UCRL-10995
University of California
Ernest 0. Lawrence Radiation Laboratory
KINETIC STUDIES OF THE PHOTOSYNTHETIC CARBON REDUCTION CYCLE
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' Lawrence Radiation Laboratory;.;: ~- ; Berkeley, California · '·\ ·· ·.. ' ...' C~ntract_No. W·74~5":'eng-48' ' . ·( ,t- ... '•'' ,. ·, 'I' ... '' ... -1 ~: ' ;, _,.
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·, KINETIC STUDIES.QF THE PHOTOSYNTHETIC CARBON REDUCTION CYCLE
., ..;.. . ,, . ~- '. J •. A. Bassham· 'I ., . \ '! .; ,_ ... \
;~ _; .... ::: Augu,st 1963 ·... 't.' ·.·' ;..
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1. : ""T'ne preparation of this paper was supported by the. Un1ted States . ' .. Atonic Energy Commission. '
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·,. KINETIC S'IUDIES OF THE PHOTOSYNTifr.."'TIC CARE'ON REDUCTION CYCLE-I.' , ' ·:··'· / (' qc·: \' #' ... • • ...... ~--/.- --1-.~ -:::"1
~ By J .A. Bassham r
La;-r~e:~ACe Radiation LaboratOI"J • University of California, Beri<:eley s Calif.
Intrcduction
The problEm of the carbon reduction pathway of photosynthef>is 1.s
considered to be solved by some plant physiologists (1). The pathv~a:y which is often accepted as being correct is the reductive pentose 9tosphate
cycle, Calvin cycle. or. as I shall refer to it, the photosynthetic carbon reduction cycle (PSCR) (2). This is the only such ,Pathhray proposed
in recent years which is essentially complete 1n its specification or
· intermediates and reaction sequences.
In the PSCR cycle, the first stable carboxylation pro:iuct
is 3-phosphoglyceric acid (PGA),(3). All the r~ining stable
intermediates are sugar phosphates and diphosphates j viith carbon
skeletons fran three to seven carbon atans in length. In the
sirnplest terrr..st the PSCR cycle consists of four stages:
1) ribulose-1,5-diphosphate (RuDP) .is carboxylated to give tvJo
~ . . . molecules of PGA• 2) PGA is reduced to tl"iose phosphate; 3) a
series of reactions convert five triose phosphate ruolecules to .. ------=--·~n#•..,·-=~~~----·=-o=-o..---..--
-2- UCRL-10995
give three pentose phosphate molecules i 4) the pantose phosph.ata
molecules are then phosphOrylated to give RuDP. carbon tfl"lich
.. . enters this cycle as carbon dioxide is la'cer "dralned off" as f
· rt;.duced carbon in the form of PGA or sugar phoaphhte!3 by secondary
photoJ:rnthetic pathways. These pathways lead ult;tmately to the
synt;h::ais of fats, proteins8 · oa:rbohydratos and other products.
1fnere arc some dis3ente.rs fran the acceptance of the PSCR
c·ycle. Stiller (4) bas· written an interesting and provocative
review of the path ot' carbon 1n photosynthesis. She arg:.Jes that
the .'P.SCR: (¥ole is incorrect 11 and proposes an alternative
path (in yJJ:lioh the first steps are not completely defined). )
l'10st of the d1fferenaes of opinion regarding the cycle
revolve around the 1nterpretat1o:l of the available experimental
datali rather than aroun::i tr.e correatness of the data itself. vJe
. have revievved the evidence used 1n formulating the cycle ( 5• 6) •
In the present article, I shall discuss sane .of the questions
which have been posed regarding the interpretation of the datai
and then shall turn to a consideration of recent experi.IP.ental.
developments which suggest sa:1e possible mcchanisLUS of reaction;;; -3- UCRL-10995
of the carbon reduction pathw-ay. The probability of the ope...-ation
j,-J.,.,. in vivo of so.'ria organized enz;r1ilic system will be considered. -~ i . .
Except for the formation of glycolic acid, secondary pathways
1 leadL~J fran the basic carbon reduction cycle will not be
discussed. These paths, leading to malic acid, a.111no acids,
sucrose and pther carbohydrates, arxi various other. products
·of · · photosynthetic oarbon reduction have been reviewed
I shr:;.ll neJ;loot the many excellent studies v1lth tlhole and
broken crJ.oroplast preparations. Such studies are most valuable
·for revealing Yrl8.IlY of the bioch...""mical capabilities of the
photosynthetic apparatus. 0•'ling to the greatly diminished
efficiency of these systc::-ms fm~ C02 reduction, it is my
opinion that little c.a11 be lem"'ned from the..11 concerning tl1e
k~etics of carbon reduction --in vivo.
T ...Jo lines or evidence have . been used. . in connection with.
. . the elucidation o{ the carbon reduotion pathway. The pri.rrr.?.I'y '-' line of' evidence consists of the kinetics of the labeling of -4- UCRL-10995
cowpOUP.ds .in the living cell following the introduction ot""
j I. labeled. substrate. 1-lo. other top}. ha.s been found far. obtaining
I. information about the identity of the intennediates in the
primary carbon reduction pathway or the ~equenc.e or reactions . . '
which link them .. One can and must criticize the metho:i and
be conscious of its limitations. If we were to disregard this
evidence, however 11 we would know alrnost nothir.g about the
·pathway,.
The second and supporting lirte of .evidence oanes fran the
stu.dy of enzymic activity of soluble am particulate fractions
isolated fran broken photosynthetic. cc:lls. The demonstration
of such biochemical capabilities L~ isolated enzymes &iv~e us
· add•9d. confidence in a reaction which we have been led to
pos·~ulate as occurring in a living syatc.T. on the basis of I ..
k:trK~tic data. The .faUure of attempts to isolate ar'S'. or
sufficient enzymic.act1v1ty for a given proposed reaction is not
an oveM'ihell:ning argument against the occurrence of that react:Lon
· as a step m an !!.!. vivo metabolic pathway. -s- UCRL-10995
The fact 1s that considerable succesn has atterrled the efforts
! to isolate from~ photosynthetic tissue of a variety of plants the
various enzymic act1Vit1es required by the :' ... PSCR oycle. Ttds .. evidence has been reviewed by V1sbn1ac --et al. (7). Stiller (4) has reViewed most of the more recent enzymic studies. Where there
has been reported ~~ inadequate activity for a given reaction
from a given orgp.nisc, we should see what this might tell u.s
about ·the .. acc~11p;Li~hment. :.. or· t11e reaction in vivo. Such . --
d:i.sc:repanc:les· sUggest that the organism may have so.nethi..""lg .tn it:;;
nu;J~:~~up ~'lhich causes the disruption or 16ss of enzymic activity
upon atte.npts tv isolate such activity. The proper interpretation ·
of such e~fects can conceivably be ill>eful in underl$ta."Uin.5 the
mechanism of the enzymic system!!:!, Y!.Y£• 'Iha Carbon Reduction Cycle --In Vivo
The carbon reduction cycle or tr.e :~ PSCR cycle is shm-m
in Figure 1. The reactions are the sazne as proposed previously
(2.5.~~6) but the cycle has been redravm to emphasize the relaticns
bet;.;een ketose phosphates, the glycolaldehyde th1am1ne pyrophoapt<£>:t:·~
addition co.npou."Xi and glycolic acid (6) •. The approximate labelinG -5.1- UCRL-10995
ATP ~OH t, Symmetrical ;f ____ _ E "'- Intermediate · · 5 's ~ .~ ~ ~ I al v02 f H CO \ al \ ·tizGOH .. 1' ----- ~E ~ E ' /\, ) -~-~ s-s ~- ---\ s'sH H I I I
\
* i-12GOH• H2COH _*Co~ -I :9o HOCH ro -I HCoH HCOH -I HCOH HCm I I ~ HCOH l-f2COP SOP I ~OP .. t ~OH FOP 'Hco ul ..I HCOH 190 ( ) H'toH H"toH I I HCOH ~OP HCOH I ....9o ~OP i-12COP R2COH _I·- ---7---- ATrY HCO / --~- fe-E-re - ...... _ RuOP HtOH // HS SH ', .L.....:.___ I tizCOP ~ NAOPH '[~.]r~~r ~OP ~OH ...~~
1 MUB-1019
Fig. 1. The photosynthetic carbon reduction cycle (PSCR cycle). Abbre viations include: FDP, fructose-1, 6-diphosphate; SDP, sedoheptulose- 1, 7-diphosphate; RuDP, ribulose-1, 5-diphosphate; TPP, thiamine pyro phosphate; E, E', unspecified enzymes or proteins. The E' (FeSH) 2 symbolizes a reduced ferredoxin-type electron carrier with a potential of -0.4 v. The asterisks denote the order of labeling (not the accurate magnitude) of various carbon atoms of sugar phosphates, and other interrpj!diates, following a short period (such as 10 sec) of photosynthesis with 'Co • Depending on pool sizes, particularly that of triose phos 2 phates, asymmetry of labeling in hexose and heptose phosphates may be more or less than shown. UCRL-10995
patt~r~ after-a few aeo photosynthesis With 1ijc~ is indicated
------/ by asteriskS._ Also shown is tbe hypothetical in vivo reduction of - -_ ·_ - - .- .. - ._-- _-_- -_ ------. \. the carboxylation product by high negative potential -(-O.ij v)
reducing agents. such as reduced ferredoxin. I shall review the
basic ki..vwtic arguments relat1n~ to this scheme. At the sa11e ti.ile r
some ne~-v discussion of the kinetics of published results will
be presented. It \'fill be assumed that the techniques of photo-
synthetic studies'with 14co2 as a tracer (5) are well knotm to
the reader.
