- Email: [email protected]
Reconstitution of the Mg2+-ATPase of the chromaffin granule membrane
Volume 103, number 2
July 1979
FEBS LETYE!=
k. Martin BUCKLAND, George K. RADDA and lalage M. WAKEFIELD Depkrrmemt of Biochemistry, Gaaiversirpof Ox,for, South Pmks Road, Orford OXI 3QU.
Engkmd
Received 17 May 1979
It has been demonstrated that the Mg”-dependent ATPase of the adrenomedullary storage vesicles dchromaffin granuies) is an inwardly directed electrogenic proton pump [14]. The active accumulation of catecholamines is dependent on ATP-linked proton transiocation [7] and it appears that both the pH gradient [8,9j and the electrochemical poten?ial [I 0,111 can drive catecholamine uptake [ 121. The proton translocatins ATPase is also responsible for active ATP transport [I 31. The similarity of the enzyme to the mitochondrial ATPase [ILs] together with the possibility that neurotransmitter storage vesicles also contain comparable ATPases [15,16] imply that a study of this system does not only help in elucidating the mechanism of neurotransmitter storage but may also lead p.oa general understanding of how ATP hydrolysis generates a proton gradient. Any detailed molecular analysis of the reaction requires simplification of the system and in this paper
Sodium cholate (Sigina Chemical Co.) was recrystallized twice from 70% aqueous eifianol after .-treatment with activated charcoal_ Cbromaffin granule ;>hospholipids were obtained from membranes by the ~:xtraction method in [18]. Soybean phospholipids we:e obtained from Sigma Chemical Co. and purified 2s ii; [173. S-13 was the generous gift of Dr B. Chance.
we report experiments biliity of reconstituting
over a period of 3 days, although dialysis of the membranes against membrane buffer at 45C resulted in RO
designed to examine the possjcoupled F.TPase activity. Such
reconstitution experiments pr&de the first step for examining the lipid specificity of the process as well as for the functional characterization of the individual protein components in the chromafdin granule membrane. The experiments
‘cholate dialysis’ technique
described rely on the
developed
[ 0 71.
Abbreviations: ATPase, adenosine ttiphosphatase.; S-l 3, N-(3Pert-butyl5-chlorosali~yl)-2-clhloro-lg-niPro~~~~~e; Wepes,
N-2-hydroxy ethyl piperazine N’-2ethane sulphonic acid
I’hcspholipids
were stored as stock solutions in
chloroform under nitrogen at -20°C. Water was twice cliszilled from an all glass apparatus and other sohnis used were distilled. Purified chromaffin granule membranes were prepared from the adrenal glands of freshly slaughtered cattle as in [ 193. The membrane pellet was suspended in a medium containing 10 mM Hepes, 10% (w/v) glycerol, 1 mM magrzesium sulphate, 0.5 mM LDTA, 3 mM P-mercaptoethanol (membrane buffer) at pi3 8.0 and kept at 4°C until use. Under these conditions the membranes lose - IO% of the Mg2’-ATPase activity
loss of activity over this period. Solubilization was carried out by dropwise addition of a 10% (w/v) solution of sodium cholate (pH 8.0) (fiiai cont. 1_5%, w/v) to the membrane suspension made 1.0% saturated in ammonium sulphate. The final cholate : protein ratio was in the range 5-7 (w/w).
After stirring at 4’C for 2Q min the suspension was centrifuged at 100 000 X gait in an MSE Prepspin centrifuge for 1 h. The supernatant (cholate extract) was decanted from th.e pellet and kept at 4OC until USC?.
Del.ipidation x&ascarried out by dropwise addition
Volume 103, number 2 of a
FEBS UTI-TEWS
July 1979
[Xl]. Protein and lipid phssphows well-eassayedas
saturated am:moniwn sulphate .sohHion to the
required findsaturation *After lOInin stirring at @CT,the suspension ~3s cenntti5qyxl at 27 000 X g,, for 20 min. The pale yeuow superndant wa:i decanted from the red pellet which was dissoh~d in membrane buffer containing 1.5% (w/v) sodium cholate at i;N 8.0 by gentle mixing with a glass BCXI. The pellet obtained at 45% saturation is designtited .45P and the supematant from this fractionation 45S. When a two stage.fractionation procedure was employed, the pellets obtained at 33% sqturation and 45% saturation are designated 33P and 33135P, respectively.
in pq.
