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The regulation of pyruvate dehydrogenase activity in rat hippocampal slices: Effect of dichloroacetate
~e~.~.~ ~e~ 3~ (:98~)4, ,46 g:~Jer/Ne~t~-Ho~..~ g-c{enth]c PubtisBers L/d,
P ~ RV v A ~
M:C:~.£-~UDNY. : -
4~
#.EN~CDNOGENASE ACT~.:g~Y
$ . .KE&StER. .. "E L I , ~~A B E'T H - K .
SM~ ~~ I-{
and GAR.v LYNCH
o
De~rtm,~:~g of t~,e~obiotogy,,$e~l of B~e[ogieet Science& O~NersiCe of Calgfotnie, frvi~e, CA 927t 7
(UoSgAU (Receives
~ h , t981; Rev:~ed v~s~on roaeivetl April 261h, I982; Accepted A.p~:~ 28th: 1982)
The effects of dicbaoroace~.ate (DCAL a~ ~nhibitor of pyruvate dehydrogenase kinase., on the phos~horylaUo~ o f the ~-subue,At of pyt-uva~e dehyd:'oge::ase and on :he activity of oyruvate dehydrogenase :pymwat~:li~arNde ~a~e (deearboxy:ating aad acceptoroace~ylating), EC t .2.4. ~, PDH) were m~s~igated in ra: hlppo~ampal slice~. ~neuhati~g hippoc~amp~ slices with ir:¢reas:r~g concentrations of DCA resulted in am i::¢re&~e ~rt the active portion of PDH, without changes in {he tota~ PDH activity, :he i~ vitro pho~phoW~ation of c~-PDH. The effe¢~ of DCA on PDH activity os~ maximal after 5 rain. These re~uI~s indicate that DCA in the htppocampM P D H kinase sad consequemty s~imulates PDH activity by decreasing its e ~tate of phoapho~,latios. Moreover the time..co'~:rse of the effect of DCA ~uggests .Cat Che tin'never rate of the phosphate gr<,mpera°tied by ~-PDH is very rapid and can be manip~;~a:ed by altering PDH kina~e acfivi~,y,
Dttring the ]last 10 years, the h i p ~ o c a m p u s has b e e n widely use{1 to stu:~y uv: mechanisms
undeAjng
v a r i o u s f'orma o f -~ynaptic p k ~ s t i d t y ,
an ptwdcutar~ the
fOi~ows b, at[ bdr:ata o f h~gh L.~quehvy fltiv'~aiatlou,
o,.e~p:e~ a K.y ~ t ~ o r ~ :.... gMa{,ng eel~u:ar m e t a b o E s m , espec{a[]y in. b r a i n w h e r e :{ represents art obb;gator2 £ e p b e t w e e ~ glucose utllizat~oa and ox~da.:ve phosp}iorySaUon arid e l e c t { o n transpor-: [~5~o P D H act:vity is regutated i~ part via p h . . @ h o r ~ a d o n - d e p L u s ~ . & o r > a t ~ o . ~ o f }is e > s u b u m t , the e n z y m e ~-- ~ ,, inactive in' ~ts o h . o s p h o ~ y l ~ e d fom:<~ ~: 1], W e s h o w e d m ~ a t c~.dange:~e*in t~.~e p h o s p h o e y ~ a t i o n c,: of ¢t~'~,g~-,o~: ce: ~re~v,~S!~{i{¢ eJeg1~:.%
caicit:m
~g:(-.: a~:tr,r:>:>:dr{a} ....... >be:e... of
!.5],
;;~'¢,"~"
eatdum -do a rapid ebmge m ~he ~ @ h ~ y l a a o of a~PDH [SL TN~ h s obvio~s~ requires thaz PDH ~etm.y " "":~" N the ~ p p o ~ p a t ~Ec~ be ~apidly a~ud m a r k ~ l y med.ified by acti~'atio~ of either PDH k ~ s e , or PDH p ~a~
