP-dependent decrease of AO absorbance. Incubation medium: 1 ml of buffer A containing 2 uM AO, 3 mM ATP in 1.75 mM Na--MES (pH 6.6) (line a) plus 30 uM (line b) or 100 uM (line c) vanadate. Reaction was started by addition of 15 pl of proteoliposome suspension (approx. 10 ug protein) (first arrow). Added at the second arrow was 1 pl of 3 M (NH,):SO 4.
193 Conclusions T h e results r e p o r t e d in this p a p e r s h o w t h a t Mg.ATP-dependent transport of protons o c c u r s in p r o t e o l i p o s o m e s r e c o n s t i t u t e d w i t h a plasma membrane ATPase partially purified f r o m radish seeds. I n t r a l i p o s o m a l a c i d i f i c a t i o n is i n h i b i t e d b y b o t h D C C D a n d v a n a d a t e : t h e concentrations of the two inhibitors required t o i n h i b i t H ÷ t r a n s p o r t are closely similar to t h o s e r e q u i r e d to inhibit t h e p h o s p h o h y d r o lyzing a c t i v i t y o f t h e p l a s m a m e m b r a n e ATPase. T h e s e results r e p r e s e n t a d i r e c t e v i d e n c e t h a t p l a s m a m e m b r a n e A T P a s e o f higher p l a n t s r e c o n s t i t u t e d in p r o t e o l i p o s o m e s c a t a l y z e s a vectorial transport of protons. A f t e r c o m p l e t i o n o f this w o r k , a n a l o g o u s e v i d e n c e and a partial c h a r a c t e r i z a t i o n o f vanadate-sensitive H ÷ transport have been r e p o r t e d for p l a s m a m e m b r a n e A T P a s e solubilized f r o m red b e e t s a n d r e c o n s t i t u t e d in p r o t e o l i p o s o m e s [ 15,16 ]. Acknowledgements T h e a u t h o r s wish t o t h a n k P r o f e s s o r E. Marrb for critical reading o f the m a n u s c r i p t a n d S. Dalla R o s a f o r t e c h n i c a l assistance.
References 1 E. Marr~ and A. Ballarin-Denti, J. Bioenerg. Bio-
membr., (1984) in press. 2 R.M. Spanswick, Annu. Rev. Plant Physiol., 32
(1981) 267. 3 M.I. De Michelis, M.C. Pugliarello and F. RasiCaldogno, FEBS Lett., 162 (1983) 85. 4 R.G. Stout and R.E. Cleland, Plant Physiol., 69 (1982) 798. 5 H. Sze, Biochim. Biophys. Acta, 732 (1983) 586. 6 A. Goffeau and C.W. Slayman, Biochim. Biophys. Acta, 639 (1981) 197. 7 M.C. Cocucci and E. MarrY, Bioehim. Biophys. Acta, 771 (1984) 42. 8 F.M. Du Pont, L.L. Burke and R.M. Spanswick, Plant Physiol., 67 (1981) 59. 9 F. Vara and R. Serrano, J. Biol. Chem., 257 (1982) 128. 10 S. Cocucci and M. Cocucci, Plant. Sei. Lett., I0 (1977) 85. 11 Y. Kagawa and E. Racker, J. Biol. Chem., 246 (1971) 5477. 12 M. Bradford, Anal. Biochem., 72 (1976) 248. 13 M.K. Pal and M. Schubert, J. Am. Chem. Soc., 84 (1963) 4384. 14 P. Dell'Antone, O. Fusinato and O. Volpato, Arch. Biochem. Biophys., 191 (1978) 413. 15 S.D. O'Neill and R.M. Spanswick, J. Membr. Biol., 79 (1984) 231. 16 S.D. O'Neill and R.M. Spanswick, J. Membr. Biol., 79 (1984) 245.