3. msk&%
The treatment of chrotiaffin granuk membranes with 1.5% &~jvj sodium cholate solubikes 94% of the membrane protein (table 1). An~monirnm sulphate (45% find saturation) precipitates protein (4§P pellet) which is relativeely poor in phsspholipids compared to the membrane and the supernatant (table I). The precipitation removes - 90% of thi phosphoBipi&.
Reconstitution was carried out by addition of the appaoptiate prcPtein friaction to a sonieated scA.lfioBI of phosgholi&da (- 10 rng/mlj in membrane buffer
The 45P fraction has a low AWase activity (table 1) in contrast to the preparation where 50% of the lipid is remowed by phospholipase digestion 1191 when only a srnaljl effect on the ATPase activity is observed (R.M.B.,G_K.W., Shennan,tinpublisPned observations). Addition sf chromaffin granule lipids to the 4SP
containing 1 _W0 Cw 1v) so.dl-mm cholate at pH S.O. This soWion was dia!ysed at &C against a 1OO-fold excess of membrane buffer at pH 7.0 with 5 changes over 36 h. At the end of this petiod, samples were removed for assay of ATPase activity, protein, and lipid
phQS.phOl-US.
fraction reactivates the ATPase (f&la), shojving maximal activity at a lipid : protein ratio of -9 -0 jJmol/mg. AZ lipid : postein WPiOS> 1.5 &umo~/mg,
ATPase activity was assayed at 30°C and pH 6.6 using an ATT regeneration system coupled to NADH oxidation, f5.A~~ being monitored continuously
the A2Tas.e actitity is stinsnulated by the addition off the uncoupler S-13 (fig.la). Dkyclohexy& carbodiimide, which is thought to Hock proton Table P
Solubilization and delipidation of the chromaffin granule membrane MgZ+-A-Pa% 70 Protein (range)
Lipid
Membranes
‘iO0
Cholate extract
94 (93-W) 54 (448-56) 43
2.05 (1.54-2.52) 2.15 (1.77-2-46) 0.22 (0.14-0.33) 4.5s
126.8 f 20.lb
(36-46)
(3.63-5.64)
-
4% 45s
: PmPein
&moUmsI (range)
AVase activitya (nmol Pi released .min-’ _mg protein- ‘) imean -+ SD)
182.5 f
47.6 5
6.3
29
a Results obtained for a typical experiment b Activ!ty (assayed before dialysis) depends on cholate I protein ratio. After dialysis, activity increases to 202.B -f 27.3 nmol/mg ’
Activity depends critically on lipid : protein ratio
Chronlaffin granule rnemhanes were solubilized and delipidated by ammonium sulphate precipitation as in section 2. .4TPase activities were assayed after extensive dialysis to remoz 324
cholate
(a) C G Lipids
(15) SB Lipids
__-_-a
0 LIPIDI
I--
Lipid I Protein ( ~moieslmg 1
a
12;:
PROTElN
Fig.2. Reconstitution of the chromaffin granule membrane &?-AT&se. 33P fraction wasreconstitured with (a) extracted
(pmoleslmg)
Fig-l_ Reconstitution of the Ghromaffm granule membrane Mgx-AT&se. 458 fraction was rsconstituted with (a) extracted chromaf~n granule pboaqholipids or (b) soybean pbospholipids as in section 2. After extensive dialysis, samples were removed for assay of aTPase activity, protein and lipid phosphorus.
chromaffm granule pbosphotipids 03 (b) soybean phospholipids as in seclion 2. After extensive dialjrds, samples were removed for assay of ATPasc activity, protei? and lipid phosphorus. (0) No additions; (0) t 1 &l S-f 3; (A> + dicyclohexylcarbodiimide (1 mg/mg protein).