on,f me a~i'd~y° I~ ~ p r o e m .~ective h:~h~bi~erof PDH M ~ : to to~al PDH a~viW~ as well ~ e~ the time~cx~ur~ of the effec~ of ~ A s~gge~g~ a very rapi~ tume~e~ of the phosphate gxo~p carried by ce.PDH, a¢ ie~,t m the rat h~ppoeamp~l ~ c e . Male Sprague-Daw~ey ra~ (150--.250 g) were ~ e d by fen arid the hipW.~campi rapidly dN~sec~ed° Transverse $iiees were cut. Mlh a Met{wain fissae chopr~r (0.42 m-~e thick) arid ¢o~te~ed in co~d K.reb~-R~nger bicarbonate buffer containing (in raM): Noel, 124; KCi, 3.33; , 1.25; MgSO~, 2.41; CaC]~, 2.45; N ~ C O ~ , 25. then transferred to I5 ~ gtas~ ~cimii~atioa vial~ were sealed with rubber ~c,p~rs atlowiag a c o ~ and were incubated at 3~'°C for the i n ~ a ~ e ~ periods of time ~nder g~t|.e shaking. A~ the end of the iacubatie~ sl~ce~ were indlvNuatly distributed in 0.2 ml of ceId 25 mM PIPES buffer, pH 7.2, eongNning t m M EDTA: they were homogenked by sonicafion for abot~t 10 sec (Virsonic cell di~ruNer)o Aiiquo~s of t~e Uice homogena~e~; were used to dete~'mi~e ac~b'e and ~otal P[kH activity. A mi×~u;e of EGTA (2 raM), DCA (2°5 raM) and sf~.di~m fi~oride (SmM) wan added to a firs~ a~iquo~ in order ~o prevent %nher PDH Nna~e and pho~pha~a~e aaivifies. This is referred to below as ~he "active' PDH activity. A ~ecor~d Niquo~ was preincubated w raM) and dinitmpg of PDH. This was activity ~¢as meas~ mCi/mmol, New E: pn~viou~ty desc~ibe~ wi~h bovine serum formed/m~n/mg protein~ .~ ~ of active. ~ " . .~e~.~.~us .. .. ~,r as rat~o tota~ enzyme. .activgyo :St~ea~~'~s t-test was used to statistically compare variou~ e ~ e r i m e m a ! greUp~, . For determination of phesphorylafion°~ of a.PDH, slkes homogepSzed by sonieafion in 2(~ ~d of 50 mM HEPES/Tris (pH 7.4)containing"50 mM s~Jium
l.w ~)o Ahq~o~ of 25 ~I (approx~ 20 ~g addieg 10 mM ,r, cor~cemra~ions), Aaer 20 sec, ~ e precipitates were wa~;hed once with e.5%
w~: ~ o p ~ e
be !C~. T C A ; 2 ! ~ sodium
43
:5% &met 10% glycere!, and myg~{ed to a 10% @ e t ~ r o p h o r e s i s g e l For autora@ograph:y the ge~ was d r b d and to a k X-ray f i ~ . Apparen~ mc~%c~aiar weights were detern'fi~ed from . bovine serum approximafi~e ,v
eon0entradons o f dichbroaeetate (DCA) were .i~ medium, and the Ne~SNion confirmed for 30 rmn. Slice~ we~e and active and rata1 P D H activiW were measured as DCA concentration progressively increases the active portion o f P D H without affecting ~ot~I PDH activity t~igo IA)o DCA has a m~t effect: at i o n a s low as 0.4 raM, and the haIf-maydmal effect N a~ aexmcentrafion of 0.8 raM. The ratio of active to term PDH activity increases from about 0o50 m 0.75-0.80 a~ the highest D C A concentration, representing a 50-60% increase. It is interesting to compare these effects of DCA in: ~ e e s to l h o ~ obtained on PDH actNity in purified mitoehondria° 7bus we previously % u ~ d that DCA, in the latter case, maximally siimu~ated PDH activity by about 50-40% with the hNf-maxkaat effect at a concentration of 0.2 mM [5]. The differences in ECso between the slice and the p~rified mkocho~adfia is probaNy due to diffusion as welt as degradation of DCA i~ the slice.
