- Email: [email protected]
Effect of phosphorylation on the size of the proton gradient across chloroplast membranes
~ O l l t l l l e 3 2", ~~ m "b e x
i
M~']973
FE-BS L E ,'lq'ERS
E F ~ i ~ c r ,o~ v n o s P r i o ~ v ~ A T m ~ o~ aa~ s~z~ o~~Z P R O T O N G R A D I E N T A C R O S S C H L O R O P L A S T ME~:CIBRANES Uri ?~C:K,Hagai ]~OTTENBEKG an,d M ordhzy A'V'RON Bio,chemis~r5~Depart'men.'. ~l~e)zm~nn'2nsn'lnzeo/Science. Re_hovot; Israel ]973
R e c e i v e ,~ a B 3 a n u a r y
U,~ng 0n~ o f z3a~m, *,.hefluoresccn::~ quvnchi~g of
1. Introduction
51u~xesc~n~ a,za~ne~ ~a t h . ~ g h i , w e ~ h o w hexe ~ h ~
The coup]hag between phoiopho~phory]n~on and pro,ton upIake in ch]orvpla~ts [], 2] ~equi~e~ '~a* ATP folma~on ~,ou],d c,~ea*e a .d~ain on the steady ~ta~te ele,c~ro~herr~ca] potentia~ formed during e~]ec:tr.~n transport. ]n ]so]ate,d ch]orophsts most ,of ~ i s potential ~eems to ,exht Ln ~ e form o f a pH gra,d~ent, and therefore, a ~tecrease in the size of ~ s :grad~em % to be .expected when phosphorylat~on ~s ~n~fia~,ed. Several meth,och for measuring the ApH formed in the ~ight ac,~oss the ~ylakoid mernblan,e were recently de~e]oped ,~n t_h~slabom.~ory I3-5].
#
,0~
ON
a , •
. -
.
.
.
.
: 5 atoM; p h o s p h a t e ,
1
.
~drme ,OFF.
" _
2 , ~ raM; 9 - a m i n o a c r i d i n e ,
Ch~]olopilasI~ f~orn letIuc.~ leaves were p.zvpmed essenti ~ah~ as previously described ]6], except fox the :~ina~,c,eni~fugadon and ~esu~p,ensi~n which were in 0.2 M sucrose, 0.1 M KCI. Fluolest'¢nc.e o f 9-maainoacr~dhae wa~ n~ea~ua-ed in an Eppenflu~ fJ~,ofi,m~ler a~ pre,,,]ou~ly de'~cribed i 4 ] .
~DN
..
.r..
.
2. Methods
~,..=
$
~,
phosphrolyaali,on ~r a~eno]y~i~ cvn,d~t~on~..~nfle~d lead to a deerease.d stvady sta~.e -ApH in the H~,ght
, ~ - -_- - ' ~ J
,,,,
"I":
-.
" .
ON
. -
"
. "OFF - .
.
1 ~uM; :raft c h l d ~ d p l a s t ~ e , o n ~ r i ~ a g . 4 $ : u g
.OFF:
. .
-
_
,OFF"
_
O/:¢Ialo~Op]~ylE '_~ .-: -
. L.. -. ~-. : - . . .
:
¥~olum9 32,,numbe~ ]
F~BS LFFFERS 3; Resets and
"Table ~}qaoxph~I~la?5on ¢.~mpox, ents ,~eefled for Lh~ ,de..ease ~n ~pJJ.
ET~p~zim~nt Cemp~n~nl No. absent
Cempon~m added None
MgCI~
f
l
i
s
e
u
~
i
May ]973 o
n
'
" "
.
.:
"
"
-
A typical ,expe~imen~ dem.mstrathagthe effect o f phosphoryhfi0n vondltlons on ihe steady stat~ ApH • . formed in ihe ]igh, is ~aown in fig. 1. The ~aversible light induced fluorescen,ce quenching o~,"9-aminoacri:~ine was mafxe0Jy xed~c~d ~fter ,the ad.3Rion o~ADY ~to an othm-W,is~ c,o.~p]ete phosphory]afi,on ~ s l e m . ~,eI~ low c.:onecn~r~tionz o f A D P were ~ d in the ]~ght, a ~m~e~ ~leady state quenchhng ~at~owas ~,m-med w]~hm ] 5--30 sec w}fi,ch in th:e light slowly returned t.o i u er,ig~nfl levd, indicating flee conversion of all ~.flie ADP. -Io ATP. The hip~her the amount of ADP added, t]:e longer R took ~o return t o / h e high quenching in :~ae ]i.ght, When the rate ,ofph0tophospbo,D.'lation was estimated from the time necessary to c,onzurae flee ADP added, values comparab]:e "toflaose eziirn~~ed .by other m~flaods wer,e obtained.