(0) No additions; (0) + 1 PM S-l 3; (A) + dicyclohexylcarbodiimide (1 mdmg proteipl).
conducting
channels [2P ,22]
and inhibit both mitochondrial and chromaffn granule AWase 121, i&bits the reconstituted system by - 50% (fig.na). Further deEipidation of the 45P fraction by repeating the ammonium sulphate precipitation step resulted in a lipid : protein ratio Of 0.02 pmol/mg and an ATPase activity of Only 6 nmol pi released min-’ _mg protein-‘. However, on readdition of chromaffin granmk phospholipids to this preparation, maximal activities of Only 44) nmol pi release .lGnpl-’ _mg prOtein_” were obtained, and it appears that the de~i~idat~o~ procedure resultsin a partially irreversible deactivation Of the enzyme. Soybean pbospholi@ds also reactivate the ATPase @g.lb), although in this case the maximal activity, obtained at a lipid : px2ein ratio of 0.6 pmol/mg is less than that with chromaftk glranwle lipids. Again activity is stimulated by addition of S-l 3, and inhibited by addition of dicydohexy~-carbodiimide, althoqh
both effects are smaller than those observed
when the system is reconstituted with chromaffin granule lipids. When the fractionation is carried out in two stages, to yield a pellet 33P (at 33% ammonium sulphate satunration),
rlz.? a ~zllet
33-45P
(at 45% ammonium
sulphate saturation) both fractions can be delipidated and reconstituted to give active preparations @g.2,3), The 33P fracti& is mo:e sensitive to both dicycfo-
hexyl-carbodiimide and S-l 3 than is the 33-44X fraction. indeed, when the 334% fraction is reconstituted with soybean phosphcjlipids, the ATPasz activity is neither significantly stinulated by addition
CblSBLipids
g 8
0
?
2
3
4
a
(ymo!eslmg
Fig.3_ Reconstitution
Mg*-ATPase. 33-49
1
LipidiProtein
2:3
4
5
1
6
1
of the cbromaffm
granule membrane
fraction was reconstituted with (a)
extracted chrom.aff%i granule phosphdipids
or @) soybeaii
phospholipidsas k section 2. After extensive dialysis, samples were removed for assay of ATPase activity, protein and lipid phosphorus. (0) No additions: (a) + 1 JIM S-13; (A) + dicyclo-
hexyl-carbodiimide
(1 mg/mg protein).
325
Vdume
103, number 2
of S-13 nor, at high lipid : protein ratios, inhibited by ~~~yclohexyf-carbod~iimide, although when yeconstituted with chrornaffin gram& ~hos~bo~~~~ds this fraction still shows residual seMtivity to both these
chrornaffin granule lipids) also contains this protein we ear6 rationlize the observation that reconstitution with this extract reestablishes the sensitivity to
compounds.
lipids does not. Ht also follows that the enhancement of the ATPasse rates by uncouplers should only be observed when this proton conducting proPeolipid is present. The results shown are entirely consistent vith this. Pn summary, coupled activilty of the chromaffin granule ATPase can be reconstituted and the resxits suggest that a search for the proton con+il;ting protein
4. Discussion We have shown
proton warnslocating. ATPase of cbromaffin granules can be solubilized in an active form by sodium cholate. The activity lost by removing the lipids is recovered on reconstitution with both chromaffiu granule and soybean phospholipids. (Substantial delipidation results in a deactivathat the
dicyclohexyl-caPbodiimide while that with soybean
component
is both feasible and logical.