3-Pho~:~phoglyceric' acid~.- 3-Pl~sphoelyceric acid (PGA) was I
four.d to be the most pra:ninently labeled cornpound after very
short periods ofph~tosynt~esis 1ri the presence of 14co (3,8). 1 . ' • ' ' 2 I_-
Kinetic_ studies shOWed that when the percentage of ~otal radioca:..""bon ' --' - I /
'fixed "''aS plotted as a_ function of t1me of' photosynthesis with 14co2,
the percentage found in POA vrhen extrapolated to zero time 1vas
more ti"..an 70% (2) and in sane experiments nearly 100~~ (3).
In thOse .experiments f'there the extrapolated percentage in PGA
at zero t:ilile Was aboat 701.; much (lQ-15%) of the remainder v:as
foun:i distributed a."lllng several sugar phosphates, particularly -7- ' UCRL-10995
fructose and ~edoheptulose phosphates •. From the standpoint of knmm
~ \ chemical or bic;>che.mcal reactions, these substances are not likely
first products of a carboxylation reaction. It was supposed th8.t
they vrere derived from the pr.ir."lary carboxylation product. wl:rlct~
W!U'l t.hought to be PGA.
14 In these same experimeilts1 other m:iall amounts of c
were fourrl during the shortest tintes. in tr.alic acid, which was
presu."ned therefore to be a product of a carboxylation reaction.
For a time it was thought that malic acid tJlight be ar1 interrnediate
in a regenerative carbon reduction cycle. HO".,~ver 11 studies with
malonic acid as an inhibitor (9) sho\'Ved that even though the
formation of mallc acid could be prevented, the operation of' the
14 carbon reauction cycle~. as ~videnced by ,the incorporation of c
into the a and a carbons of POA was not impeded.
Such information was obtained from the cheuical degradation
of phosphorc;lyceric acid (9). A kinetic study of the. appea.."'a.'1Ce of
·radiocarbon in the 1ndividual atans of phosphoglyceric acid sho•ted
that radiocarbon. ~ppeared first 1n the carboxyl carbon and later·
equally 1n the a and B carbon atar~B. After 60 sec of photosynthesis ·' .. ·,., ·.· .·.· UCRL-10995
· .111 the presence of f114co3• the distributiOn. of 14c among c~boxyl)
': ....
a;. and B cai>bori atans' of PGA \\"aS' about 1n .t'be. ratio 2:i:l. i
respectively.
Tnis finding had anin1portant consequena·e. It suggested -~
that the a and B carbon ato.113 of PGA are not formed de novo • 0 ' ..,_.. .,.,..._..
·.. : . ' .· . . . . . ' . ' . . .. · from co?• but rather ~that tk'lSy ar~ derived. from.the carboxyl . - . .
carbon atom via ·a.. oyolic process~· Only the aarbOXyf· carbon
·. reaction.o' ·
. . . Th~ possib:tlity that unstable intemediates proceed the...... : ' . ' . " . ' '• '
observed stable'products of carbon fixation long has been . . ' ' . . . .
recognized. A careful search (10~11) failed to reveal the
· existencG of such early unstable products to date. Hhile by
definition the possible existence of such uqstable intermediates
. I' never can be elim1nated completely • certain limitations can be . / ) I;
. . I . . set upon the concentration and stability of. such subs~es by
tl''..e experimental .results thus far obtained. . .
' . ·. ; . \1e have b~en able to show by· means ,of kinetic studies (12) ,. . ' ... ·' ...... , . ,y UCRL-10995
4 . ' . or ~he photo&$tnthet1o fixation ~f l co2 during steady state ..... ,_· ..
. . :~ '
..• ... \ ...... : ..... ,. ·.,.' -'~~ ., ··:,:.·,·more ~han l.5 mt~ol.ea· of carbon--Pel' cm3.or Chlorell.a aelb,- !::; "'\ ,;: 1:~/'f:J'·~~~;i/. ~~. •' . 1 :. ~- ·.~. ':·_ ._· •. ':', _.,: • !l: .:. ' .. • . • . '·::. _,·;: > < • ,!:·j/'. I • . ~ • · praiueta ot fixat1on.·'Unier the ·expeir1mehtal-oond1t1ons used ·'· ;'' . · ~~ ' • • I • .. .. ,• :· this was equivalent~~ s-z~.or-~~~~thew._ In ®r .: · · . ::· ·y:):~~-f>~_-)m;lli::!l!;:l!!; 1 :j}:j~?Lr :. .. ·. _- . · ,.;.' . .··~pWOn -the most reasOnable aaauq,t:!l.orl' asf~;~::tbe :nature or most :·; _ _ . ·· · ·· : ;_: :::;:::;r:lill::;!l1ll:tt}f]Y!!::V. · _ . . />J1V ... _,,,,.:~-,,~.O:.thls =~-~~~le~ ~ c,: -~~~~--~~j;~JV",a pool or_·_', . -;~~~!~;-._- i 1::;_;:: :· ·· _.... ;, dissolved carbon dioxide. 'ana biellJ,r~~t~:;,~~~~ ,the CrJ.orella <:ells•::'Jii,' , t ·•. 3 . , >_' ______, ,: •. !:~i:t;' :l:~ ~iii i ! :llY~ , . :~~;(;;_ii!: - .. _. -) j ·- "· ·- · .. :In tne same report• .we .l1$ted · ~~adY: ~~~:e concentrations. ~ Jii~·H!:::.,.-~:·.<_.,.,;;.-- · :· J~ 1 ~:.· : . . · -. . .. . _. . . _. _ !:;;l~il\;::: ;irll:i:·:ii;i:%:1H 1 :1J::!:;:i . ·. , . ·: --" .. ~<:~·~t::~~lf~i:J~r-->·.:_·, . · ...: .11 .··_,, '. or a tltUilber ot intemediate oo;ar.io~::iot''i:~l:>.$:i:cSl'bon redu<:tion ~ ~:: ·;;;.)!:!:;: ·· · · :f 1 "i • · . -···. . · :~ :·~,:.U:r;l::t!t:m:.1:l;i lr]iil1-iilli·l:li:pi;1l!;(. . . ··.·.. ·:\:._·::-:- __ ;::\~l1t:·. . · :1 . •·(.,:. : cycle. These concentrations ·of. active;ll:l:t!Jriidii$\0\'er pools were.,. ,-_-,;·.~~~:, . _·-·. · : .•. :l . ·.:, >~~·by·~ t~c~que ;s~~~g~~~li!L .. a:~~. :h[ ·---. __ -..• _· ~~~~ , ' . , ~ ' " , ; , '!;· .';;, :'iq • ··~~i:'jll,l;;.J '·i ' · • ;: ',: ~ I • •' ' ·, '; , l 1 · , • • - • ·' · ~- , ,._ ·:_ -~· .•·}rl;j(:; ~( 1 !:::;l!il~;;:,i:n::lJiiiili,·U1:;Jm:l;);;: ;. . · . · · , :~ ... ·: · · ·.. -~--·-.·.~ .::::~~<-~:·. .. . -- -- ·- ·~ .. i 1 with}4c ·during steady state p11o~;;}~~~~~i~ l4co2 ot'eo~tant y:~1'}i.: -· :) :::!]__ 1~- '> ... _ ... . _ ...... ' :·o::tr.i ;~_:::;~~!!P:ntf~W~1~i;l:m\l!:;;J\;i!(- _ . : ··,... .·.; ·:_.~>;:~>'~"-:;:\;!;;·:.; ·. '.- ·.. spee1fic radioactiVity (S) (la) •. : -~Jth~;:·~~~ l4C, 1n .ea0h' ,·. .:i;:i·. ccmpourxl $t satumUon (A),. (C)-"~: 2i::'~j 2:~ eooh , ·":: i~:\;j;: ' . ' ' ' i.. . ' _,t't'' '1'. .:- l' . . . / " ' : . !' ,, •·. -: .. ~-·· :::;~: ·.. · << :'::;(,·' :", :(:.!;: w);_~·. : .: :. .~. . . .. ; . . ' '.; ;:}:'. :: ' ,- .reservoir (ill units .sitch'aa ~~lSs/cm~:~et Pa~k~-~) can ··:::;F · --: ·. i"'·:". ·. . . ;.'·': .... ·.·.•;;' ··,;' ' . . ·\;·, ·:~~'.<_\t;t.·-c ~i~J,~ . : <::; . '1 ::,··:..: .:·- ... •:;jjl..:~'. :··" .-~··'·;;~'.:b.~ aaJ.oulated_aooOrding .to.t¥ ;~~;.Q,,~;~.\!s·~:A·~~l~.o~suc~:.)l\~~'· •-·- ,_,_ .~:e#:< :_;,~:7~::,;:~,~ :~;(;:~2~~~~{-~::~,;~ ~)l· 1~~~.· -' - - -- :- -_ -_._ ,':,; ,-;'\:r ::- ~· :',('J{tsr:: ,: , --- ·:~: ·,;l ,;;::Jr ., , •. '~- 'i J_:{ (hj ·;.·. •1: '. i '·
•.. · .·'· ' ' ''. In the exper~nt Ju~t quo~ ,Cla)"ttte h1te ot now .. (~) .ot···:·\ ·' ,· ~. ' •. :!' 4 • : • • . • '. . • • ' • . • • .; '' -~ • ' • •,!· ·. . .. ~- . . . • . : ~- .''' . ":, i;.