~pH measured
3.72 3.5,~. . . . . 3.,58 II Azsenzle None 3.74 5 ram A r ~ n z t ~ 3,24 2.3 mM Y~ospha~e 3.21 Ill ADP None 3.~1 ~.4 m~,f ADP 3.41 0.4 mM ATP s.7~6 .The eornplet,~~'~em con~ned m a ~9ta] "~olum~:of 3.0 ml Tr]eine~glycin~.NaOH,pH ~.O, 50 raM; KCI, 50 ~M; M~]=, 5 haM; arsenate, pH 8.0, 5 rnM; ADP, ~.4 raM; pyocymahae, l
.
5 mM MgCJ 2 5 ham CaC}2
5 ~M; and ¢hloz~Splasls e~nxai~fm.~ 4 t ~Ig o f Cyd~rophyl].
11~1~rn~_at~on o~" th~ osmotic spaces of ch]oro-
M,easurementz and valculalions of ~he o ~ ,tSc volumes of ¢h]oroplasl fragmem~ fr,on~ the disi:ribution o f ~a4.C]Sorbi~ot and ~3H] H 2 0 were ,done :as de. scribed 13]. ApH values were determined flora .'the osrn,~fic volumes and fluorescence quenching ~ e s according t0 SchuJd~ner e~ el. 14].
rl
p]as,~ d d n,orrev~a] any significant cliff,lento in ~he p,~esenc~ or ab;enc~ o f ADP. These resul~ ruble ou~ shfinka2: ~f the thfiakoJds as ~ e czuse for ~ e d,ecr,easer~ flu,me~cence quen.ch~g ratios under phosphory]a d.oa conditions. In a lyp~ca~ experi',ment, in the absence and presence of ADP, osmotic spaces of ] 2. l
i
rD.~
1 ,D •
•
"
..
0.'3
"
0.4
"
•
"
-
(
"
"L
.
I
.0
~2 0 0
"
•
;300
400
" "
~ 0 0
•
-
.
FEBS LETTERS
¥olume 32, numbez '1 .
-
May 1973
. -
"%0
/ ~
4
!
OF~
~/,~
t / .~:
o/
1
OPF
9-omino-
~,
OFF
oc~]ine
0.5
g
v
I=
/
J_
. t/ 7
OFF
\ .B
9
~t t0
Fig. 4. p H dependence of ,the de~ca~ ~ ApH unde~ phosphozyhfion cond~tk)zs. The zeacfion mktu~e con~a~nera, hu a to'lal roll.me of 3.0 ml: ~r.icine-:m~ea~te-NaOH. @A ~ e intLicated pH between 6.85-8.9), o_~lrJcine-gfycine-NaOH (pH 8.45-
! 9-=~-
"\
OFF
9.8), 50 .z~M; KC],50 .naN; K3FelCN)6 , ~.l raM; MgCI2, 5 raM; phe~ha~¢, 2.3 raM; 9-aminoacz~din~ 1 - 2 ~M; and chlo~ophsls ¢9~ahuing 3~ oz 72 #g of ch.lo~ophy!!. Wheze indicated, A D P . 13.4 I n N , w a s ~ d . :d.
OFF C
TaNe ] shows thai N1 hhe componenls needed for photopho~phorytation {el a~senoly~is), were a~so needed "to obNin the decrease m £.uores~noe: quenchLng in aloe ] . ~ t E i ~ e : p h o s p h a t e or a:senate we,ze . n e e d e d , w]fl~ a h a l f maximal effeC ~ observed around 1
mM and ~naxLm~Aeffe¢¢ beyond ] 3 mM (no~ sho~m). A D P could nol be ~ep]aced by ATP, and Mg 2÷ could not be zep]aced by Ca 2+ . The Mg2+ effec~ was hhe smalle~I, p~obaMy due 1o ~ e endogenous Mg 2+ in flae OF; .