tion which is only partially reversible.) Both prepara&ons are stimulated by uncouplers above a certain lipid : protein ratio, in common with the native membrane. We believe this stimulation is dependent on the formation of ‘closed vesicles’, which is also responsible for the decrease in ATPase activity at high iipid : protein ratios-We have examined some of our preparations by eiectron microscopy and the results are consistent with this interpretation. Dicyclohexylcarbodiimide,which is thought to act in mitochondria By blocking the proton conducting channel [2B ,221 inhibits Pbe reconstituted chromafik granule ATPase as it does in the native membrane [2]. The two stage fractionation reported here results in the separation of one fraction (33P) which is relatively more sensitive in the reconstituted system to uncouplers and dicydohexyl-carbodiimide than the other fraction (3345P). This is particularly evident when activity is reconstituted with soybean phospholipids. The significance of these observations is best considered in the light of the proton trans!ocating ATPases of mitochondria, chloroplasts and bacteria, which are, of course, involved in ATk
synthesis. In these systems a proteolipid which binds dicyclohexyl-carbodiimide has been found [23-25]. This component can be extracted by chloroformmethanol mixtu;es [23,24] and has been implicated in the process of proton trans]ocation. We suggest, therefore, that by analogy, the proton translocating AT&se of chromaffin granules contains a similar protein component which is more abundant in the 33F than in the 33 185P fraction. Bf the chloroformmethanol extraction (used for the preparation of 326
R.M.B. thanks the Medical Research Council for a Research Studentship. ‘I;;& WGrk was supported by
the Science Research Council.
[I] Bashford, C. E., Radda, G. K. and Rfchie, 6. A. (1975) FEBS Let*. 50.29 -24. [2] Bashford, C. L., Casey, R. P., Radda, G. K. and Ritchie, G. A. (1976) Neuroscaence I) 399-412.
[33 Casey, R. P., Njus, B., Radda, G. K. and Sehr, P. A. (1977) Biochemistry E6,972~977. [4 ] Njjus,D., Sehr, P. A., Radda, 6. K., Ritchie, G. A. and Seeley, P. J. (1978) Biochemistry 17,4337-4343. 15) Hatmark, T. and Ingebretson, 0. C. (1977) FEBS Lett. 78,53-56. [6] Pollard. H. B., Zinder, O., Hoffman, P. G. and Nikodejevic, 0. (1976) 9. Biol. Chem. 251,4544-4550. 171 Basiiford,C. k., Casey, R. P., Wadda, G. K. and Rite&e, G. A. (1975) Biochem.J. 148,1X-155. IS] Phillips, J. W. (1978) Biochem. J. 170,673-679. [9] Schuldiner, S., Rshkes, H. and Kanner, B. I. (1978) Proc. NatL Arx8. Sd. USA 75,3713-3716. [lo] Njw, D. and Radda, G. K. (1979) Biacbem. J. in press. [ If] Drake, R. A. i., Harvey, S. A. K., Njjus, D. and Radda, 6. K. (1979) Neuroscience, in press. [I 21 Njms, D. and Radda, G. K. (1978) BiocMmm.Biophys. Acta 463.293-244. 1131 Aberea, W., Kostron, H., Wuber, E. and Winkler, H.
(11978) BiochL:m.9.172,353-360. [14] Apps, D. K. and Glover, L. A. (1978) FEBS Lett. 85, 254-258. [I51 Toll, L., Gundersen, C. B. and Howard, B. D. (1977)
Brain Res.. Pxi, 59-M.
Volume 103, number 2 [I63
1171 d [I 8) [19] 1201
Toll, L. and Howard, B_ D. (1978)
333~ i9?9
FEBS LETTER§ Biochemistry 17,
2517-2523. Kagawa, Y, arrd Racker, E. (I 971) 9. Wiol. Chem. 246, 5477-5487. High, E. 6. and Dyer, WI. 1. (1959) Can. J. Biochem. Physiol. 37.911-917. BuckIand, R. %=I.,Radda, 6. K. and Sherman, C. D. (1978) Biochim. Biophys. Acta §13,321-337. Rosing, J., Harris, D. A., Kemp, A. and Slater, E. C. (1975) Biochim. Biophys. Acta 376,13-26.
[21j
Mitchell. P. (1973)
FEBS Eett_ 33,26?-274.
1223 Mitchell, P. (3974) FEBS Lett, 43, X89-194. 1231 CatteU, K. J., Eindop, C. R.,Knight, LG. and Beechey, R. B. (1971) Biochem. J. 125,169-;?‘I. i24] Sone, N., Yo&ida, hf., Hirata,H. and Kagawa, Y. (19786 Proc_ Nati. Acad. Sci. USA 75,4219-4223. 125 ] Nelson, N.. Eytan, E.. Noisani. B., Si_gist, H.,
Sigrist-N+on, K. and Gitbr, C. (.1977) Proc. Natl. Acad. Sci USA 74,2375-2378.