189
RECONSTITUTION OF P R O T O N PUMPING ACTIVITY OF A PLASMA MEMBRANE ATPase P U R I F I E D F R O M RADISH
MARIA CECILIA COCUCCI, MARIA IDA DE MICHELIS, MARIA CHIARA PUGLIARELLO and FRANCA RASI-CALDOGNO Cen tro di Studio del C.N.R. per la Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia "L. Gorini', UniversiM di Milano, via G. Celoria 26, 20133 Milan (Italy) (Received May 24th, 1984) (Revision received September 14th, 1984) (Accepted September 14th, 1984) Plasma membrane ATPase partially purified from radish seedlings (Raphanus sativum L.) (2.4--3.5 umol Pi min 1 mg I protein) has been reconstituted in proteoliposomes by the cholate-dialysis technique. Proteoliposomes are able to acidify their internal volume in the presence of Mg : ATP. Mg : ATP-dependent proton pumping is prevented by N,N'-dicyclohexylcarbodiimide (DCCD) and by vanadate at the same concentrations which are effective on the phosphohydrolyzing activity of the plasma membrane ATPase. Key words: proteoliposomes; plasma membrane ATPase; proton transport; H*-ATPase; vanadate; radish
Introduction
The existence of an electrogenic proton pumping ATPase at the plasma membrane of higher plants has been postulated on the basis of evidence coming from in vivo work [1,2]. Direct evidence that the plasma membrane ATPase drives an electrogenic transport of protons comes from the partial inhibition by vanadate of Mg : ATP-dependent electrogenic transport of protons measured in microsomal vesicles from radish, oat and t o b a c c o [3 --5]. Plasma membrane ATPases of yeasts and fungi have been purified and their capability to drive electrogenic transport of protons demonstrated both in native vesicles and in reconstituted proteoliposomes ([6] and references therein). Partial purification has been
achieved also for the plasma membrane ATPase of higher plants [7--9]. The partially purified plasma membrane ATPase from oat roots has been reconstituted in proteoliposomes and ATP-dependent electrogenic transport of protons has been shown to occur in such proteoliposomes [ 9]. However sensitivity to vanadate or other specific inhibitors of plasma membrane ATPase of this ATPdependent transport of protons has not been reported, thus weakening the conclusion that the observed proton transport is really driven by the plasma membrane ATPase. Here we show that plasma membrane ATPase partially purified from radish seeds [7] and reconstituted in proteoliposomes is able to drive transport of protons as shown by the sensitivity of ATP-dependent transport of protons in such proteoliposomes to vanadate. Materials and methods
Abbreviations: AO, acridine orange; DCCD, N,N',dicyclohexylcarbodiimide; FCCP, carbonyl cyanide p-trifluoro methoxyphenylhydrazone; HEPES, N-2 hydroxyethylpiperazine-N'-2-ethanesulphonicacid; MES, 2-(N-morpholyno)ethanesulphonic acid.
Plant material Radish seeds (R. sativum L. cv. Tondo Rosso Quarantino, Ingegnoli, Milan, Italy) were germinated for 24 h as described [ 10].
0304-4211/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
190
Solubilization and purification o f A TPase Plama membrane ATPase was solubilized and purified as previously described [7], except that the homogenization of seedlings was carried out in the presence of 1 mM phenylmethylsulfonyl fluoride and 5 mM ATP. The last step of the purification procedure (solubilization with lysolecithin) was omitted. The specific activity of the ATPase used in this work ranged from 2.4 to 3.5 ~mol Pi min -~ mg -1 protein, when the activity was assayed in the presence of 100 ~g m1-1 lysolecithin in the standard conditions described [71. Incorporation o f the purified A TPase in liposomes The cholate-dialysis m e t h o d of Kagawa and Racker was used [ 11]. Phospholipids (100 mg) (II S type, Sigma} homogenized in 3 ml of 50 mM NaC1, 50 mM KC1, 6 mM MgC12, 3 mM K -N- 2 -hydroxyethylpiperazine-N'- 2 -ethane sulphonic acid (HEPES), 0.035%o ~-mercaptoethanol (pH 6.4) (buffer A) were sonicated to clarity under nitrogen stream, then 0.8 ml of 10% K-cholate (pH 7.5) were added. A b o u t 0.2 ml of enzyme preparation (approx. 1.8 umol Pi min -1, when assayed in the presence of lysolecithin) containing 0.5--0.75 mg protein were brought to 0.5 ml with buffer A and mixed with 0.5 ml of the phospholipids-cholate solution. The mixture was sonicated, in ice under nitrogen stream, with a microtip for 2 min and dialysed against 500 ml of buffer A for 23 h at 4°C with three buffer changes. A TPase assay The ATPase activity was assayed in buffer A in the presence of 0.02% phospholipids, with 4--6 pg protein in a final volume of 0.5 ml. The reaction was started dy addition of 3 mM ATP and was run for 15 min at 30°C. The released Pi was assayed as previously described [ 7]. A TP-dependent changes o f A O absorbance Proteoliposomes (15 pl) were incubated in I ml of buffer A i n the presence of 1 or 2 ~M
