kinetics oflight-dark co2 fixation and glucose assimilation · 2006. 2. 28. · journal of...

JOURNAL OF BACTERIOLOGY, Nov. 1976, p. 633-643Copyright C 1976 American Society for Microbiology

Vol. 128, No. 2Printed in U.S.A.

Kinetics of Light-Dark CO2 Fixation and GlucoseAssimilation by Aphanocapsa 6714

R. A. PELROY,l* GERRI A. LEVINE, AND JAMES A. BASSHAM

Laboratory of Chemical Biodynamics, University of California, Berkeley, California 94720

Received for publication 23 August 1976

Cells of Aphanocapsa 6714 were subjected to alternating light-dark periods(flashing-light experiments). The corresponding activation (in the light) andinactivation (in the dark) of the reductive pentose cycle was measured, in vivo,from initial rates of 14C02 incorporation and also by changes in the totalconcentration of 14C and 32p in soluble metabolites. Two principle sites ofmetabolic regulation were detected: (i) C02 fixation was inactivated 15 to 20 safter removal of the light source, but reactivated rapidly on reentering the light;(ii) hydrolysis of fructose-1,6-diphosphate (FDP) and sedoheptulose-1,7-diphos-phate (SDP) by their respective phosphatase(s) (FDP + SDPase) was rapidlyinhibited in the dark but only slowly reactivated in the light. The time requiredfor reactivation ofFDP + SDPase, in the light, was on the order of 20 to 30 s. Asa consequence of the timing of these inactivation-reactivation reactions, newlyfixed C02 accumulated in the FDP and SDP pools during the flashing-lightexperiments. Changes in the concentrations of the adenylate pools (mainly inthe levels of adenosine 5'-triphosphate and adenosine diphosphate) were fast incomparison to the inactivation-reactivation reactions in the reductive pentosecycle. Thus, these regulatory effects may not be under the control of theadenylates in this organism. The activation of C02 fixation in the light is at leastin part due to activation of phosphoribulokinase, which is required for formationof ribulose-1,5-diphosphate, the carboxylation substrate. Phosphoribulokinaseactivity in crude extracts was found to be dependent on the presence of strongreducing agents such as dithiothreitol, but not significantly dependent on aden-ylate levels, although adenosine 5'-triphosphate is a substrate.

The reductive pentose cycle is the sole meansof photoautotrophic C02 fixation by the unicel-lular blue-green alga Aphanocapsa 6714 (16,19). When cells are placed in the dark, C02fixation ceases and oxidative metabolism ofglucose-6-phosphate (G6P), via the oxidativepentose cycle, begins immediately. G6P oxida-tion in the light is prevented by high ratios ofreduced nicotinamide adenine dinucleotidephosphate (NADPH) to NADP (18) and also byinhibition by ribulose-1,5-diphosphate (RuDP)(19).

In the present study, our objective was toidentify the principle sites in the reductive pen-tose cycle that are inactivated when cellschange from photosynthetic to respiratory(dark) metabolism. In a series of flashing-lightexperiments, the initial rates of 14CO2 fixationand changes in levels of the metabolite pools atthe reductive pentose cycle were measured. Inthese experiments, inorganic phosphate labeled

I Present address: Biology Department, Pacific North-west Laboratory, Battelle Memorial Institute, Richland,WA 99352.

with 32p was administered to provide a secondtracer for sugar phosphates (and related phos-phorylated metabolites) and also to permit ob-servation of changes in the adenylate pools.

MATERIALS AND METHODSGrowth. Aphanocapsa 6714 was grown on a min-

eral base medium, BG-11, supplemented as de-scribed previously (17). Cells were grown from smallinocula in the presence of approximately 20 ,uCi ofcarrier-free 32p per ml (17), ensuring complete satu-ration of the tracer in phosphorous-containing me-tabolites. When cells had grown to an optical den-sity of 120 optical density units, they were harvested(500 ml per side-arm flask), washed in phosphate-free BG-11 medium, and resuspended in 50 ml of BG-11 medium minus phosphate. Sodium bicarbonatesolution was added to make the cell suspensionabout 30 mM, and then the cells were placed in thesteady-state machine, as previously described (15,17).