July 1979
FEBS LETYE!=
k. Martin BUCKLAND, George K. RADDA and lalage M. WAKEFIELD Depkrrmemt of Biochemistry, Gaaiversirpof Ox,for, South Pmks Road, Orford OXI 3QU.
Engkmd
Received 17 May 1979
It has been demonstrated that the Mg”-dependent ATPase of the adrenomedullary storage vesicles dchromaffin granuies) is an inwardly directed electrogenic proton pump [14]. The active accumulation of catecholamines is dependent on ATP-linked proton transiocation [7] and it appears that both the pH gradient [8,9j and the electrochemical poten?ial [I 0,111 can drive catecholamine uptake [ 121. The proton translocatins ATPase is also responsible for active ATP transport [I 31. The similarity of the enzyme to the mitochondrial ATPase [ILs] together with the possibility that neurotransmitter storage vesicles also contain comparable ATPases [15,16] imply that a study of this system does not only help in elucidating the mechanism of neurotransmitter storage but may also lead p.oa general understanding of how ATP hydrolysis generates a proton gradient. Any detailed molecular analysis of the reaction requires simplification of the system and in this paper
Sodium cholate (Sigina Chemical Co.) was recrystallized twice from 70% aqueous eifianol after .-treatment with activated charcoal_ Cbromaffin granule ;>hospholipids were obtained from membranes by the ~:xtraction method in [18]. Soybean phospholipids we:e obtained from Sigma Chemical Co. and purified 2s ii; [173. S-13 was the generous gift of Dr B. Chance.
we report experiments biliity of reconstituting
over a period of 3 days, although dialysis of the membranes against membrane buffer at 45C resulted in RO
designed to examine the possjcoupled F.TPase activity. Such
reconstitution experiments pr&de the first step for examining the lipid specificity of the process as well as for the functional characterization of the individual protein components in the chromafdin granule membrane. The experiments
‘cholate dialysis’ technique
described rely on the
developed
[ 0 71.
Abbreviations: ATPase, adenosine ttiphosphatase.; S-l 3, N-(3Pert-butyl5-chlorosali~yl)-2-clhloro-lg-niPro~~~~~e; Wepes,
N-2-hydroxy ethyl piperazine N’-2ethane sulphonic acid
I’hcspholipids
were stored as stock solutions in
chloroform under nitrogen at -20°C. Water was twice cliszilled from an all glass apparatus and other sohnis used were distilled. Purified chromaffin granule membranes were prepared from the adrenal glands of freshly slaughtered cattle as in [ 193. The membrane pellet was suspended in a medium containing 10 mM Hepes, 10% (w/v) glycerol, 1 mM magrzesium sulphate, 0.5 mM LDTA, 3 mM P-mercaptoethanol (membrane buffer) at pi3 8.0 and kept at 4°C until use. Under these conditions the membranes lose - IO% of the Mg2’-ATPase activity
loss of activity over this period. Solubilization was carried out by dropwise addition of a 10% (w/v) solution of sodium cholate (pH 8.0) (fiiai cont. 1_5%, w/v) to the membrane suspension made 1.0% saturated in ammonium sulphate. The final cholate : protein ratio was in the range 5-7 (w/w).
After stirring at 4’C for 2Q min the suspension was centrifuged at 100 000 X gait in an MSE Prepspin centrifuge for 1 h. The supernatant (cholate extract) was decanted from th.e pellet and kept at 4OC until USC?.
Del.ipidation x&ascarried out by dropwise addition
Volume 103, number 2 of a
FEBS UTI-TEWS
July 1979
[Xl]. Protein and lipid phssphows well-eassayedas
saturated am:moniwn sulphate .sohHion to the
required findsaturation *After lOInin stirring at @CT,the suspension ~3s cenntti5qyxl at 27 000 X g,, for 20 min. The pale yeuow superndant wa:i decanted from the red pellet which was dissoh~d in membrane buffer containing 1.5% (w/v) sodium cholate at i;N 8.0 by gentle mixing with a glass BCXI. The pellet obtained at 45% saturation is designtited .45P and the supematant from this fractionation 45S. When a two stage.fractionation procedure was employed, the pellets obtained at 33% sqturation and 45% saturation are designated 33P and 33135P, respectively.
in pq.