acridine orange (AO) and 3 mM ATP in 1.75 mM Na--2- (N- morpholino ) ethanesulphonic acid (MES) (pH 6.6).. Changes in the ionic composition of the incubation medium always resulted in a lower degree of ATP-dependent proton pumping activity, probably due to a relatively high permeability of proteoliposomes to ions, including protons. Assays were performed at room temperature (22--25°C). AO absorbance was measured at 492 nm with 550 nm as the reference wavelength by a Sigma ZWS II dual wavelength spectrop h o t o m e t e r equipped with a Philips PM8222 recorder. The instantaneous change of A A 4 9 2 - - s s 0 n m due to the addition of ATP or of proteoliposomes has been corrected in all the traces. All the experiments were performed at least three times with three replicates; traces are from one representative experiment. Protein determination Protein content was determined by the Coomassie blue dye-binding procedure [12] with bovine serum albumin as a standard. Chemicals Sodium orthovanadate and A~) were purchased from BDH. AO was recrystallized according to Ref. 13. All other chemicals were purchased from Sigma. Cholate was recrystallized according to Ref. 11. Results
A vanadate-sensitive ATPase from radish seeds has been purified to a spec. act. of 13 pmol Pi min-1 mg -1 protein by Cocucci and Marr~ [7]. The enzyme has a pH o p t i m u m at pH 6.6, is insensitive to oligomycin and azide, highly specific for Mg:ATP, phospholipid-dependent (with a marked preference for lysophosphatidylcholine), activated by monovalent cations, insensitive to NO~ and other anions, inhibited (in addition to vanadate) by Ca 2÷ and DCCD. Biochemical characteristics of this ATPase closely resemble those reported for plasma membrane ATPases purified from yeasts, fungi and higher plants [6,8,9], thus
191
indicating that this enzyme is a plasma membrane ATPase. In the experiments described in this paper the plasma membrane ATPase from radish was purified as described by Cocucci and Marr~ [7], b u t the last step of purification (solubilization with lysolecithin) was omitted. The characteristics of the ATPase activity at our stage of purification are reported in Table I. The specific activity is lower than that reported for the lysolecithin-solubilized enzyme (approx. 13 pmol Pi min-1 mg -1 protein [ 7 ] ), nevertheless contamination by mitochondrial and tonoplast ATPases appears negligible: in fact the ATPase activity is insensitive to oligomycin and to NO3. The ATPase activity is severely inhibited by vanadate and DCCD. The concentration of DCCD required for maximal inhibition of the ATPase activity is somewhat higher than that reported for the lysolecithin-solubilized enzyme [7], b u t this difference might well depend on the high concentration of phospholipids in our assay conditions. The capability to drive transport o f protons of the ATPase reconstituted in proteoliposomes by the cholate-dialysis technique was
checked. Intraliposomal acidification was monitored by measuring absorbance changes of AO, a dye which has been extensively used [14] to monitor inside acid transmembrane ApH (a decrease of AA492- ss0 nm reflects an acidification of the intravesticular space). Figure 1 (line a) shows that addition of ATP to reconstituted proteoliposomes determines a net pumping of protons into the liposomes, which reaches a steady state after 3 to 4 min. This process is substantially unaffected by oligomycin (line b). ATP-dependent pumping of protons is immediately reversed by the addition of the ionophores carbonyl cyanide p-trifluoro methoxyphenylhydrazone (FCCP) or gramicidin or of (NH4)2SO4. Moreover no ATP-dependent
ATP
a
~
c ~
Table I. E f f e c t of inhibitors on ATPase activity. ATPase activity was assayed as described in Materials and methods. E t h a n o l ( 0 . 2 % ) w a s present in all assays. Specific activity in the presence o f 100 ug m1-1 lysolecithin was 2.97 u m o l Pi rain i mg-~ protein. No pnitrophenylphosphate hydrolzying activity was detectable. Data represent the mean of t w o experim e n t s run with three replicates. Addition
None 30 ~M vanadate 100 uM vanadate 2 ug m1-1 o l i g o m y c i n 30 uM DCCD 100 uM D C C D 300 uM D C C D 20 mM K NO3
FCCP
ATPase activity
d~
u tool Pi min - ~ mg- ~ protein
Relative activity
2.28 0.82 0.30 2.30 0.99 0.66 0.24 2.21
100 36 13 100 43 29 10 97
ATP
~,
T
(NH4)2S04
Fig. 1. A T P - d e p e n d e n t decrease-of AO absorbance. P r o t e o l i p o s o m e suspension (15 ul) (approx. 10 ug protein) was incubated in 1 ml of buffer A containing 1 uM AO. Line a, c o n t r o l ; line b, plus 2 ug m1-1 oligom y c i n ; line c, plus 0.2 uM valinomycin; line d, plus 10 uM FCCP. All samples c o n t a i n e d 0.2% ethanol. R e a c t i o n was started by addition o f 10 ul of 0:3 M ATP in 175 mM Na--MES (pH 6.7). A d d i t i o n s of 1 ul of 5 mM FCCP, 4 mM gramicidin or 3 M (NH4)2SO 4 were made at the indicated points.