),'' ,., I
-.·"',.., -.:.
·. ~ ...... '· ;_,
'· ::" •\ ·; 1 ",.. { ; • i r -~-.~ ;.. ¥<: . J::;_;;·-·· : ...• . ' . ;;~ ...... ' .. . . . '.; ' -'. . . ~'
·~~ .. ~: ·t· .· .0.,36 micromoles 1 or.. o. 01, tnioromoles per carbOn at~ .. ~itfon• while .·,' ...· ...... · ···-;. . 1··.~k~:·;~~r . , . . ... _· , .. ;, . . . .. , ~i· .·. - .. :·· .. ::::~: :;,:...... ~· ·:·'J .'.· .. ;·::.>.~:.<~~k -.. ~:.'_: ... /}f:t\'·::;,·: ·.. the corresponUng values ror PGA.were 3,0 and l.~o. ::re.speotiveli~ • -~. ~ ·j ;~(~;·· ,:·;I':-/ -~ '.. ! ·-,-~·:'I :,..,i" .. · .~.·· . :~.:":; . ., r: ,> '·1 •• ') r.,· .... ·.- '~ ·, .? ': ~ ,.
:l -~-. ,·! I. ,, '', ,: It is a o~ ~t st~•s ~'·~~· l4c .., !~ ., )• '·l; ... _•.,._ ,. '', ~ I • • r-' ,/· '• . \ ; .. • •, :; ; ·r~~~·~~~:.·~.:.~;i.7i~;::~::·~,./->:~.1ntrcduced by CarboX1:lation of-.RuOP would .pass throUgh PGA carboxyl. ·ilj ·. ·>_· .:.}:~-t~~f;}~J;::~?::;· ~~-~;:f~ t· .· ... :~.. >' ~.. ·. ·.• .•.. :~.;: '.·. c: '>~i.:~ :>·.:f~.:. ' :·_. ~"; :.~-~.': .:.~}:::_:~{ ·. ' . ·:: .. f'::):'', :.>, :. ' . ~. '• :'-~~-.-·.::.t~{'~,;~ ... ·:;.:,.·;::-: t •.• ; trioSe-~ one .earb~·Rul)p carbori'atem·~· 3:··Qi'ld· then PGA -~ ~ - ... {. .. ,, ~ . ~·- .. ,_, :· ;· .. ~-· ~ ~' i •· • ..,..;,·_~· ..• ~:'".c ..
;.. /:... i ,. \ .' • ~ f. .r:· . . ~ . ,.· .\ .. '!·' -11- UCRL-10995
·}.
tbroui;h the "~~table pool" j thence through RuDP c&""'i>on atcr.ns
... l and 2; the C4 and· s carbon atoms of PGA, car~on at'o.w 2 and
3 of triose phosphate, carbon a tans 4 ant 5 or RuDP and back into
the PGA a and B carbon atoms.
Thus the carboxylation carbon passes thr"ough one set of em··b;:J;,
atcxr.s ~~hose total concentration we ca..'l. denote by c1 and is equal t;()
1.0 + 0 .. 27 + o.or = i.34 micromoles. ?ne "diose" carbon pa:::ses different through ,a. I set of carbon atcm positions which l>Je can denote by
c2 3. Including the unstable pool (\>vhose upper llrrJ.t in size 1:) p ,· ''
L5 miCrol1lOle.s), c2.o3 is equal to 1.5 + 0 .. 14 + 2.0 + 0.54 + 0.1~ =
4.32 m1cro:noles.
A precise calculation of the degree of laQeling, particular.~1y
at times leas than 30 sec, would l"'equire consideration of each
and co::nponent pool in c and c hmuld be rather complicated, due . 1 2 J 3 I
to the cyclic nature of the process. \ole can obtain. a reasonably
good approxination of the average degree of saturation by 60 sec
of c and of c by ~reating them~ single pools. The degr-ee of saturation of 1 2 3 ' , . . UCRL-1.0~ 95
By a. similar calculation degl."ee of satUJ:1at1on of c 1:-:: O.,BlL; the 2
rt· tnen 'foll9\>1S that the degree of saturation of ~he entir''~ :FX~A
' ·' \ - . - pool at 1 min'wouJ.d be [0 .. 95 + 2(0.84)]/3~=·o. 188. It can be calcll.J.a.ted
from e1.-peri:nerital data in referenc~ 12 (at 59 sec) as 2.29/~ , = o. ?8.
The deiVee of satUr-ation of the ribulose diphosphate11 by calculaM::m
!
whereas the calculation fran tha ,.exp':rirr.ent is (at 59 sec)
0.225/.36 = 0.625 .. · i.
A slovter rate of eatu..-...a,tion of PGA and of' RuDP. \'Tould result .
,) .
frorn .the operation of t~ f P?CR.i~i C:.ycle because of the larger
total pool. of carbon in the r_PSCR :;; c;ycle. The total concentration
. . of carbon in intermediates of the .. c I!SCR; c;ycle in~ the above ,, . . exper.i.'nent is C "'~' 12.2,. ·When R· = 12 and t 1:11 1, x = l - e-1 = 0.632.
This average value for ail the carbon positions 1n the cycle is
thus in good agreement \'lith the experimentally calculated value
·for RuDP (0 .. 625). The carboxyl carbons of PGA will be nearly
; saturated by one min so the estimated value f"or the three carbon \i
. .· atcms of PGA would be (1 + 2(0.,632) )/3 = 0. 755. also close to- .
the experimental value .of 0. 78 o These agreements between calculated -13- UCRL-10995
CL'1d experimental results prooably are better than should be
·eXpected, copsidering the approxi:natio."1S used.; They do
illustrate that some cyclic pl~cess, involving intermediates or
substances in rapid equilibriunl with intermediates, whose
total concentrations are about those of substances involved in
the . ·. PSCR cycle. is require-d to fit the available kinetic data.
In her review ( 4), Stiller asserted (in support of her
proposal) tr~t our studies (12) indicate that "the carbon
atans of pentose phosphate nttain a higher specific activity tha11
those of the phosphoglyceric acid !'.rom \'lhich they are presumably
deriv<::.' : . . 14 .beginn:inr; of tr.e period of steady state synthesis with co2 until saturation of the intermediates the specific activity of the .PGA was always considerably hig.'ler than that of RuDP. ' :, ' / · If the carboxylation of RuDP results 1I1 the formation of ti'TO molecules of PGA, then the ca!'CH')Y".Yl grou9 of 1 of every t'iiO PGA molecules will contain t:r.a.e nei'rly incorporated lllc. 'ro test this modelj \ · an. amount or 14c radioactivity vlt'.ich 1rould cor.raspond ·to the r::1dio- · UCRL-10995 activity expected in this carboxyl if the mcxlel l·lere correct. Siooe . i. ·' tr.d.s Carboxyl 'goup wbUid rapidly saturate 1f the model were correct$ tb.is involved subtractinS l/2 x l/3 = l/6 of ~he saturation level . . . . . ~ of radiocarbon in PGA after about 30 sec. The remaining c which would have to be derived from the RuDP carbon atan.s was compared v1ere found to be labeled to a higher degr-ee of sat\l;I'ation than the average of the five atans of RuDP, suggesting that this model (2 molecules of PGA per carboxylation) was 1ncorrect. Using a different ~el L~ which only one PGA molecule was formedj and in which it ·was farmed from the newly mcorporatecl 14 . .. . co2 and carbon atoms 1 and 2 of RuDP» a si!.uiJ?..r calculation shot'it::!d that the residual carbon atoms of PGA were not .labele:i mare rapid~' tha11 the average of the five P.uDP carbon atoms until after 50 sec. In this case there "~>Ias no contrad1ctio."1 bet~reen model a.'1d data even after 50 sec; since we kno\'l tr.;at carbon ata11s l and 2 of RuDP are. more quickly labeled than the carbon. ato:ns 3, 4 and 5 r:: ( 2) • From this data and reasoning, we concluded that the carboxylation of RuDP leads to only one c:olecl,lle of PGA in equilibrium nith the. PGA pool. The other three carbon ato."!lS from RuDP appear to have been -15- UCRL-10995 converted either to a form of bound PGA not in equilibrium \!lith the pool~ or\ to some other molecule~ We speculated that if the .. in vivo reaction were reductive, the other w~lecule mi~~t be -- ' triose pbospr~te. '• It was noted earlier that the radioactivity in PGA does not alv.rays extrapoJ.a:te to 100% at zero t1.11e (2). Sometimes such extrapolation gives 10-15% radiocarbon 1.'1. suzar phosphates at zero tirr.;e. This flrrling SUf!Eests that it is not only the tl".l!'ee I ' carbon moiety derived fran carbons 3, l+ am 5 of Ru.DP 1'tlhich may not be · 1.l1. equilibrium 'i'rith the PGA pool. It appears th:it some of the PGA labeled i"lith the ne:r1ly lncorporated 11~c in the carboxyl group is also bouroP pe.rhaps to an enZ',J1ne, an:l converted to sugar phosphates Vii thout freely equilibratint; ·,.Ji th the PGA pool. Reduct1on :of~PGA to ~r phosphates. Calvin a.'1d Benson (13) proposed that PGA is reduced to triot')e phosphate and. tl1at triose phosphate then condenses to ma.ke hexose phosphates. 