.
-~--= ~ I ~nin
OF;=
:Fig. 13, :EtIe~ of ,nelg~ ~rmast-ezinhib.~iols on ~he ,de~s.ease in & p H dae ~,oph0sphozyhtion. The ~eacfion nfix'~uree o n ~ n e d in ~ io~alv,0lume Of 3.0 r~l:~ficine-glycinc-NaOH, pB 8£5, 59 raM; KC"t,.20 rnSt;.MgCt 2, 5 raM; az~,¢n~*e 1 mM;pyocya~ae, 5 ~M; 9:aminoacridine, 1 jam and ,chlozoplasts contain,J'~g 30 ,ug.~of:c~o~phyli.'; W3a~leinfli~ated,ADP, 0.4 raM; Did9,. ~0.5 mg]x~; orK~Se04., l@ m M, ~e~e added. .
pr,epa~a~d~u.
r
""
Fig. 2 4:emons~ates lhe depcndene2 of the decrease in ApH on lhe cone~ntzat~on of ADP. A half. maximal effeC~ was o b e y e d mound 5,0 oz 80/~M ADP wifl~ phe~sphate ,o;msenm.e0 re~pecti~e!y. ADP concemmfions-beyond ] ann had m b~ avoided~nee
,I-lhese,concen~ra¢]onS.ADPby
at itselfcaused s o m e quenclaang o~ .hh.~..fluo.~escence b~?9 ~ a ~ o ~ c r ] d i n e . . .... These ~ s u | ~ s c.oT~latc ~ell:.vc]~ ~e dependence of .
• and 1_.18 ~1H2.0 X mg ~hlomphytl- ,and apU Vatue~. " ATP dklnot lead to a s i n ~ ae~rease in ,apH-umler . ~. o f 3 , 5 4 ~ d 3.:t 2 were. deie~miaed, ~-especii~ely~::;. ?::;; :. .. ~esaan¢-vefi,ffi~i~ns:..: : : ::-: J:-:"j.-. :!./_ ..- -....-. ::-
feci of.~n.ergy t:ransfe.:mh~b~tors;.F:g::3 ~h.ows thai
" ph0spla6:l:t!~n~ : d :,~e:r~s::e;,effei~:tifadded a f t : D~o~9:~ ] . when added:,n~:-.AD:P,.c.omp~e~.~y~ : . : the ph~spho~la'tion,r~a:gen~s, : . : ": sl,o:ed ~:eMg.h '..fl:~ao~eS~:en..~.e~uen~hmg.ob*a!n,:edin.. : . : _ . . . - . - - :: :: : . i" :--i..'.:.) :[: : ...." :
11~ above: i~s ~ I : ~ : a ] viflu,e i n ~he ':ight : 9 ] . Also, addil:onofADP~£1e,ID]o-9hadr~beffe,c~:0n'~e~n-er~se,d.ApH: S.elenme, .:ee.ent~]y-found t o a,ct ~s an e n - ~r:gy ~ransfer ,~nhib~I:o: o f p~ho'~ophosphoryht~on I]O], led to a sim:lal ~ffecl w h o a pr..esenl[in ex¢ess o w r azsena~e (probably ~ u e to ~:o:npetifion b e t w e e n ~:them). The pH i e p e n d e n e e o f ~ e d.~.~ea,~in A p H u n d e r phospholyls;tion eondR~Ons ,:~ ~ho~m ,:n fig. 4 J T h e ef~ec'~was obvious o @ wi:~ ~ e pH zange- o f 7.5--9.5 in ~i~e~ noncyclic felri.~yan]d:e dependent photophos, pho:y]ati,~n OlW~fl: .~'¢~C phoi~phosph,olyhfion with pyo:cyan~n~(no~t shown). ~ i m a l e f f ~ Wss oB~e~ed he~=n p H $.5-9J0, s i m B ~ .~o the d ~ p m a d ~ n ~ of p]i01ophosphory]afi,0n on naed~ra p H ~ ] ] ]. In o~ne]usien: 0 Pho:~ophosphoiy]aIion eau~s s ~ # f i c a u ~ d,ec~,~a~e~n $_h~ZxpH £o~me,d in ~ e ]~gh~t aeross ~ e ~ y l a k O i d ~ c m b ~ a n e u n d e r S~eady s t a ~ eon.d~1~,ons.This ob~r~a'don is in a,~r,eemen~ w~Lh if.hesnggestion tha~ phdSp,h o r y ] a ~ o n acc.e]erates ~ e ~fl]ux. o f
' :i .