3. msk&%
The treatment of chrotiaffin granuk membranes with 1.5% &~jvj sodium cholate solubikes 94% of the membrane protein (table 1). An~monirnm sulphate (45% find saturation) precipitates protein (4§P pellet) which is relativeely poor in phsspholipids compared to the membrane and the supernatant (table I). The precipitation removes - 90% of thi phosphoBipi&.
Reconstitution was carried out by addition of the appaoptiate prcPtein friaction to a sonieated scA.lfioBI of phosgholi&da (- 10 rng/mlj in membrane buffer
The 45P fraction has a low AWase activity (table 1) in contrast to the preparation where 50% of the lipid is remowed by phospholipase digestion 1191 when only a srnaljl effect on the ATPase activity is observed (R.M.B.,G_K.W., Shennan,tinpublisPned observations). Addition sf chromaffin granule lipids to the 4SP
containing 1 _W0 Cw 1v) so.dl-mm cholate at pH S.O. This soWion was dia!ysed at &C against a 1OO-fold excess of membrane buffer at pH 7.0 with 5 changes over 36 h. At the end of this petiod, samples were removed for assay of ATPase activity, protein, and lipid
phQS.phOl-US.
fraction reactivates the ATPase (f&la), shojving maximal activity at a lipid : protein ratio of -9 -0 jJmol/mg. AZ lipid : postein WPiOS> 1.5 &umo~/mg,
ATPase activity was assayed at 30°C and pH 6.6 using an ATT regeneration system coupled to NADH oxidation, f5.A~~ being monitored continuously
the A2Tas.e actitity is stinsnulated by the addition off the uncoupler S-13 (fig.la). Dkyclohexy& carbodiimide, which is thought to Hock proton Table P
Solubilization and delipidation of the chromaffin granule membrane MgZ+-A-Pa% 70 Protein (range)
Lipid
Membranes
‘iO0
Cholate extract
94 (93-W) 54 (448-56) 43
2.05 (1.54-2.52) 2.15 (1.77-2-46) 0.22 (0.14-0.33) 4.5s
126.8 f 20.lb
(36-46)
(3.63-5.64)
-
4% 45s
: PmPein
&moUmsI (range)
AVase activitya (nmol Pi released .min-’ _mg protein- ‘) imean -+ SD)
182.5 f
47.6 5
6.3
29
a Results obtained for a typical experiment b Activ!ty (assayed before dialysis) depends on cholate I protein ratio. After dialysis, activity increases to 202.B -f 27.3 nmol/mg ’
Activity depends critically on lipid : protein ratio
Chronlaffin granule rnemhanes were solubilized and delipidated by ammonium sulphate precipitation as in section 2. .4TPase activities were assayed after extensive dialysis to remoz 324
cholate
(a) C G Lipids
(15) SB Lipids
__-_-a
0 LIPIDI
I--
Lipid I Protein ( ~moieslmg 1
a
12;:
PROTElN
Fig.2. Reconstitution of the chromaffin granule membrane &?-AT&se. 33P fraction wasreconstitured with (a) extracted
(pmoleslmg)
Fig-l_ Reconstitution of the Ghromaffm granule membrane Mgx-AT&se. 458 fraction was rsconstituted with (a) extracted chromaf~n granule pboaqholipids or (b) soybean pbospholipids as in section 2. After extensive dialysis, samples were removed for assay of aTPase activity, protein and lipid phosphorus.
chromaffm granule pbosphotipids 03 (b) soybean phospholipids as in seclion 2. After extensive dialjrds, samples were removed for assay of ATPasc activity, protei? and lipid phosphorus. (0) No additions; (0) t 1 &l S-f 3; (A> + dicyclohexylcarbodiimide (1 mg/mg protein).