'Ihls pror:osal was supported by their identificatio."l of PGA, triose phosphateP and hexose phosphates as labeled products follO'v\ling short periods 14 of photosynthesis i.."l the preser>.ce of co2 (14). The reduction of' PGA to hexoses via a pathlilay roser:1.'ll1n;!: tl1e reverse of glycol:ysts \ft.::> . :. -16- UCRL-10995 ind:Lcated by degradation of hexose a.l"ld of PGA from the same e;cperimen~ (3) ~ For example, after 15 sec photosynthesis • ·\ l • . • by barley the distribut1o~ of ra1i~arbon 1n. the carboxyl a~ a· &"'ld S carbons· of PGA was roughly 50%, 25% and 25% raspectivcd.. y, also 50%, 25% and 25%• respectively. This degradation of hexose derived from sucrose did not differentiate bet\ieen the carbon atoms 1,2 and 3 of the hexose ;'f.s canpared with carbon atans l.i,S. end 6. The Gibbs effectt bad it beE!n preseryt, t-.'"OU.ld not have been observed. Kandler a."ld Gibbs (lG) later ! ,4 found that following pbotos;ynthesis Wit~ • .._ CC2 ~ both glucose phosp1·nte and the glucose moiety of. starch \'iere asyrrmetrically labeled. carbon atom 4 was more highly labeled than carbon 3, while carbon atQ!".2 1 and 2 lm~re more highly labeled .than carbons 5 and 6. It is not difficult to propose a ·reasonable aA-planation of this Gibbs effect which is consistent with the .PscR·: cjcle (2:5) • . ·. The difference bet;·reen 3 and 4 could be predicted .in terms of the . . ' PSCR · , cycle on the basis of the !i'.easurable pool of dihydrox.,vacetone -- -phosphate. T'nis pool dilutes_ the tracer caning into it. rrhen phosphoglyceraldehyde is converted to dihydroxyacetone -17- UCRL-10'J95 p!10-3ph.ate. Dihydroxyacetone phosphate then reacts "td.th re\••ly foro~d phosph:oglyceraldehyde which t.as a higher specific i~.ctivity. -'·· ·... : ;~ ,._,.,; '!'his reaction results in the fomation of hexose rnore hi€)"lly .,. labeled in carbon atcm 4, derived fr011. the phosphoglyoeraldeh;;de ~ than in caroon ato"!'.s 3, derived from the dih.ydroxyacetone phosphate (2) {See Fig.-1). rrhe higher labeling of carbon atorns 1 and 2 as compared with 5 and 6 can be readily e:~>plained in ter:as of the reversibility of the transketolase reaction (5) (See Fl,s. 1). Careful consider.:itlon of both of these explanations will sb.o-;.1 tha.t the size of the effect or i!".deed -its existence will depend to a large extent on pool size5 arrl the rate of the net for1~ard reaction as compared with the rate of the reverse reactions. It in not surprising there;.'ore if sornetiri'.es the Gibbs effect is cbser-ved and scn1eti:-nes it is not. It is· true that we bave repo.rtc' synthesis with Sc{~nodesmur::, the fructose phOsphate \,.as symmetrically labeled ~Jith most of the radioactivity being in carbcn atcms 3 and 4. It will be noted, ho~:evcrj tr.at sedoheptulose phosphate obtained fro~ t:--..at san10 sa:nplt~ containe:l mozt of lt:.> radicx.t.ctivity equally -18- UCRL-10995 distributed between carbons 3,4 and 5 .. Since carbon atcms 4 and 5 · · or sedoheptulcse ?hOsptv"lte are d~rived 1l•Ot'1l carbons 3 ~nd 4 cf ... }· in In the srurJ.C report we fOl.md tr1at; sedoheptul030 phosphate ..' ...... • • ' .~ '.. ; ~ • j ; ·.·. short tirnes (est.; 0.4 sea),cai'bonatan 4 conta:L•wd only 8% \'}f ·.the rad1ocar•bon while the total of ..carbons 1,2 and 3 >:iJ).S 33% and the · · · (from other\ result~; in that report) total or carbons 4,5 and 6 ~.,;a:s 57%. It is evident/ that carbon · 3 by it~elf eotltain.ed most of' .th<·~ 33% and Ca,j,4Jon 5 cont.unad 57% carbon atcxns. 4 a."'rl 5 derived tro:n cal·"'bon atoms 3 and 4 of fructosti" phosphat~ accord.ing to the PSCR . cycle v;as observe-d. Howevsr; in that particulal" sample 11 only the t;m halves of the h;;;xose ·-,. were compared. in degradation stud1e5 and We.J.""e found to he '· approxima.tel,y ~..}~l. r.r carbon atom 1 a"ld. 2 are more labeled ' than carbon 5 and. 6, while carbon a.to:ll. 3 1~ less labeled than \,. I I,_ carbon ato:u 4; the two ~~lves _(l + 2 + 3 and 4 + 5 + 6) ·o~.·hexose can be equally labeled fortuitou!lly. ~while St.llle!• ill.d not ·dist::uss the labelln;:;; or se<.lchcptulo.;;e -19- UCRL-10995 phosphate wrJ!ch would result if her hypothesis were correct~ it can be re$.dily predicted. It is a consequence of ·r.er hypothesis that carbon atoms l. and 2 of pentose phosphate rust altiays be •' equally labeled. Sedoheptulose phosphate fo:rroed by transketolase from such a pentose would therefore always have carbon ato:ns 3 an.d '· 4 equally labeled. This requirement is clearly contradicted by our published dev:oada.tion of sedoheptulose from the 0. 4 sec soy bean le~l.f (2) (See above). In tr..e study or the kinetics of 14c labeling of interme-diates during steady state photosynthesis (12/)15,17 ,18) 1 tvfo classes · of co.rrpoun::ls can be distinguished.. One class of compounds includes those \'fbich becane saturated with 14c \llith1n 3 or 4 min after 4 its i..Yltroduction as 1 co2• Tl:is satUFd.t1on is the result of the fact that their pool sizes are relatively small and that they are for the most pa.."'"t intermediates in the basic cal... bon reclucttort cycle so that the net rate of flow of carbon throur:.h these· '' - compounds is large. All of the. interm.;.;diates postulated for the PSCR: 1 c}ycle plus phosphoenolpyruVic acid and glucose-6-phospl"Jate fall in this category. -20- UCRL-10995 A second class of co.'TipOunds includes such substances I I . ·as malic ·acid, sucrose, alardne·, etc., which do not become I saturated \rl.th lltc for uany min 'after its 1ntroouct1on 14 as co2• It. is <;:lear that these compounds cannot lie in the cyclic . path of caroo:1 reduction. No 1ntenned1ate in the cyclic path of carbon reduction can become satur·ated bet''ore a'1.y other intermediate. It is tr-J..e that a subFJta.nce such as phosphoenolpyruvic acid or glucose-6-phospha'ce ca.'1 be in such rapid equilibrium Hith at1 .it1tennediate in the. cycle that it ca'1 saturate at essentially I I the same rate al3 i..nterm~:xilatf~s in the cycle. It would be possible that by tl:"t.is criterion fJ."'Uctqse-1,6-dipbosphate is not an 1.nterm.ediate in the cycle ( 4) but mer.ely a substance ·in rapid equilibrium ·~vith such intermediates as triose pl10Si)hate. However,.. if trus were the case, one must dis:m:tss. ttle argurnent ·-. that there is: an 111.0.dequa.te a'110unt of enzymic actiVj.ty such as aldolase to form f'ructose-1,6-diphosphate at .a rate consistent with its being an intennedia.te in the cycle ( 4). As mch enzy-r;Uc activity is needed for the rapid equ1U.bration of a co:npound i"''i.th I l ' . : to be itself al'l intenne-J1ate in the cycle. ./ -21- UCRL-10995 ·,"' I ·J) :.: As 't'le pointed out ten years azo (2) there ls little difference in the rates of' labelifi-S ~-aong the vurious intermediate;u. in the cycle except PGA~ The more rapid labe.llng of PGA is of' course due to 1ts carboxyl group vihich contains the moat recentl,y incorpor-ated· carbon. Thus except in the case of PGA, the position:>· . of intem..ediates in the cycle 1t1ere not determined by sequence of labeling of the 1nte:rmed~tes as has been sug;::;e~ted ( ~). The sequence of cycle intermediates vtas determined by degradation studies~ transient chans~s in concentrations of labeled c~~~ounds . . ' and a consideration of kno•vn biochmniaal reactions (2"5) • Conversion of triose phosphate to pentose Ehospr.r.tte. The degt"adation data supporting the steps given by the PSCR."'