.
.
.
.
-
Referenees " : :..
,. " .... . :l]-R-Mi,Ichell, N ~ n ~ 1~91(1961) 144.. " . -. . ~2] A~T¢3agend~z:f sndE. Ufibe, B:rookh~v,en Syrup; Bi~0L19. (:t96:~)2:5. ' ....../. "....... ,~3] H. Rot~nbe:~,.T, G ~ , m . ~ : ~ M. A ~ , Eu~ope~ J. B:o:cham: 25 (I972) 54. • -
::
. " .:
..
.
.
]. Bioch~rn. 26 --~i] 14. Rott~nb~3~ and T. Gam~:dd, Em,oy~an J. B~o,e~m. 25 (t972) 7.2. . . . . ". . . . " .
.
( 3 9 7 2 ) 6 4 .
~6] M. A~on, Bi~h~m. Biophy$. A¢la 4~ (]960)257: .{7]:~. Avzon, Anal. Bioehe~n. 2 (]96~) 353.. 18] R ~ . M=C~r~y; RJ. ,G~il,]ory.madE. Rsc]:~;,J. Bio~h Cheln..:24L0{~9~5) PC 4B22. ~9] E.Sk B a ~ g ~ , B. Ro~,e~ab~.g emilM. A%~on, E~rop=an ~. Bioeh~na,,~ ~p;ass. 1~] ~d.A~r~On, in: P~o~. 2xad/~In~'lCorl~ress on Pho~tos~n~sis, zfls. G. Vo~fi ei sL Onnk N.Y., Th~ Hsgu~, ~972) p.
:.;i;:i!:;;;,5i:
i
;/ii17
_
[:
"
//.,
f
-
i
M~']973
FE-BS L E ,'lq'ERS
E F ~ i ~ c r ,o~ v n o s P r i o ~ v ~ A T m ~ o~ aa~ s~z~ o~~Z P R O T O N G R A D I E N T A C R O S S C H L O R O P L A S T ME~:CIBRANES Uri ?~C:K,Hagai ]~OTTENBEKG an,d M ordhzy A'V'RON Bio,chemis~r5~Depart'men.'. ~l~e)zm~nn'2nsn'lnzeo/Science. Re_hovot; Israel ]973
R e c e i v e ,~ a B 3 a n u a r y
U,~ng 0n~ o f z3a~m, *,.hefluoresccn::~ quvnchi~g of
1. Introduction
51u~xesc~n~ a,za~ne~ ~a t h . ~ g h i , w e ~ h o w hexe ~ h ~
The coup]hag between phoiopho~phory]n~on and pro,ton upIake in ch]orvpla~ts [], 2] ~equi~e~ '~a* ATP folma~on ~,ou],d c,~ea*e a .d~ain on the steady ~ta~te ele,c~ro~herr~ca] potentia~ formed during e~]ec:tr.~n transport. ]n ]so]ate,d ch]orophsts most ,of ~ i s potential ~eems to ,exht Ln ~ e form o f a pH gra,d~ent, and therefore, a ~tecrease in the size of ~ s :grad~em % to be .expected when phosphorylat~on ~s ~n~fia~,ed. Several meth,och for measuring the ApH formed in the ~ight ac,~oss the ~ylakoid mernblan,e were recently de~e]oped ,~n t_h~slabom.~ory I3-5].
#
,0~
ON
a , •
. -
.
.
.
.
: 5 atoM; p h o s p h a t e ,
1
.
~drme ,OFF.