12
g
f;_
Z
,
£
t
~
*
S/"a:
°
'r
! 1
!
i v Fig, IA: eff¢~ of various dgeNe.~oace~te (D%'A) ¢oncen~rafier~s o~ 'active' and ' ! e t a ' P O H aciivit:, H i p ~ a m ~ a ! @ee~ were g~r~pared as e:escr~ed a~d pre~ncuba~ed el. 37~C for N . mi~ . ~. d e .f a. cem~.an~ ~tre~m o f O s C ¢ % @5:5}. ! , ' a r ~ 2 s co,~.~et,~°auo~ ~.... ~a of DCA we.~:e ia~rodaced in' the ir,eabatioa~ ~-~,edi~m a M the iee~ba~o.~ e o m i ~ e d fo:~ 30 mia. Active {c~osed eirciea) a~d H;~ai (opec
p~M,~ o f Nine (~h~ ~o~N >rei~c~#~a~:~cr~a ~
kv;a:~agk>e ~eric4 ~a~ kep~ co~s~ar~t aa ~ h)~ Results
of th:e a.O~.,O Mr
(TaSte Ia. Severa~ grQu~pshave d e a = o m ~ ,
~h~ t~g ~ , - ~
phe
~s
thv reh:
af a: T iatr~ is very ~°tpi~, being ¢~ose to ma~dmal a:ft~s S mla,
.
~oPDH :~.ho~phoryht~on and P D H activity are ¢oas.,.steat w, tR. tb-a ~ d ~ *:~at ~XTA. ~n the slice ~:~eparafiono ra~}id~y (i~e. ~ess thau 5 ~ i n ) mh~gg.~ =~ aetivky ' ~ ' P D H ~'i ~z~a.m
v
L~ d
OF ~ A O N
THE °~2ST-HOC ~ PHOSPHO~YLAT:ON OF VARiOU9 PROTEINS
sti,ccs v-'<~eiucubated with ~0 mM DCA fo~-30 :~.in d e Exp~rimenl b as descr{b~d iv. the ~egend a f fffgo 2 or ~or 5 mi:~ (i n Extra,men . . . . . e 2}.. Ae, vor~.~d:owaphs we~c seam:e4 ,~mg a~ E-C
¢olein~
12,,a±0.~
=%
~2.1±0.4
0
58 51. 40 (e=:PIH)
4. ~-GI .t1. . . 7. .,.-.1 5 1 0 . 9 ± l,O
Ii.3±0.4 ~304 ~-=I o2 11.6 ±0.8_
-~ 13 + 15 + 34
ms, a,so ! < ~,0.,
Exp~d~m 2 40 (~-PDH)
5.2 cO.5
+ fi2
P < 0.0'2
~
~
...
.
and that the PDH phosphata~ sust
m
wry
7.9 ± Q,d '~
tus.
~emovcs t h e phospimi:e g.~oup(s) ,-.~<=~ ...., .,.~i,,,~ b y th~ a
" "- . . Fh~,~; . . . It~I~g[)' I t g g e s t s that- the t u r n o v e r o f this ==;ho~,p=~;~/e . . . . . . ,- • e/fsct~v¢Iyo
r a t i o o f active to t o t a i act~%ty0 This is par~ictdafiy i n ~ e r e a ~ g ~:~ ~dew c [ o~y~° previousty e d h y p o t h e s i s tha~. high f r e q u e n c y e~ectic~-fl stkc, u~at~on of hi
8i ~ a t h w a y s car~ mea~uiate the ph~aphoc~)_ai~ou o f ce~PD[~l, ~u this ~:a~,¢ t h e e f f e c t vca:.~ p r e s e n t at 2 ~;~hl f£d~o'whlg {he ai~m:~ation bt~t v~xa ~o t©~gw i 5 m}~ a f t e r the Kai~ [6]. ha ~ a ¢ | ~ s i o a , t h e present d a t a sh©w ~hat b~ the rat blpF:}can'~psl ~!icc preparad¢:~, t h e t s m o v s r o f the phosphate: g r o u p carr~ccj by ~he c~-s;~b:~s-~R o~ ~,-q)~{ -~ the
de~cctab~¢
;) or ygco
ng c b a u g e a in the ~a~ hig!>,frequevocy P D H aGi, v l ~ due i t s c>s
tbu
it [6]o
a z : r evea
,
m:gbt b e ~ useful m o d e l )M a c t l v l t ? o T h i s b e c o m e s e v ~ fi~zs.!. ~0£ 0~;:~]( h}gh i;'cquc~>::)' d¢ctAc~i •a t i o ~ ,
A 53gL
u ~ z i n g the ~'inc~4e of ~rocein~e bi~d~g, ~%:'~lyt: B~c~m.~ 72 (i97~ 2z~{}=254.