.::cycle for the convemion of five triose phosphate molecules to 3 pentose phosphate molecules (see Fig. l) have been amply discussed before (2b5.~~6) o1 A detailed diagram of the distribution of label through the · PSCR ;. ·, c·ycle was recently ty~bllshed (18). It has been suggested .sornetl.TUes that sedoheptulose-1. 7-diphospYate is not an interJ"rLodiate in the. cycle and that sedoheptulose-7-phospr.fite UCRL-10995 .•. ,_ \. postulated intermediates· of the\ PSCR cycle) during steady 14 state experin:~.ents with co2.. I11o.t•ecve.r, like these other dip!losphates; s~:doheptulos~ diphoaphate sh~-:a a t"apid transient 6.rop 1.'1. concentration 111t~n the li$i1t is ttll"ned off (20) .. necentl~l' t:r:?l.. n.:saldolcJ.se activity is ti10:r'e difficult to fin! in adcqus.te anKY.mts in se 14 .· synth~si~~ .in the pr~sence of co2 point to a lisht-stim..llr.ttC;: earlier, Calvin and r.assi.-r:U. (15) found that upon turning off the light, the concentration or ribulose diphosphate dropped rapidly . . \ i to iero 1n contrast to the behaVior of most or the other su~ phosphates. The dark-i.."'lduced drop to zero in·RuDP concentration haS been observed 1n subsequent studies (20,23), but in one of these (20) fl"Uctose diphosphaw concentrations also dropped .close to zero. 'I'he drop in concentration of RuDP \'laS attributed to a cessation in its fo:r"iletion in the dark fro'TI ribulose-5-phosphate by a ~:inas·~ reaction requiring ATP producej by 11&1Jt reactions. At the sa1Ile tirne. the carboxylation reaction re--y,-uiring no lib}lt-produve:l COfactors \'laS presuzned to Oontinue in the dark:, t!"..US depleting tpe Hu.DP. The carboxylation reaction .. Calvin an:1 ~{ilson (17) found tbat when the co2 pressure irla.S lcr.·mred to a fe~·; thousandths of a per• 14 · cent follotdng a period ot photosynthesis with co2, the most 14 no ticeao · 1e t rans1 en~.~.. ch langes 1·1ere a rap~"'d r is"'e in c.-rt "'uDP · a'1G.·· a· .' . rapid drop in l4c-PGA •.Tl1is stron[;ly sugg;,~sts that RuDP is the . . , . . . ~ carbozylation subst,..-...a_te in the reaction leading to the formation of ?GA. It ·.~as mentioned earlier th-J.t several studies (3,20,23) of the -24- UCRL-10995 transient ber..a.vior of labeled inte~ates follcr~tring turning I \. . . off the light showed a rapid rise in PGA concentration al'Jd a . \ rapid ·drop in RuDP as t>tell as in :eruc.tose and· sedoheptulose diphosphate •. In an attempt to determine whether one or tv10 molecUles of· PGA t'lere fore.ed per carboxylation reaction, we studied the rate of increase of PGA after tUI"n.1..'1g off the Ug)'Jt f.oll()Wing steady . state photosynthesis ( 23) • During the fir·s t ' 10 ;:;ec after the light ·vtaz turned off somewhat more than one · molecule of PGA appeared per ·molecule of H.uDP which disappeared. This \>las tair..en as evidence that in the dark two molecules of PGA were formed _pe~ carboxylation of RuDP • ., fn an effort to learn more about the role of lieht-produced. cofactors in the £!1 ~ carbon reduction reactions.; t'le nude a kin;;· tic study of the synthesis of frorn 14 by Chlorella in the COhl~)ound.s co2 · dark folla.\'ing preillumination (20) •. Benson and Calvin (13) had fc~.md that the dark fixation of _14co2 by Chlorella ·is greatly sti:r.u1:::tE~d by preillU11li..rs.tion. They found that principal. products after 15 s.sc in the dark following preillumination ar~ PGA:. sugar phosp1-.ates, malic acid» alanine, and aspartic acid (14). · -25- UCRL-10995 to reach a:steady state of photosynthesis with unlabeled. carbon. T'c;en, without interrupting the flail of carbon dioxide, we 'Gurnf~d off the., light an1 !rrmediately SW"ltched from ordinary car·bon Some of the results of this experi.'Tlent r:_aJ be seen in Fig. 2. There vras cu"l initial rapid uptake of 14co?.... during the first 15 sec • 14 m•;.:.nen ....v•lere .occurrc.,.A.. a t ~aporary 'd ecreaBe 1n c· • Pr.. esurnao . 1 y some o1'' ·the newly incorporated l4c to1as respire(l. After- this drop, a slight > 14 loss of newly incorporated C in such products as sugar mono- phosphate occurs during the period from 15 to 30 sec,. Thereafter; 14c in these co.np(.)tt.nds remains es:Jent·J ·~ ·11y co:Lst;;.nt •. Tr.U.s. behavior strongly su&:;ests a mecr.tanlsm whereby tr:e ne;r.rly photosynthesized sugar phosphates r..re prevented fro:n b~lng oxidized by respiratory reactions. Frcrn tho usual concept of the PSCR cofactors were er..hau.sted it:l the dark l> the triose phosphate dr;;:bydrogenase reaction ~vould be reversed and the newly fom~~J SUf:P-l" phosphates rapidly oxidized. -25.1- UCRL-10995 14c Fixed Alanine 14 Time after Addition of co2 (sec) 14 Fig. 2. Fixation of c into compounds in Chiarella following preillumination. Sugar monophosphates include monophosphatEf~ of fructose, glucose, sedoheptulose, ribose and ribulose. "Total C fixed" includes all radio active compounds, including those on the origin, found on the chromatogram. -26- UCRL-10995 Another very interesting point abOut .these results is the rapid rate of labeling or sugar monophosphates· between 5 and 10 sec 0 This rate is eq:\lal 1n 1.ag."litude to the rate of labcl.L'1.g of PGA . . . . · d<1St.Jlte :the fact that at that M.me· the ?QA carboxyl gt">Oup can in all probability be no more than 20 to 30% saturated (l2) .. This appears to ,be: the mest direct ev1dertee yet ror the reduction o.f some bou."ld forill of newly incorporated l4co2 not in equilibrium nith the ?GA pool.· ·I. i These results are not, as it might at first a.:opear fi inco~~istent ~~th the rapid .increase in Blli\ cor~entration in the 14 of the c saturatect pools .. l:i the pre1lluraination experiment inter. 14 the c label.:ttlg of a pool. ~'he size o.t the pool may be cha.."'lginr_.:; 14 . :l,n the sa.,-e ·or opposite direction as the C laoeli~'to ~rhe t<'!O experiments are consistent if one ~')poses tl"lat t,;hen the lir~1t ia turried off AT? is the first eofector depleted leading to an .1rn:!leu1ate or.set of tt-.£: drop .tn T.:DP conc~ntratiot:. ~t.V1d UCRL-10995 :h11mediate cessation of reduction of the free _PGA. One must at the ' same time_ suppose that the reduction of so:ne of the bo.md, newly 14 _ incorporated c ("bound PGA") proceeds th..""'Ugh the mediation of ~~ stronger reducing agent than r.t.ADP; since no ATP is present. Tllis could be a reducing cofactor of the strength of reduced PPNR (chloro- plast ferredoxin) • The Carboxylation EnzyrrLS In Vitro Carboxydismutase- (RuDP carboxylase) converts one molecule I j ~ • ,, of RuDP and one molecule of _C02 to t~ ( 24-26). The enzym has been prepc.red from intact chloroplasts by Lit.tleton and Ts 'o (27) and by Pon (28). P...lthourr)1 ma.r,y different procedures have been employed for the pur'ificat1on of carboxydisnutase / (25;28,29); only abwt 10 to 20 fold increase·.in ,specific activity of trle enzyme has been achieved (25~28). Ultracentrifue;ation of .i ..... purified carboxydism:utase haS often given t'r.fO protein components .. One of these components with a cedll~~ntat1on constant of 18 S to 19 s constitutes the major part of the protein (25:~28u 29~ zn. ':'his fraction contains the carooxydis:iiUtase activity (29). Ultracen.tri~tion of sohtble leaf protei'fl.S-'(30-32) g:::rm~ a. '\' ; ., . ... ,;- ·.'i- 1 . '' '.