" _
2 , ~ raM; 9 - a m i n o a c r i d i n e ,
Ch~]olopilasI~ f~orn letIuc.~ leaves were p.zvpmed essenti ~ah~ as previously described ]6], except fox the :~ina~,c,eni~fugadon and ~esu~p,ensi~n which were in 0.2 M sucrose, 0.1 M KCI. Fluolest'¢nc.e o f 9-maainoacr~dhae wa~ n~ea~ua-ed in an Eppenflu~ fJ~,ofi,m~ler a~ pre,,,]ou~ly de'~cribed i 4 ] .
~DN
..
.r..
.
2. Methods
~,..=
$
~,
phosphrolyaali,on ~r a~eno]y~i~ cvn,d~t~on~..~nfle~d lead to a deerease.d stvady sta~.e -ApH in the H~,ght
, ~ - -_- - ' ~ J
,,,,
"I":
-.
" .
ON
. -
"
. "OFF - .
.
1 ~uM; :raft c h l d ~ d p l a s t ~ e , o n ~ r i ~ a g . 4 $ : u g
.OFF:
. .
-
_
,OFF"
_
O/:¢Ialo~Op]~ylE '_~ .-: -
. L.. -. ~-. : - . . .
:
¥~olum9 32,,numbe~ ]
F~BS LFFFERS 3; Resets and
"Table ~}qaoxph~I~la?5on ¢.~mpox, ents ,~eefled for Lh~ ,de..ease ~n ~pJJ.
ET~p~zim~nt Cemp~n~nl No. absent
Cempon~m added None
MgCI~
f
l
i
s
e
u
~
i
May ]973 o
n
'
" "
.
.:
"
"
-
A typical ,expe~imen~ dem.mstrathagthe effect o f phosphoryhfi0n vondltlons on ihe steady stat~ ApH • . formed in ihe ]igh, is ~aown in fig. 1. The ~aversible light induced fluorescen,ce quenching o~,"9-aminoacri:~ine was mafxe0Jy xed~c~d ~fter ,the ad.3Rion o~ADY ~to an othm-W,is~ c,o.~p]ete phosphory]afi,on ~ s l e m . ~,eI~ low c.:onecn~r~tionz o f A D P were ~ d in the ]~ght, a ~m~e~ ~leady state quenchhng ~at~owas ~,m-med w]~hm ] 5--30 sec w}fi,ch in th:e light slowly returned t.o i u er,ig~nfl levd, indicating flee conversion of all ~.flie ADP. -Io ATP. The hip~her the amount of ADP added, t]:e longer R took ~o return t o / h e high quenching in :~ae ]i.ght, When the rate ,ofph0tophospbo,D.'lation was estimated from the time necessary to c,onzurae flee ADP added, values comparab]:e "toflaose eziirn~~ed .by other m~flaods wer,e obtained.
~pH measured
3.72 3.5,~. . . . . 3.,58 II Azsenzle None 3.74 5 ram A r ~ n z t ~ 3,24 2.3 mM Y~ospha~e 3.21 Ill ADP None 3.~1 ~.4 m~,f ADP 3.41 0.4 mM ATP s.7~6 .The eornplet,~~'~em con~ned m a ~9ta] "~olum~:of 3.0 ml Tr]eine~glycin~.NaOH,pH ~.O, 50 raM; KCI, 50 ~M; M~]=, 5 haM; arsenate, pH 8.0, 5 rnM; ADP, ~.4 raM; pyocymahae, l
.
5 mM MgCJ 2 5 ham CaC}2
5 ~M; and ¢hloz~Splasls e~nxai~fm.~ 4 t ~Ig o f Cyd~rophyl].
11~1~rn~_at~on o~" th~ osmotic spaces of ch]oro-
M,easurementz and valculalions of ~he o ~ ,tSc volumes of ¢h]oroplasl fragmem~ fr,on~ the disi:ribution o f ~a4.C]Sorbi~ot and ~3H] H 2 0 were ,done :as de. scribed 13]. ApH values were determined flora .'the osrn,~fic volumes and fluorescence quenching ~ e s according t0 SchuJd~ner e~ el. 14].
rl
p]as,~ d d n,orrev~a] any significant cliff,lento in ~he p,~esenc~ or ab;enc~ o f ADP. These resul~ ruble ou~ shfinka2: ~f the thfiakoJds as ~ e czuse for ~ e d,ecr,easer~ flu,me~cence quen.ch~g ratios under phosphory]a d.oa conditions. In a lyp~ca~ experi',ment, in the absence and presence of ADP, osmotic spaces of ] 2. l
i
rD.~
1 ,D •
•
"
..