5 Brovmmg, M., Bau~ry, Mo, B e a ~ , W. and Ly~ch~ O., t~he~horyla~on-me~ar.ed ¢h,~aag~ ~n ~y~wea~e .~,~aydrog~z~e ~d~6ty ~.nftuert¢¢ pyr~w~e~sa~ned ¢a]cium accumulation ~y ~ n mit~h~adria, 3. Ne,ar~:hern., ~6 ( ~ H ! ~ 2 - 1 ~ . 6 ~rowr~h~g, t4L, Baudry, tyroarid Ly~cb, G.~ Ev~en~c ~ha~"t:d~h4reque~%v~ m u ~ o n ~f~ragaces the phe~ekoryladon ,.~~ p~uva~e dehydrogeaa~ ~ad ~bat the ac~Jvg), or" th~ ~ }~~h~cd/o:m~tochtrndiSa] cat¢i~m ~ u e ~ r ~ i c m . tn A. R o m t e a ~ ropro~ein~: Char~edzat~,o~ and F~mc~on, F ~ . 7 ~rowaing, ~ . , Besmear, W.. Ke]l::a, P. ~ Lynch, 0,~ The ~0.¢~ ~ r bra~n ir~fluen,.~ by high fi-eqnen<¢ ~yr~apt/.,:~imu~atloa i~ .~.~ea~ph~ ~t~bu~i~ ~f p~uvate ~ ]
r~e'm
8 Conwa:¢, RoC. aad Romten~erV~ A°, Endogem~u.~: pho~pho~'Ia.~io'a ~n v~tro~".,¢~tive ef~et~ of %, ~,acriDce me~hod~ on ~pecffic br~hl cro~e~n~ B~a~n Re,., ~39 {]97g} 364J-37~, 9 .k~pe, R. as~fl t~ss, J.P., The r,eg~.~la~onof pyruvate dehyctroge~a~e in bra~r~~n v~v~, J. N . ~ e h ~ a . . 26 {~97g} 7f~).o7~4. ~O Le~e~'. A.B.. Weinberg., M., ~sohashi., Fo, U~er, M.F. aria Lin;~, T,. h~p t ~ w ~ pho~pbory~a~an a~A ~ctivi~yof py~uva~e dehydroge~na~¢h~ :at Rv~ mh~e~,~dr~&aad ~ "~ae~_eeof
an¢
~2 Ly: Co Holland, Amsterdam, l~7~,..~p, l~-~2go 13 Lynch, G. and Schubert, P., The use of ~n vitro b~ain s ~ for muR~d~l~nary ~tugie~ of ~yaap~ic function. Ann. Rev° Neuro~¢i.o 3 (19~) I - 2 L t 4 Magi|en, G., G~don, A., Au, A. a~d Diamo~,d, t.;. tae~df~cation of a mi~oehom~M ~hmph~:rc/~im in brain syna~tic m~.~nbra~¢ preparations, $. N , 3 6 {~8|} 1 :.: {5 McHw~dn, H. a ~ gm:heM~d, Ho° B ~ a c . ~ i ~ g and ~he Cen~,] Ne,~ou~,iS~te~, 4ih ~ ¢ - C h ~ c h f l b Livings~o~ae, Ed~aburgh, 197l° ~6 . . . . . . . . . . . . . . . .
I7 |8
P ~ RV v A ~
M:C:~.£-~UDNY. : -
4~
#.EN~CDNOGENASE ACT~.:g~Y
$ . .KE&StER. .. "E L I , ~~A B E'T H - K .