- • " :--., •· UCRL-10995 Unstable Fi~st ~oducts Moll~s an1 Calv~ (38) 1Bol.E:..ted tv.ro co:npzy.mds tentatively id;crati.f.ioo ! ' Shkolnik and /Dor;JZU"J (f.IO) reported 3everal yoru... ~ ¢1.2.'0 tt10 l'oraation. of an unotabl.e ., terr~J,ie:t"ature k11llns of the plarit uzed and. lo••i teriper·atur•e cra"'matogx•a;;hy .. te:upe.~,"'"ature.o Further report$ Ofl tf'.e id<"::ritity an:i kinetiC behavior Of ,· this co.r:~pourd are· awaited \-lith ~~~t; intere:.:;t. -30- UCRL-1.0995 Galmiche ( 41) reported an arelysis of' the prod~cts of a fe¥1 i i· ' 14 sec photosynth~si.s with co .. In his experi.rrients, the plant \'iaS . \· 2 ldlled in isopentane at -160°C and extracted in the cold \iith the · aid of forrn.amide. T'ne extract was analyzed. at 0°C Hith ·, hipJ1 .~ voltage electrophoresis .. \11:"-.ile ~l"'..s identifications are reported to be not yet completed 11 those compoun::ls wrJ.ch were identified include<;i, in addition· to diphospho;J.yceric acid and 11a:nino 11 acid phospr..ates , compoun:ls \'lhich have been reported in previous studies ~"lfith 14co~ fixation durinu; photosynt~esis;. in ·. ' vib..ich the a."lalysis \as perfo:r>m.ed by two dLilenzional paper cbro:n- atot,Taphy.. Thus j) the gentler methoo of ancilysis has thus far not revealed any ne~i radioactive ccrapounds that are likely to be unstable first products. Inhibition Studies Kandler and Liesenkotter ( 42) have reported studies on the i fixation of 14 during photosynthesis by Siti co2 CP~orella chloroplasts in the presence of various ini".tibitors. Studies v;ere also ma.de viith labeled gltlcose in t.t.:.e ]j4j1t and :L."l the dar.i<:. Ti:.c:y · reported that loiti co~·:centre.tlons ( lo-5 l:•I) of monoiodcacc~tic j ' / UCRL-10995:: I • 1, . ~- ~ .. ' . ·i. l ': I , .. · ·_ . . .: · ··· reduction in· Whole cells. They consider«i tb1a aa evidence ..' . '· l ~ • . . . ~ . ; . • ·!' . .' 0 • • • • • : :;· .-··:~-( ': '. • I , ;,; i ' i , ; :."<•'. ,. ;. ' ·::/~:>;; >>· · ·· . that there wdst.a & pat11. of carbon red.uetioh·Whic~ b~ses ., • : ,· ' ' ~ :· ;. • .' ~ ••. .1 -~ . :_· • ~ . -r!-~-! , ... _-. ·.. ·. ... '.', "'\: ; . ~ -~ '' !· ' . '•' t . { ' ' ' ·' i·· i·· ' . . ·r •• ·'.. lo • ,• :·. '· ·' ' ' ' · · '· A basic drawback _to ma&t ot tneae · ~>erimente seems to be ' ' .. i. . • .. l.' . -:·t:r:.• : ;. '''i i ··'I I· i, . i~ • ~ <'.: • • . • ·.. ·.; /'!> c.:lla with the· inhibitor. Thus ths chango$ 1n labe~ ~oduced . '' ; ., by' the 'inhibitor are sanewhat d1i."'f1cult to interpret. ' • • ' . l• ; ·. -: . ..., ' • 'r, -· ., ·._.; ' : , f : \' ''I .. ( '· ; . '. f . '. :'' · '· · ~ ._ ~ · . · · ·. · ertect ot KCN on the. 1ntenoodiatti cooo~ntrati'ons :4 O.hlorella. . ~: ' ' 11', _.: .\ '!··. \- \, .' . \.'· .. · r ·. '. .: ~ . ' ~...... '~ ... I .. · ~\ .- . ''.' , ·''. .. .. t:·- .: f ...... ~ .. . -33- UCRL-1.0 995 PGA rray be reduced by a reversal of glycolysis. They further . ·; sugeest that\, the origin of the free PGA pool ljes in t;lycoly~ls of labeled sugar phosphates formed by photosynthesis. The:,r suppos ~ the rate of this glycolysis to be accelerated when the l16f•t is turned off due to an increased supply of inor~nlc phosphate. They postulate that co2 deprivation causes increased birding of phosphate leading to a decrease in inorganic phosphate ani an inhibition of glycolysis. Trds would in turn result .in a decrease in the level of the tree PGA pool. In view of the kinetic experiments already. mentioned (2j20) the direct reduction of some neHly incorporated co2 to the level of sugar phosphates without eq,uilibrium \ilith the main PGA pool is an attracti,re hypothe::;;is. Its attractiveness does not depend on acceptance of the idea that the main source of the free pool of PGA is glycolysis of SU§ar phosphates forrr£d directly from co by photosynthesis. The free PGA pool mig.ht be derived 2 from the "bound" PGA. An explanation of transient ef.fc..:>Cts on PGA pool size based -34- UCRL-10<)95 · upo-a the level of inorganic phosphate is intrigui.'1'J;. It .must be noted, hO'I'leVer, that We haVe Observed (20) SUCh tr-ansient increases in the PGA pool on turning off the light in experiment:~ . i with Chlorella in which the medium eonta1ned 2. 0 z:n~, phospha~~(~. One wonders \'ihether the concentration·of phosphate in the chloroplast can become rate llmiti."'lS uJ·uer the~e con::iitions. \ requires no preincubation (21). ~~'hen 8-rnethyl lipoic acid (tf!LI\.) is added to a suspension of Chlorella gyrenoidosa \m.ich have been photosynthesizing under steady state con:litions vJith 14co2 for a sufficient tirr~ to saturate the ir~ermediates of the PSCR · cycle3' sudden an:i dra.l.na.tic char~ses 1n the concentrations of l;iome .of the inter.r.ediate compounds occur. At the sa.-11e tilw~p. carb~:m dioxide uptal'e and oxygen evolution are completely iru-.U.bited. This inhibition is partially reversed after some 10 to 20 mino -· During the first ~5 z.ec after addition of MLA~ the concentration of labeled PGA drops at a rate ccn-:1parable to / t:r.e rate of uptake of l:co2 fl"om the external me. sa.'!W time, the concentrations of 14c labeled t:'r...lctose-1»6- \ -35- \./ diphosphate and sedoheptulose-lao 7-diphosphat~ rise very rapidly3 whHe the concent...nation of their respective noo~Jhosprm.tes decrease I ', appreciably. sJ,.prisingly the concentration of RuDP undergoes only. \ . relatively small ,cl.!ar'..ges and le¥els off at a value slightly less .than. the · steady state photosynthesis concen.tration. Pollowlng the in1t1a1 t.'t"a."'!Sient cl:".t.."lr'<$;eS there i.s a.n ucce:Lt.:·:r-at.icn' of the form3.tion of labeled suc.t"'se t~Z...tich climbs to a ver-:J hi(;h 'Ja.lu.e a.'1d then drops rapidly dur'inf~ the period of recovery from i.nhibitionQ The ir.t.dbition of oxygen ev·olution arrl co2 uptake by I>f!.A could. be e:q,lain.ed in terrr.s of a.n intt-..rference of the :ln.'llbitor with elcctra.'1 tra~sport fro:n PPNR (chlorcplast ferre:Ioxin) to the I carbon cycle. If so,~~ it is very inter.estir'.I{J; to note tl'l--J.t the conccn- tration of PGA drops rapidly, t-rhereas interference with its reduction v.,rould tend. to cause its ocncentraticn to increase. The relatively w.all response of the concentration of labelej RuDP O!'l addition of NIA co:n~"klred to the f_9:'eat char..(~e in the level of PGA is note'l\'O...~hyo hmt ~-:pears to be a.'"l 1n.Y).ibition of the convel'"Sio.n ,_ \ of fructme and sedoheptulqse diphosphatcs to their respective \ monophospbates rnust also be expla.ined., pe..rlup:3 in terms cf .. , phos- ·phatase in.l:db::l.t1cn.. It 1r; pr~1nt'lt'.lre to speculate on t11e basis ,...... •· ~ ., i'ifC..'""e a.'Jout the oper--o;~.tion of the carbon .reduction cycle !!1 y,iyg_, .. ! 