0.'3
"
0.4
"
•
"
-
(
"
"L
.
I
.0
~2 0 0
"
•
;300
400
" "
~ 0 0
•
-
.
FEBS LETTERS
¥olume 32, numbez '1 .
-
May 1973
. -
"%0
/ ~
4
!
OF~
~/,~
t / .~:
o/
1
OPF
9-omino-
~,
OFF
oc~]ine
0.5
g
v
I=
/
J_
. t/ 7
OFF
\ .B
9
~t t0
Fig. 4. p H dependence of ,the de~ca~ ~ ApH unde~ phosphozyhfion cond~tk)zs. The zeacfion mktu~e con~a~nera, hu a to'lal roll.me of 3.0 ml: ~r.icine-:m~ea~te-NaOH. @A ~ e intLicated pH between 6.85-8.9), o_~lrJcine-gfycine-NaOH (pH 8.45-
! 9-=~-
"\
OFF
9.8), 50 .z~M; KC],50 .naN; K3FelCN)6 , ~.l raM; MgCI2, 5 raM; phe~ha~¢, 2.3 raM; 9-aminoacz~din~ 1 - 2 ~M; and chlo~ophsls ¢9~ahuing 3~ oz 72 #g of ch.lo~ophy!!. Wheze indicated, A D P . 13.4 I n N , w a s ~ d . :d.
OFF C
TaNe ] shows thai N1 hhe componenls needed for photopho~phorytation {el a~senoly~is), were a~so needed "to obNin the decrease m £.uores~noe: quenchLng in aloe ] . ~ t E i ~ e : p h o s p h a t e or a:senate we,ze . n e e d e d , w]fl~ a h a l f maximal effeC ~ observed around 1
mM and ~naxLm~Aeffe¢¢ beyond ] 3 mM (no~ sho~m). A D P could nol be ~ep]aced by ATP, and Mg 2÷ could not be zep]aced by Ca 2+ . The Mg2+ effec~ was hhe smalle~I, p~obaMy due 1o ~ e endogenous Mg 2+ in flae OF; .
.
-~--= ~ I ~nin
OF;=
:Fig. 13, :EtIe~ of ,nelg~ ~rmast-ezinhib.~iols on ~he ,de~s.ease in & p H dae ~,oph0sphozyhtion. The ~eacfion nfix'~uree o n ~ n e d in ~ io~alv,0lume Of 3.0 r~l:~ficine-glycinc-NaOH, pB 8£5, 59 raM; KC"t,.20 rnSt;.MgCt 2, 5 raM; az~,¢n~*e 1 mM;pyocya~ae, 5 ~M; 9:aminoacridine, 1 jam and ,chlozoplasts contain,J'~g 30 ,ug.~of:c~o~phyli.'; W3a~leinfli~ated,ADP, 0.4 raM; Did9,. ~0.5 mg]x~; orK~Se04., l@ m M, ~e~e added. .
pr,epa~a~d~u.
r
""
Fig. 2 4:emons~ates lhe depcndene2 of the decrease in ApH on lhe cone~ntzat~on of ADP. A half. maximal effeC~ was o b e y e d mound 5,0 oz 80/~M ADP wifl~ phe~sphate ,o;msenm.e0 re~pecti~e!y. ADP concemmfions-beyond ] ann had m b~ avoided~nee
,I-lhese,concen~ra¢]onS.ADPby
at itselfcaused s o m e quenclaang o~ .hh.~..fluo.~escence b~?9 ~ a ~ o ~ c r ] d i n e . . .... These ~ s u | ~ s c.oT~latc ~ell:.vc]~ ~e dependence of .