SM~ ~~ I-{
and GAR.v LYNCH
o
De~rtm,~:~g of t~,e~obiotogy,,$e~l of B~e[ogieet Science& O~NersiCe of Calgfotnie, frvi~e, CA 927t 7
(UoSgAU (Receives
~ h , t981; Rev:~ed v~s~on roaeivetl April 261h, I982; Accepted A.p~:~ 28th: 1982)
The effects of dicbaoroace~.ate (DCAL a~ ~nhibitor of pyruvate dehydrogenase kinase., on the phos~horylaUo~ o f the ~-subue,At of pyt-uva~e dehyd:'oge::ase and on :he activity of oyruvate dehydrogenase :pymwat~:li~arNde ~a~e (deearboxy:ating aad acceptoroace~ylating), EC t .2.4. ~, PDH) were m~s~igated in ra: hlppo~ampal slice~. ~neuhati~g hippoc~amp~ slices with ir:¢reas:r~g concentrations of DCA resulted in am i::¢re&~e ~rt the active portion of PDH, without changes in {he tota~ PDH activity, :he i~ vitro pho~phoW~ation of c~-PDH. The effe¢~ of DCA on PDH activity os~ maximal after 5 rain. These re~uI~s indicate that DCA in the htppocampM P D H kinase sad consequemty s~imulates PDH activity by decreasing its e ~tate of phoapho~,latios. Moreover the time..co'~:rse of the effect of DCA ~uggests .Cat Che tin'never rate of the phosphate gr<,mpera°tied by ~-PDH is very rapid and can be manip~;~a:ed by altering PDH kina~e acfivi~,y,
Dttring the ]last 10 years, the h i p ~ o c a m p u s has b e e n widely use{1 to stu:~y uv: mechanisms
undeAjng
v a r i o u s f'orma o f -~ynaptic p k ~ s t i d t y ,
an ptwdcutar~ the
fOi~ows b, at[ bdr:ata o f h~gh L.~quehvy fltiv'~aiatlou,
o,.e~p:e~ a K.y ~ t ~ o r ~ :.... gMa{,ng eel~u:ar m e t a b o E s m , espec{a[]y in. b r a i n w h e r e :{ represents art obb;gator2 £ e p b e t w e e ~ glucose utllizat~oa and ox~da.:ve phosp}iorySaUon arid e l e c t { o n transpor-: [~5~o P D H act:vity is regutated i~ part via p h . . @ h o r ~ a d o n - d e p L u s ~ . & o r > a t ~ o . ~ o f }is e > s u b u m t , the e n z y m e ~-- ~ ,, inactive in' ~ts o h . o s p h o ~ y l ~ e d fom:<~ ~: 1], W e s h o w e d m ~ a t c~.dange:~e*in t~.~e p h o s p h o e y ~ a t i o n c,: of ¢t~'~,g~-,o~: ce: ~re~v,~S!~{i{¢ eJeg1~:.%
caicit:m
~g:(-.: a~:tr,r:>:>:dr{a} ....... >be:e... of
!.5],
;;~'¢,"~"
eatdum -do a rapid ebmge m ~he ~ @ h ~ y l a a o of a~PDH [SL TN~ h s obvio~s~ requires thaz PDH ~etm.y " "":~" N the ~ p p o ~ p a t ~Ec~ be ~apidly a~ud m a r k ~ l y med.ified by acti~'atio~ of either PDH k ~ s e , or PDH p ~a~
on,f me a~i'd~y° I~ ~ p r o e m .~ective h:~h~bi~erof PDH M ~ : to to~al PDH a~viW~ as well ~ e~ the time~cx~ur~ of the effec~ of ~ A s~gge~g~ a very rapi~ tume~e~ of the phosphate gxo~p carried by ce.PDH, a¢ ie~,t m the rat h~ppoeamp~l ~ c e . Male Sprague-Daw~ey ra~ (150--.250 g) were ~ e d by fen arid the hipW.~campi rapidly dN~sec~ed° Transverse $iiees were cut. Mlh a Met{wain fissae chopr~r (0.42 m-~e thick) arid ¢o~te~ed in co~d K.reb~-R~nger bicarbonate buffer containing (in raM): Noel, 124; KCi, 3.33; , 1.25; MgSO~, 2.41; CaC]~, 2.45; N ~ C O ~ , 25. then transferred to I5 ~ gtas~ ~cimii~atioa vial~ were sealed with rubber ~c,p~rs atlowiag a c o ~ and were incubated at 3~'°C for the i n ~ a ~ e ~ periods of time ~nder g~t|.e shaking. A~ the end of the iacubatie~ sl~ce~ were indlvNuatly distributed in 0.2 ml of ceId 25 mM PIPES buffer, pH 7.2, eongNning t m M EDTA: they were homogenked by sonicafion for abot~t 10 sec (Virsonic cell di~ruNer)o Aiiquo~s of t~e Uice homogena~e~; were used to dete~'mi~e ac~b'e and ~otal P[kH activity. A mi×~u;e of EGTA (2 raM), DCA (2°5 raM) and sf~.di~m fi~oride (SmM) wan added to a firs~ a~iquo~ in order ~o prevent %nher PDH Nna~e and pho~pha~a~e aaivifies. This is referred to below as ~he "active' PDH activity. A ~ecor~d Niquo~ was preincubated w raM) and dinitmpg of PDH. This was activity ~¢as meas~ mCi/mmol, New E: pn~viou~ty desc~ibe~ wi~h bovine serum formed/m~n/mg protein~ .~ ~ of active. ~ " . .~e~.~.~us .. .. ~,r as rat~o tota~ enzyme. .activgyo :St~ea~~'~s t-test was used to statistically compare variou~ e ~ e r i m e m a ! greUp~, . For determination of phesphorylafion°~ of a.PDH, slkes homogepSzed by sonieafion in 2(~ ~d of 50 mM HEPES/Tris (pH 7.4)containing"50 mM s~Jium
l.w ~)o Ahq~o~ of 25 ~I (approx~ 20 ~g addieg 10 mM ,r, cor~cemra~ions), Aaer 20 sec, ~ e precipitates were wa~;hed once with e.5%
w~: ~ o p ~ e
be !C~. T C A ; 2 ! ~ sodium
43
:5% &met 10% glycere!, and myg~{ed to a 10% @ e t ~ r o p h o r e s i s g e l For autora@ograph:y the ge~ was d r b d and to a k X-ray f i ~ . Apparen~ mc~%c~aiar weights were detern'fi~ed from . bovine serum approximafi~e ,v
eon0entradons o f dichbroaeetate (DCA) were .i~ medium, and the Ne~SNion confirmed for 30 rmn. Slice~ we~e and active and rata1 P D H activiW were measured as DCA concentration progressively increases the active portion o f P D H without affecting ~ot~I PDH activity t~igo IA)o DCA has a m~t effect: at i o n a s low as 0.4 raM, and the haIf-maydmal effect N a~ aexmcentrafion of 0.8 raM. The ratio of active to term PDH activity increases from about 0o50 m 0.75-0.80 a~ the highest D C A concentration, representing a 50-60% increase. It is interesting to compare these effects of DCA in: ~ e e s to l h o ~ obtained on PDH actNity in purified mitoehondria° 7bus we previously % u ~ d that DCA, in the latter case, maximally siimu~ated PDH activity by about 50-40% with the hNf-maxkaat effect at a concentration of 0.2 mM [5]. The differences in ECso between the slice and the p~rified mkocho~adfia is probaNy due to diffusion as welt as degradation of DCA i~ the slice.
12
g
f;_
Z
,
£
t
~
*
S/"a:
°
'r
! 1
!
i v Fig, IA: eff¢~ of various dgeNe.~oace~te (D%'A) ¢oncen~rafier~s o~ 'active' and ' ! e t a ' P O H aciivit:, H i p ~ a m ~ a ! @ee~ were g~r~pared as e:escr~ed a~d pre~ncuba~ed el. 37~C for N . mi~ . ~. d e .f a. cem~.an~ ~tre~m o f O s C ¢ % @5:5}. ! , ' a r ~ 2 s co,~.~et,~°auo~ ~.... ~a of DCA we.~:e ia~rodaced in' the ir,eabatioa~ ~-~,edi~m a M the iee~ba~o.~ e o m i ~ e d fo:~ 30 mia. Active {c~osed eirciea) a~d H;~ai (opec
p~M,~ o f Nine (~h~ ~o~N >rei~c~#~a~:~cr~a ~
kv;a:~agk>e ~eric4 ~a~ kep~ co~s~ar~t aa ~ h)~ Results
of th:e a.O~.,O Mr
(TaSte Ia. Severa~ grQu~pshave d e a = o m ~ ,
~h~ t~g ~ , - ~
phe
~s
thv reh:
af a: T iatr~ is very ~°tpi~, being ¢~ose to ma~dmal a:ft~s S mla,
.