'rhe fornat1on of labeled ·glycolic acid can perhaps be by a::·tli'leial electron accepto.r·s ha~:> been der.1cnstroted (46). i' in the light$ prem.:lm::lbly by actiYation of t;,lycolic acid vlith ATP (see l~.ig.; 1) .. Ac~.ini~terro 14c lr.lbeled glycolic acid is convcrte-1 to u and $ carbons of' PGA by ~hoto5ynthesiz:\r4.S pla..'lts. This conversion acccm.pa.nle:i b;')· rmidcz~~5.zation of label /'.-:;) .: ;' '/~-' I -37- I co:npou.orn is \inVolved. l / ') cay result in part from oXidation of RuDP (which increased acid .. 'r!'t..16 cor.<..ocund in· turn c~ul.d be r.,ydrolyzed b:t glycol.ic ac:l.d · phosphatase (51) .. An· OrV'....rJ.zcd Enz;,;ro.e System? . I have rug;;eztcd elsel.;he.t"0 (451)~~0) that .the carbon aru t.heir correctness is n.ot essential to. the basic concer>t of the or&P,nized multifunctior.a.l SJ'"Stem. An ~zsential feature of' the propoaal was the existence of ; . .~; ;i· ,·,1 •' .. .. ~ ~ :. •• •. \I ~ '( . . ' ... ·. ~ \ .. ~. '\.. '. . ..,; "'-~ ..,·. ·, -~· -_ .... _ .(. :--.~: . .. . . ~~ . -l-'., '.· .: ~ • I; -. .~ .... •. ... ;·. ;;'!:_ ·;. '; -: I '· _.. ·: ;f ·."1. >: -~.{ J. ';-· .. ,. . ". --, ~- - ~- .. . ' '! 1. '')." :. \. .t. ·: ;. ·. --~ ' ·, . '' ' .. . '> :· ~- _.,.·.:_·: .,,,;,.,:;~;·,:;_: ._.;:,·::.ilnpr<)ved -~qa~· fer Viewing tho system !n situ;·: ~ ·.. · ' ! . . ·'·· .. / .. In evanto DW4t be0 j \:' . '' . ' . . ): ./;,;::f~~~'( ~ .~ ~ ~ ~-.a, ··. :.:·, 1 . ' • 't :,_:> ·, ~; :__ : ti:~.;\)~ ~ ·· ;: ·.. the· kio:fot ·ayr.;tem reapOns1bl" tor tatty Ac.td synthesiS .tAnd' tMt . t~ ·:;(/~·,,;.: 'iLJ:{ "·~~i~-~~:;...... ·< . . . : . .; ~ . · . : ._.. ~ r:_··-~,:_::' ;_!{; :.::,;·::·.>:'faro the PSCR cyole. The latte:; .b1osynthet1c 'f)Qt~ .requires r.l(;IJ:WE': . ~. :· i~;.: :-: :_: ··: .. ;:;1;::··:~~·-:·}r:: : · . _ _. ~ . · -,. · _ . _ · · · . . ~:::·:: :.;_:r:-,.·;:;:;.I:.,:;: ::·.·.straps and 1a~~~'i to include a nut~ber· or meuuraole tree pom <)f ·:·· .. ·:. ··~~·CF:x~:;~~,:(.;;( . . :- ·: .. :.. · · .· ·. ·> :·. · .·. . ·i·- ...... ,: · ,. __ ::.. • 1 . · >>: >_<.f.',:·-·~'::,·.\!· l~to ~s-.- the PScR oyolo coUld ft. ·mad1ated .. 1by ,an. , . .·,. ,::·· ·~::}~::··T:·.~~~:-::-:.::.::1· ._. . . . ; -...... ( .·~ ; ·.. ·; ·_:_ ·~}- :... ·· ~'· ·. ·... : · arg;lni.Zed aystom or. 2 to Ja multll\w..ct1onal ~. each no largm• ., ·• .. ' . ·. ', . . I .' .:"! ,_.: • ·, . ~ •• • i lo:'·, .-:<' :· ; ( ~--. ~ ~; .• \ 4' ,, • . ." ..:,.! ·~ '. t. ,;;..: . : .· . ~ ! • ...... • • ,~-:· ',, .: ....:·:_·than fioactton'I Part1Cles• ~ s*t.t.clea oould.be ~ed along . ':t·.: .-·.:\~;'::.--;::::.;<;_'.' ___ ·_; .. : .. :' . . '., ';! ~-~- .· .· ' ' . ·. .!' '~-\~·! .:' . . ·i.' .:.: :\! :: ::' ' · .. :~':.·~ .. ~-~ .•~-.:.; a~ surface or w1tb1n me:nb~losed·c~tmenta •. ·The -.; .. - ... ' . -: ~;- -..~:· •-. ; ~.. ~ . . - . . . ~ , ...... - . . "': . . -~: '4. . ..· ~ . . ~ • ; I ' '~ • o ~- f' o 0 f • ·- • • 't . ' ; < ~. • .. .. i· .. . •.. . l '. ';~~ UCRL-10995 '' ,· f',-.,-..• .., rll"' 14,.,. Df"i). . - ...... ""'" \1-J...... e>,.:;10rlt~:mts (;.,:!)) was discu3::Jed 11.1 tht.:t previous- ~eoti0!1. ~c1:1. a. block ' ,, -41- UCRL-10995 '\llould require some mechanism beyond that v-Jhich would be available ,. if the interconyersion between PGA ~~ sugar phosphates were accomplished only by .triose p~'1ospr..ate dehydrogenase and the cofactors ATP/ADP and NADPWNADP+. Such a block can be readily visualized if one supposes the carboxylation.reaction to lead to a bou."'ld form of glyceraldehyde phosphate v1hich is not rapidly oxidized --in vivo (45). 4. The absence of measurable a.'1lounts of' 3-phosphoglycer·a.ldeb,yde and of 4-phosphooF,fthrose, in contraat to the measurable amounts of. E.ll the other postulated intermediates in the cycle, sug,sests that these moieties ri1. bourld by enzyme sulfr()'dryl i;I'OUps ~ as HS-Ebz-S-CO-R. 5. The fact that no one has been able to isolate and identify ( an 1ntennedia4.e in the carboxylation reaction to date must be ', { .. · considered as sugge~tive evidence for the existence of enz~ne-bound intermediates through at least two stag;es of the· carboxylation reaction. ,6e The inadequacies and variabilities in the activities of isolated enzymes Which would catalyze the var~ous reactions of .. the carbon cycle ~i~~t be best explaL~i ~1 terms of a ~ultifunctional . ~ -..:· ':.> ~:\ \/·~i/;j_'\f i, ··- \ .Jf2""' ,; ·uCRL·~-~,~,9~~ .. !. ,:_>;~\·';'J~ -~·<;·~~::j~~ ~r;f:; .''\ f i :· _; '; '\ . · , , . _ . . ; . ". ?,:: ;:;.'·:, ·• ·' · •-. ··:-: ', ·,)'::/·~ ·Brta, $1Cli) a large and ea.l1ly disaggrega.tGf!t e~ ·S1Stettl ,. " ' :~~~;{v:¥~i~J~1:i~ ·~ ~ ~;~~ ~~late. 1ri*~· xt .1s ·~ .t~ ~:!'.:~;t:':: ~·-:'·.~ :··· :~~-:./;.~:; ./ ,. ·-i:}:;r;(}:_~·:~::;..,Organizatian ot the .lameuae ard strom systems ot chlOroplasts .:.~- · .~·· ~-~~-~~~ r:;Ai~.:?~:>~:~~i;:;,. i. - . -, .· -: , . . ·.; ._-·- ... . - ...... - .. - . . -_ - .r - ' ~~!~::~~-'~,,::-~>-·.Varies -oQria1dei'abl1 ~-One. Qrga.nian to anothbr. So aiso· do thEt 1 • ' ',. ;•f; 1.sJit~~~~.: ;:tee~~:~ .to. bren :cen waua ·~ ~~te '80m~ or ··~ :~·. . ~ · L~·~::~k:~·-.::--·:":<;~1~~-t.ractiO'ns-t'rc.b 1ntac~ cell$_. -It would not. be,.aurpM.aing· .-r~rr~-~--::.,:~;/_.-.;- --- -_.- ,- :-,._ -···:. :: ._,_ ·-- -, ___ ;___ ~- · "'- .:._._ · ... · ))~;;':{- ;-r;,··~. :-· t~re.t"ore 1t ·1:he .uounts ·or ~senoe. ot. $pec1tlo. e~ ·. ac~1v1t1es .. > r· ~(-·;::t:~~,:<:: - - . ;;. . . 1 , . . . , . .. . : ,.\ ,_. 'l>' •• ,;.'::.{:~'·:.·", '· '.a v ··.~ ... ~·~· ·.... .~~ _,,~. :t:::y' ,isolated&~ ,~uah .• ro\.lltit\motioM.l orge.n~ :sYstem w~d vary '· .- -·- ,· ·::::·~ . ·, _... -,' . ·\ ·,• ·.. '. ~. ·grea~ f:ron1 one: Grf!..aniSm·to attother• . ,· ..... ·.,l· ' ,. •;.. :; .. .. ; ~. . ·' -~ . .'" . .. '1, "t· ~~-: . '· -t \' "'1: . ' '-'•·:·.. .'I.' .. -:: ··~ .··. ; i ··; ;~~;.}( ·~ 01~~ lll!all~iJ!unat1~ enzyme· GYatem rcr the baa~ earboo ' ... ·· .... ':::::~;:~ •. ,"·:~·;;:15;":_'> ;.·;-\ rcduotiOn cycle, an organitim 1n Sat». cues may not require a : ~~-< ~:.~: ;::.~~-}\~::~\(.,}~--~: :,~? _; . . . . . ' . ' :' ...... ,, ~. ,, . \i~1~0!:3'.;';_~pec~;·fu!Qton 4ip!x)sP~ ald~' ~"~'~ by ~:Venti~· ;_;'::;-:~:,:::··;:.~, me~s. Thiw,. the abseno4t ot mwh.ari ~-.in speoi-tic organlmm ··.· ... ·. ·_.: . ·:~ .. . . · ···. :· ·.~· .: ·--~- := . f..'( .. ·····. \-..-·;.; •... ·'·· ''t·l:::·· · ... ,.: ,; ...._ .. , ... ..··- ., . '. ·~ .. . . -~- -~ "'1 ..... ;·~: -:-.;-. ·.,·:·;·.>:::<:-(::~,:>. f ;··· ·.·' <_.r~·- '~);~'i',{;~::;:: '-~-· '· - :;..t.· .\ ;:· ;$ '·1. : · ..•~ ~ :-- "> ~ -' :-· ~-~-:~.~{:·t\-.'. ~ ..· ...... f..: .· ... ' 1.:.•...... +.,·:_· ~ :-:. ~ ... 'If•' "I '. ..;_ ·-~~ f \ ··." .... '.< ':''"··· .f·• ·1 .:...... •'. -43- UCRL-10995 {54)55) may mean merely that that organism had no need to \ ' form PGA '~ the chloroplast uy oxidation of fructose-J.,t; •. ' dipbosphate. Conceivably 5uch ort,:l!lis.ms ::ra~~e all the FJA they need by liberation of 11 bourl.d l?'Jl~cf formed in the pr.L-nary carboxylation reaction. 