(0) No additions; (0) + 1 PM S-l 3; (A) + dicyclohexylcarbodiimide (1 mdmg proteipl).
conducting
channels [2P ,22]
and inhibit both mitochondrial and chromaffn granule AWase 121, i&bits the reconstituted system by - 50% (fig.na). Further deEipidation of the 45P fraction by repeating the ammonium sulphate precipitation step resulted in a lipid : protein ratio Of 0.02 pmol/mg and an ATPase activity of Only 6 nmol pi released min-’ _mg protein-‘. However, on readdition of chromaffin granmk phospholipids to this preparation, maximal activities of Only 44) nmol pi release .lGnpl-’ _mg prOtein_” were obtained, and it appears that the de~i~idat~o~ procedure resultsin a partially irreversible deactivation Of the enzyme. Soybean pbospholi@ds also reactivate the ATPase @g.lb), although in this case the maximal activity, obtained at a lipid : px2ein ratio of 0.6 pmol/mg is less than that with chromaftk glranwle lipids. Again activity is stimulated by addition of S-l 3, and inhibited by addition of dicydohexy~-carbodiimide, althoqh
both effects are smaller than those observed
when the system is reconstituted with chromaffin granule lipids. When the fractionation is carried out in two stages, to yield a pellet 33P (at 33% ammonium sulphate satunration),
rlz.? a ~zllet
33-45P
(at 45% ammonium
sulphate saturation) both fractions can be delipidated and reconstituted to give active preparations @g.2,3), The 33P fracti& is mo:e sensitive to both dicycfo-
hexyl-carbodiimide and S-l 3 than is the 33-44X fraction. indeed, when the 334% fraction is reconstituted with soybean phosphcjlipids, the ATPasz activity is neither significantly stinulated by addition
CblSBLipids
g 8
0
?
2
3
4
a
(ymo!eslmg
Fig.3_ Reconstitution
Mg*-ATPase. 33-49
1
LipidiProtein
2:3
4
5
1
6
1
of the cbromaffm
granule membrane
fraction was reconstituted with (a)
extracted chrom.aff%i granule phosphdipids
or @) soybeaii
phospholipidsas k section 2. After extensive dialysis, samples were removed for assay of ATPase activity, protein and lipid phosphorus. (0) No additions: (a) + 1 JIM S-13; (A) + dicyclo-
hexyl-carbodiimide
(1 mg/mg protein).
325
Vdume
103, number 2
of S-13 nor, at high lipid : protein ratios, inhibited by ~~~yclohexyf-carbod~iimide, although when yeconstituted with chrornaffin gram& ~hos~bo~~~~ds this fraction still shows residual seMtivity to both these
chrornaffin granule lipids) also contains this protein we ear6 rationlize the observation that reconstitution with this extract reestablishes the sensitivity to
compounds.
lipids does not. Ht also follows that the enhancement of the ATPasse rates by uncouplers should only be observed when this proton conducting proPeolipid is present. The results shown are entirely consistent vith this. Pn summary, coupled activilty of the chromaffin granule ATPase can be reconstituted and the resxits suggest that a search for the proton con+il;ting protein
4. Discussion We have shown
proton warnslocating. ATPase of cbromaffin granules can be solubilized in an active form by sodium cholate. The activity lost by removing the lipids is recovered on reconstitution with both chromaffiu granule and soybean phospholipids. (Substantial delipidation results in a deactivathat the
dicyclohexyl-caPbodiimide while that with soybean
component
is both feasible and logical.
tion which is only partially reversible.) Both prepara&ons are stimulated by uncouplers above a certain lipid : protein ratio, in common with the native membrane. We believe this stimulation is dependent on the formation of ‘closed vesicles’, which is also responsible for the decrease in ATPase activity at high iipid : protein ratios-We have examined some of our preparations by eiectron microscopy and the results are consistent with this interpretation. Dicyclohexylcarbodiimide,which is thought to act in mitochondria By blocking the proton conducting channel [2B ,221 inhibits Pbe reconstituted chromafik granule ATPase as it does in the native membrane [2]. The two stage fractionation reported here results in the separation of one fraction (33P) which is relatively more sensitive in the reconstituted system to uncouplers and dicydohexyl-carbodiimide than the other fraction (3345P). This is particularly evident when activity is reconstituted with soybean phospholipids. The significance of these observations is best considered in the light of the proton trans!ocating ATPases of mitochondria, chloroplasts and bacteria, which are, of course, involved in ATk
synthesis. In these systems a proteolipid which binds dicyclohexyl-carbodiimide has been found [23-25]. This component can be extracted by chloroformmethanol mixtu;es [23,24] and has been implicated in the process of proton trans]ocation. We suggest, therefore, that by analogy, the proton translocating AT&se of chromaffin granules contains a similar protein component which is more abundant in the 33F than in the 33 185P fraction. Bf the chloroformmethanol extraction (used for the preparation of 326
R.M.B. thanks the Medical Research Council for a Research Studentship. ‘I;;& WGrk was supported by
the Science Research Council.