~oPDH :~.ho~phoryht~on and P D H activity are ¢oas.,.steat w, tR. tb-a ~ d ~ *:~at ~XTA. ~n the slice ~:~eparafiono ra~}id~y (i~e. ~ess thau 5 ~ i n ) mh~gg.~ =~ aetivky ' ~ ' P D H ~'i ~z~a.m
v
L~ d
OF ~ A O N
THE °~2ST-HOC ~ PHOSPHO~YLAT:ON OF VARiOU9 PROTEINS
sti,ccs v-'<~eiucubated with ~0 mM DCA fo~-30 :~.in d e Exp~rimenl b as descr{b~d iv. the ~egend a f fffgo 2 or ~or 5 mi:~ (i n Extra,men . . . . . e 2}.. Ae, vor~.~d:owaphs we~c seam:e4 ,~mg a~ E-C
¢olein~
12,,a±0.~
=%
~2.1±0.4
0
58 51. 40 (e=:PIH)
4. ~-GI .t1. . . 7. .,.-.1 5 1 0 . 9 ± l,O
Ii.3±0.4 ~304 ~-=I o2 11.6 ±0.8_
-~ 13 + 15 + 34
ms, a,so ! < ~,0.,
Exp~d~m 2 40 (~-PDH)
5.2 cO.5
+ fi2
P < 0.0'2
~
~
...
.
and that the PDH phosphata~ sust
m
wry
7.9 ± Q,d '~
tus.
~emovcs t h e phospimi:e g.~oup(s) ,-.~<=~ ...., .,.~i,,,~ b y th~ a
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5 Brovmmg, M., Bau~ry, Mo, B e a ~ , W. and Ly~ch~ O., t~he~horyla~on-me~ar.ed ¢h,~aag~ ~n ~y~wea~e .~,~aydrog~z~e ~d~6ty ~.nftuert¢¢ pyr~w~e~sa~ned ¢a]cium accumulation ~y ~ n mit~h~adria, 3. Ne,ar~:hern., ~6 ( ~ H ! ~ 2 - 1 ~ . 6 ~rowr~h~g, t4L, Baudry, tyroarid Ly~cb, G.~ Ev~en~c ~ha~"t:d~h4reque~%v~ m u ~ o n ~f~ragaces the phe~ekoryladon ,.~~ p~uva~e dehydrogeaa~ ~ad ~bat the ac~Jvg), or" th~ ~ }~~h~cd/o:m~tochtrndiSa] cat¢i~m ~ u e ~ r ~ i c m . tn A. R o m t e a ~ ropro~ein~: Char~edzat~,o~ and F~mc~on, F ~ . 7 ~rowaing, ~ . , Besmear, W.. Ke]l::a, P. ~ Lynch, 0,~ The ~0.¢~ ~ r bra~n ir~fluen,.~ by high fi-eqnen<¢ ~yr~apt/.,:~imu~atloa i~ .~.~ea~ph~ ~t~bu~i~ ~f p~uvate ~ ]
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~2 Ly: Co Holland, Amsterdam, l~7~,..~p, l~-~2go 13 Lynch, G. and Schubert, P., The use of ~n vitro b~ain s ~ for muR~d~l~nary ~tugie~ of ~yaap~ic function. Ann. Rev° Neuro~¢i.o 3 (19~) I - 2 L t 4 Magi|en, G., G~don, A., Au, A. a~d Diamo~,d, t.;. tae~df~cation of a mi~oehom~M ~hmph~:rc/~im in brain syna~tic m~.~nbra~¢ preparations, $. N , 3 6 {~8|} 1 :.: {5 McHw~dn, H. a ~ gm:heM~d, Ho° B ~ a c . ~ i ~ g and ~he Cen~,] Ne,~ou~,iS~te~, 4ih ~ ¢ - C h ~ c h f l b Livings~o~ae, Ed~aburgh, 197l° ~6 . . . . . . . . . . . . . . . .
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