7. The C-3 and C-4 difference part of the Gibbs effe<~t r:e:3 been explained earlier in terms of a srr.all pool of glyceralder~·yde- 3-phosphate and a large pool of dihydroxyacetone phosphate. Such a difference in pool size v:ould be re~ily eJ\-plained in terms of the multifunctional enzyn:e system, if the glyceraldehyde- · 3-phosphate moiety 1s enzyme bound. The other pa..""t of the GibN> ·effect, discrepancy between carbons 1 a."ld. 2 as comp;:.1red i'1i.th 5 and 6, could also readily be explained. In this case, one would suppose that the enzyme binds a ccx1:rr.on 'pool of C-l arxi C-2 t"r~11. transketolase reactions on fructosc-&-;phospha.te, eedoheptulose- ..... 7-phospr.ate and xylulose-5-phosphate. Thus one could account far the reflection of pentose c-1 and C-2 labeling back i::1to hexose C-1 and C-2 labelingo (See Fig. 1.) -44- UCRL-10995 mu.itif'unctiona.l organized enzyme system for biosynthetic pathways a±;e' 1mpol.. tant. Nore efficient utiliZation of enzyme ' \ . functions.- prevention of excessive energy loss due to . hydrolysis of chemically active functional group::> such as acid a,'1.rzydrides, and avoidance of compet~tive inhibition by naturally occurrine~ interzru..~iates are som.e of these advai1tages. In vim.v of th..e knOvm efficiency and rapidity of the reactions of the carbon reduction cycle durine pnotosynthesis, one is led to suspect the existence of such a system .for this process. Its basic import~~ce as a p~i~~ury blosynthetic reaction and its_ iorig evolutionary history \vould seem to i / require that carbon reduction 1n photosynthesis be Ir..C-diated by the most efficient system possible in living cells. t•'' ''iJ -45- UCRL-10995 LITERATURE CITED lo Bonner, ~. :J AIBS ~·, 13, 20, (1963). \ \, ' 2. Bass.ham1 J.aA., Benson~ A.A.~~ Kay, L.D., Hru:Tis; A.Z .. , . \ Wilson, A.T., and Calvin, M., J. Am. ~· ~e,76, 1760 (1954). 3. Calvin, M., Basshaffi, J.A.~ Benson~ A.A., Lynch, v., Ouellet, Schou, L., Steplr...a., H., and Tolbert, N., Sy:;-!posia f?.£. the f I "··-· Society £2£. EiperL'il·sntal BioloGY, No. V, p. 284 (1951). 4. Stiller, f/Iq Ann. ~.. Plant Pr..ysiol., 13, 151 (1962). 5. Bassham, J .A., and Calvin, r-1., ~Path£!:. Carbon in Photosyn~hesis, ·(Prentice-Hall, Englewood Cliffs, N.J. 11 1957). 6. ~alvin, M,, and Bassr..a:n, J .A., 'lne Photosynthesis £f. Carbon Cc:!:B;oundslf (;·l.A. Benjamin, Inc., New York, N.Y., 1962). 7. Vishniac » ·~v. , Horecker, B.L. 11 and Ochoa, S. , 1n Ad van. · Enzymol. , 19, 1 (Nord., F.F., Ed., Interscience Publishers, New York-London, 1957). 8. Calvin,· r"l., ar.d Benson, A.A.; Cold Spri~ Harbor Symposium ~~ r,~antitative Biology, Vol. XIII, p. 6 (1948). 9. iiassham, J.A., Bcn.son, A.Ao, and Calvin, H., ,J. Biol.- '781 (1950). -46- UCRL-10995 11. Galvirl, t~1 .. , and 1\aspryzyk, z., ~· Nat. t'l.ca,(i. Sci. :g_.§... Jl 45~ . 9~ (1959). ·:. 12. Bassham, J.A., and Kirk, !•1., Biochim. Eiopbys. {\eta, 43, 44'7 (1960). 13. Galvin; f-1., and Benson; A.A., Scienpe, 107, 476 (19!t8). 14. Galvin,. i1., and Benson, A.A., Science, 109; 140 ~l9!i9). 15 .. Calvin, M.~ and i'1assini, D -·t:.Xoer 1 en.·t·i a.., .·rr-_II,1, • 4...11 .r:;.., (10'\.':i)._,_,G. _ ~ . ' J .A., oiocillm. Biophys. Acta, 48, 299 (1961). 19. Bassha:n, J.A., Scientific A""ilerican,.. June,' 1962, p. 3. 20. E-a.ssbaJ1l; J .A., and Y..irk, )•!J., I·,1icroal;:r.ae CllYl Pllotos.:rnti'letic Bacteria (special issue of P1arit a.trl ~ Physiol. > Japan) ~93 (1963) •.. 21. Bass:r...a:n, J .A.~ Egeter, H., Ed.i:-tonston, }1"'., and .Kirk, ! to be published. \ 22. Peterkofsky ;' A.:~ c:uxl Hacker, Plant Pi1ysicl., 36, 4G9 (1961). - ...... _._...... - \ UCRL-10995 \. -47- 23. Bas3ham, J .A._ Shibata, K., Steenberg, K.. , Bourdon, ....T , arld dfv1n, M.; l· ~· ~· §.2E_., 78, 4120 (1956) ." ~- ·24. Jakoby, \v.a., Br-•.unmond, D.O., and 0011.0a, s • .; l· Biol • . 218, 811 (1956) .. 25. l1eis3bacb, A., Horecker, B.L., and Ii~1tz, J., J. ~· ~·> 218, "{95 (1956). 26. Racker, E., Arch. Biochem .. Sioohys., 69, 300 (1957). 'Y1 .1 •• an(~ T~{ ...0 .0.·.!::~., ,~,.C~1· ):;l'O•"h"""" <;.j" r.\Fttl·.,to·Jj <,;; n J v_r.1 . • , - - 'o #• ~· ...... - ...... '-"·!'<·to·')hy 73) 120 (1958). 20. Pon, N.G. (Doctoral r.rhesis, f.lio-0r&:'U1ic Chemistry Group, / Acta, 23, 342 (1957). 30. Sing~r, Cm11.pbell» J .H., and 'viildrw:m, J Bl" ol. •"h"'m . ~·, 197, 233 (1952) .. 31. Sp;grra~, :;;.L. (Doctoral Tt!e31s, Call.fornla Institute of 'l\:chnolor;y, Pasadena, Calif., 1953). Singer, :S.J., i>Jildman, S.G., ;[_ .. Bio1. 205, 969 (1953). -48- UCRL-10995 . '· . \· ·. ' . 34. Park, ··ft.B., and Pon; N.G ... J. ~· §!E!., 3, l (1961). 35. Van Noort, G., (Doctoral 'l'l1esis, University or California; . Los Angeles, ~962). ...-~7. Ako··iJ"··n·"""""o-o-...... c.,,, '"'''~ G... , ~..,,...,...... (~"1'"~~ ..... v ...... , :•J.,·• .ubi oc.em.h · _.,z ~n_press.· · 38. i"bses, V., and Calvin, N., ;>.!"oc. --Nat. Acad. Sci. U.3~, I \ (1958). ·.' ':'i: Ferrier, R.J., and CC:.lvin, -Nat. Acad. ~· u.§.., 43, 1644 (1962) ~ '40. Shlcolniki' !\1. Ya., and Dornan, N.G •. , Biokhimiya, 25, 276 (1960). 41. Galr:iic:t.e; J .!•1., Conmt. ~·, 254, 1:1.69 ( 1962). 42. f..:1.r.dlerj 0. 11· ar..d Liescnkottcr, I., ~· Vth Internatiorr2Ll p. 326. Biochemical· Conf~ess, l'f:.osco:;:, 1961. Vol.. VI/(Sissakian, N~f"i., Ed.). I~ 3. Ea.ndler; 0. , an::l Liesenl.-:otter, I. , !./iicroalp;ae and Fhotosynt.!}t.~tic Bacteria (special issue of Plant and Cell Physiol.) Japan) 513 (1963). ' ''· \,:.;)i 0')')17~ . '- Jj I.,..,. Res~ Cor·1IT?.. ,, J.. -·-· ~ 376 (1962). / I \_ -49- UCRL-10995 4;5. Bassham 11 J .A. 1 Mvan.· Enzvmo1. ~ x:t:J ~ 39 (L"lterscie."1Ce Publ1sl;lers f) - t·!ev; York-London i 196 3) • 46. da Fonseaa-\•Zollheim, F • ., Bock~ K.v:., ahd Holzer, H., Bioch6n. Biophys. ~· ~., 9, 466·(1962)~ PhysiolQ Plantaru~~ 3~ 487 (1950). LISo l3$nson:> A.A.~ and Calvin, f-1 .. » ![_ • .Exr>. Botany~' 1~ 63 (1]49). !I_. Exp. Botany~ 13ll 176 (1962). 1285 (1961). ·52o L)rnenlli F., Faderation Proc.:i 20 (lJ).~~ 934 (1961). -----....-~ :;,3. Richter., G.~ Nattn"Hisscnsc!l<:t.ften~ 2llil 604 (1959). 37~ 402 (1962). 36, supp1o ~( (1961). This report was prepared as an account of Government sponsored work. Neither the United States, nor the Com~ mission, nor any person acting on behalf of the Commission: A. Makes any warranty or representation, expressed or implied, with respect to the accuracy, completeness, or usefulness of the information contained in this report, or that the use of any information, appa ratus, method, or process disclosed in this report may not infringe privately owned rights; or B. Assumes any liabilities with respect to the use of, or for damages resulting from the use of any infor mation, apparatus, method, or process disclosed in this report. As used in the above, "person acting on behalf of the Commission" includes any employee or contractor of the Com mission, or employee of such contractor, to the extent that such employee or contractor of the Commission, or employee of such contractor prepares, disseminates, or provides access to, any information pursuant to his employment or contract with the Commission, or his employment with such contractor. ' ". •...:. ..,;.. ,. !I ;, n I.1. \.,/ I I