192
a
c/f I
0.001 /tA 492-550 I
I 1 min
Fig. 2. Effect of DCCD on ATP-dependent decrease of AO absorbance. Experimental conditions as in Fig. 1, except that buffer A contained 30 pM (line b) or 100 pM (line c) DCCD.
t
I 2 min
To.o02
T
p r o t o n p u m p i n g is d e t e c t a b l e if p r o t e o l i p o s o m e s are p r e t r e a t e d with FCCP (line d). A d d i t i o n o f v a l i n o m y c i n to the assay m e d i u m (Fig. 1, line c) o n l y slightly increases the rate of ATP-dependent proton transport, t h u s suggesting t h a t in o u r e x p e r i m e n t a l c o n d i t i o n s (50 m M NaC1 plus 50 m M KCI) t h e p e r m e a b i l i t y to chloride or to Na÷ a n d K ÷ is large e n o u g h to collapse t h e A~I, built u p b y the electrogenic transport of protons. A T P - d e p e n d e n t t r a n s p o r t o f p r o t o n s is s t r o n g l y i n h i b i t e d b y 30 ~M D C C D (Fig. 2, line b) a n d b l o c k e d b y 100 p M D C C D (Fig. 2, line c). Figure 3 s h o w s t h a t v a n a d a t e drastically reduces ATP-dependent transport of protons w h e n s u p p l i e d at 30 p M (line b) a n d m o r e so at 100 p M c o n c e n t r a t i o n (line c). H o w e v e r in t h e p r e s e n c e o f 100 p M v a n a d a t e a n d o f A T P a d r i f t in A O a b s o r b a n c e i n d e p e n d e n t o f p r o t e o l i p o s o m e s o c c u r s which r e n d e r s it v e r y difficult to measure ATP-dependent transport o f p r o t o n s in this c o n d i t i o n .
492-550
t
Fig. 3. Effect of vanadate on ATP-dependent decrease of AO absorbance. Incubation medium: 1 ml of buffer A containing 2 uM AO, 3 mM ATP in 1.75 mM Na--MES (pH 6.6) (line a) plus 30 uM (line b) or 100 uM (line c) vanadate. Reaction was started by addition of 15 pl of proteoliposome suspension (approx. 10 ug protein) (first arrow). Added at the second arrow was 1 pl of 3 M (NH,):SO 4.
193 Conclusions T h e results r e p o r t e d in this p a p e r s h o w t h a t Mg.ATP-dependent transport of protons o c c u r s in p r o t e o l i p o s o m e s r e c o n s t i t u t e d w i t h a plasma membrane ATPase partially purified f r o m radish seeds. I n t r a l i p o s o m a l a c i d i f i c a t i o n is i n h i b i t e d b y b o t h D C C D a n d v a n a d a t e : t h e concentrations of the two inhibitors required t o i n h i b i t H ÷ t r a n s p o r t are closely similar to t h o s e r e q u i r e d to inhibit t h e p h o s p h o h y d r o lyzing a c t i v i t y o f t h e p l a s m a m e m b r a n e ATPase. T h e s e results r e p r e s e n t a d i r e c t e v i d e n c e t h a t p l a s m a m e m b r a n e A T P a s e o f higher p l a n t s r e c o n s t i t u t e d in p r o t e o l i p o s o m e s c a t a l y z e s a vectorial transport of protons. A f t e r c o m p l e t i o n o f this w o r k , a n a l o g o u s e v i d e n c e and a partial c h a r a c t e r i z a t i o n o f vanadate-sensitive H ÷ transport have been r e p o r t e d for p l a s m a m e m b r a n e A T P a s e solubilized f r o m red b e e t s a n d r e c o n s t i t u t e d in p r o t e o l i p o s o m e s [ 15,16 ]. Acknowledgements T h e a u t h o r s wish t o t h a n k P r o f e s s o r E. Marrb for critical reading o f the m a n u s c r i p t a n d S. Dalla R o s a f o r t e c h n i c a l assistance.
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