[I] Bashford, C. E., Radda, G. K. and Rfchie, 6. A. (1975) FEBS Let*. 50.29 -24. [2] Bashford, C. L., Casey, R. P., Radda, G. K. and Ritchie, G. A. (1976) Neuroscaence I) 399-412.
[33 Casey, R. P., Njus, B., Radda, G. K. and Sehr, P. A. (1977) Biochemistry E6,972~977. [4 ] Njjus,D., Sehr, P. A., Radda, 6. K., Ritchie, G. A. and Seeley, P. J. (1978) Biochemistry 17,4337-4343. 15) Hatmark, T. and Ingebretson, 0. C. (1977) FEBS Lett. 78,53-56. [6] Pollard. H. B., Zinder, O., Hoffman, P. G. and Nikodejevic, 0. (1976) 9. Biol. Chem. 251,4544-4550. 171 Basiiford,C. k., Casey, R. P., Wadda, G. K. and Rite&e, G. A. (1975) Biochem.J. 148,1X-155. IS] Phillips, J. W. (1978) Biochem. J. 170,673-679. [9] Schuldiner, S., Rshkes, H. and Kanner, B. I. (1978) Proc. NatL Arx8. Sd. USA 75,3713-3716. [lo] Njw, D. and Radda, G. K. (1979) Biacbem. J. in press. [ If] Drake, R. A. i., Harvey, S. A. K., Njjus, D. and Radda, 6. K. (1979) Neuroscience, in press. [I 21 Njms, D. and Radda, G. K. (1978) BiocMmm.Biophys. Acta 463.293-244. 1131 Aberea, W., Kostron, H., Wuber, E. and Winkler, H.
(11978) BiochL:m.9.172,353-360. [14] Apps, D. K. and Glover, L. A. (1978) FEBS Lett. 85, 254-258. [I51 Toll, L., Gundersen, C. B. and Howard, B. D. (1977)
Brain Res.. Pxi, 59-M.
Volume 103, number 2 [I63
1171 d [I 8) [19] 1201
Toll, L. and Howard, B_ D. (1978)
333~ i9?9
FEBS LETTER§ Biochemistry 17,
2517-2523. Kagawa, Y, arrd Racker, E. (I 971) 9. Wiol. Chem. 246, 5477-5487. High, E. 6. and Dyer, WI. 1. (1959) Can. J. Biochem. Physiol. 37.911-917. BuckIand, R. %=I.,Radda, 6. K. and Sherman, C. D. (1978) Biochim. Biophys. Acta §13,321-337. Rosing, J., Harris, D. A., Kemp, A. and Slater, E. C. (1975) Biochim. Biophys. Acta 376,13-26.
[21j
Mitchell. P. (1973)
FEBS Eett_ 33,26?-274.
1223 Mitchell, P. (3974) FEBS Lett, 43, X89-194. 1231 CatteU, K. J., Eindop, C. R.,Knight, LG. and Beechey, R. B. (1971) Biochem. J. 125,169-;?‘I. i24] Sone, N., Yo&ida, hf., Hirata,H. and Kagawa, Y. (19786 Proc_ Nati. Acad. Sci. USA 75,4219-4223. 125 ] Nelson, N.. Eytan, E.. Noisani. B., Si_gist, H.,
Sigrist-N+on, K. and Gitbr, C. (.1977) Proc. Natl. Acad. Sci USA 74,2375-2378.