• and 1_.18 ~1H2.0 X mg ~hlomphytl- ,and apU Vatue~. " ATP dklnot lead to a s i n ~ ae~rease in ,apH-umler . ~. o f 3 , 5 4 ~ d 3.:t 2 were. deie~miaed, ~-especii~ely~::;. ?::;; :. .. ~esaan¢-vefi,ffi~i~ns:..: : : ::-: J:-:"j.-. :!./_ ..- -....-. ::-
feci of.~n.ergy t:ransfe.:mh~b~tors;.F:g::3 ~h.ows thai
" ph0spla6:l:t!~n~ : d :,~e:r~s::e;,effei~:tifadded a f t : D~o~9:~ ] . when added:,n~:-.AD:P,.c.omp~e~.~y~ : . : the ph~spho~la'tion,r~a:gen~s, : . : ": sl,o:ed ~:eMg.h '..fl:~ao~eS~:en..~.e~uen~hmg.ob*a!n,:edin.. : . : _ . . . - . - - :: :: : . i" :--i..'.:.) :[: : ...." :
11~ above: i~s ~ I : ~ : a ] viflu,e i n ~he ':ight : 9 ] . Also, addil:onofADP~£1e,ID]o-9hadr~beffe,c~:0n'~e~n-er~se,d.ApH: S.elenme, .:ee.ent~]y-found t o a,ct ~s an e n - ~r:gy ~ransfer ,~nhib~I:o: o f p~ho'~ophosphoryht~on I]O], led to a sim:lal ~ffecl w h o a pr..esenl[in ex¢ess o w r azsena~e (probably ~ u e to ~:o:npetifion b e t w e e n ~:them). The pH i e p e n d e n e e o f ~ e d.~.~ea,~in A p H u n d e r phospholyls;tion eondR~Ons ,:~ ~ho~m ,:n fig. 4 J T h e ef~ec'~was obvious o @ wi:~ ~ e pH zange- o f 7.5--9.5 in ~i~e~ noncyclic felri.~yan]d:e dependent photophos, pho:y]ati,~n OlW~fl: .~'¢~C phoi~phosph,olyhfion with pyo:cyan~n~(no~t shown). ~ i m a l e f f ~ Wss oB~e~ed he~=n p H $.5-9J0, s i m B ~ .~o the d ~ p m a d ~ n ~ of p]i01ophosphory]afi,0n on naed~ra p H ~ ] ] ]. In o~ne]usien: 0 Pho:~ophosphoiy]aIion eau~s s ~ # f i c a u ~ d,ec~,~a~e~n $_h~ZxpH £o~me,d in ~ e ]~gh~t aeross ~ e ~ y l a k O i d ~ c m b ~ a n e u n d e r S~eady s t a ~ eon.d~1~,ons.This ob~r~a'don is in a,~r,eemen~ w~Lh if.hesnggestion tha~ phdSp,h o r y ] a ~ o n acc.e]erates ~ e ~fl]ux. o f
' :i .
.
.
.
.
-
Referenees " : :..
,. " .... . :l]-R-Mi,Ichell, N ~ n ~ 1~91(1961) 144.. " . -. . ~2] A~T¢3agend~z:f sndE. Ufibe, B:rookh~v,en Syrup; Bi~0L19. (:t96:~)2:5. ' ....../. "....... ,~3] H. Rot~nbe:~,.T, G ~ , m . ~ : ~ M. A ~ , Eu~ope~ J. B:o:cham: 25 (I972) 54. • -
::
. " .:
..
.
.
]. Bioch~rn. 26 --~i] 14. Rott~nb~3~ and T. Gam~:dd, Em,oy~an J. B~o,e~m. 25 (t972) 7.2. . . . . ". . . . " .
.
( 3 9 7 2 ) 6 4 .
~6] M. A~on, Bi~h~m. Biophy$. A¢la 4~ (]960)257: .{7]:~. Avzon, Anal. Bioehe~n. 2 (]96~) 353.. 18] R ~ . M=C~r~y; RJ. ,G~il,]ory.madE. Rsc]:~;,J. Bio~h Cheln..:24L0{~9~5) PC 4B22. ~9] E.Sk B a ~ g ~ , B. Ro~,e~ab~.g emilM. A%~on, E~rop=an ~. Bioeh~na,,~ ~p;ass. 1~] ~d.A~r~On, in: P~o~. 2xad/~In~'lCorl~ress on Pho~tos~n~sis, zfls. G. Vo~fi ei sL Onnk N.Y., Th~ Hsgu~, ~972) p.
:.;i;:i!:;;;,5i:
i
;/ii17
_
[:
"
//.,
f
-