Double-labeling experiments: 14C and 32p. Twotypes of labeling experiments were conducted. Ininitial-velocity experiments, the cells were prela-beled with 32p, and the 14C label was introduced toinitiate the experiment. In steady-state experi-

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634 PELROY, LEVINE, AND BASSHAM

ments, the cells were also prelabeled with 32p, butthe 14C label (as '4CO2) was added 30 min beforebeginning the experiment. Since '4C enters interme-diary metabolism much more quickly than 32p, it ispossible to achieve steady-state concentrations inthis comparatively short time.

In initial-velocity experiments (i.e., where therate of 14C assimilation was measured) the specificactivities for carbon were: 14C02, 10.02 ,tCi/gmol;[U-14C]glucose, 8.4 ,uCi/,umol. The concentration of12CO2 plus 14CO2 in the gas phase was 0.5%, vol/vol.For the adenosine nucleotides, the value of 32P wasdivided by the number of phosphorous atoms permolecule - i.e., to give nanocuries or microcuries permicrogram-atom of phosphorous - a measure of con-centration (14, 15). In steady-state experiments, inwhich cells were prelabeled with both phosphorousand carbon tracers, the specific activities of 14CO2and 32P were: 22 /uCi/gmmol and approximately 20,uCi/,umol, respectively. The amount of labeled ma-terial is expressed as concentration of label per mil-ligram (dry weight) of algae in all figures.To begin an initial-velocity flashing-light experi-

ment (see below), 14CO2 or [U-14C]glucose was intro-duced into the steady-state system. Cells initiallycontained no 14C. Phosphate groups in metaboliteswere already fully labeled with 32P. In time, 14C/32Pratios approached constant values that are charac-teristic for a given metabolite. Thus, 14C/32P ratioswere a measure of specific activity of a given metab-olite.On an equivalent molar basis, 3-phosphoglycer-

ate (PGA) and fructose-1,6-diphosphate (FDP) con-tain a ratio of phosphorous to carbon of 1:3, which istwice that of the hexose monophosphate G6P. Thus,at any given time between zero time and whensaturating (steady-state) concentrations of 14C arereached, the 14C/32P ratios of these two classes ofmetabolites will be related by a constant factor. Forcomparing compounds on an equivalent basis, wearbitrarily chose to make all comparisons relative tohexose monophosphates. Thus, 14C/32P ratios forPGA and FDP were multiplied by a factor of 2 tomake them equivalent to 14C/32P ratios for G6P whencomparisons of specific activity in the different poolswere to be made.

In our initial-velocity experiments, we wereforced to make assumptions about the FDP + sedo-heptulose-1,7-diphosphate (SDP) and G6P + sedohep-ulose-7-phosphate (S7P) pools. The diphosphates,as well as the monophosphates, tend to move to-gether in the solvent systems used for paper chro-matography (15). These compounds can be separatedby secondary chromatography; however, this re-quires hydrolysis of the phosphate groups followedby rechromatography and radioautography of 14Cremaining in the dephosphorylated sugars. Thiswould have been difficult or impossible in the earlystages of the initial-velocity experiments becauselevels of 14C were quite low. However, over thecourse of many experiments, we have noted thatconcentrations ofFDP were always two to four timesthe level of SDP, whereas the concentrations of G6Pwere three to nine times the concentration of S7P.

Thus, only a small error was introduced into specificactivity calculations by assuming all phosphorous inFDP + SDP was in FDP (3 carbons per phosphorous)and all phosphorous in G6P + S7P was in G6P (6carbons per phosphorous).

Flashing-light experiments. To begin a flashing-'light experiment (initial velocity) 14CO2 or [U-'4C]glucose was added to cells prelabeled with 32P, asdescribed above. At the beginning of the experi-ment, cells had been in continuous light longenough for steady rates of photosynthesis to be es-tablished. As the '4C tracer was added to the system,the lights were turned off for a half-cycle of 45 s andthen turned on again for the second half-cycle of 45s. This schedule was followed through several cy-cles, with samples taken every 15 s. Samples wereimmediately killed in 80% methanol, and the radio-activity in soluble metabolites was determined bytwo-dimensional paper chromatography followed byradioautography, as previously described (15). Ra-dioactivity in the separated metabolites on the chro-matograms was determined by gas flow spectrome-try on an automated counter, described in detailelsewhere (14, 15). In the steady-state experiment,where both carbon and phosphorous tracers in thereductive pentose cycle were prelabeled to steady-state concentrations, the schedule consisted of 40-slight-dark half-cycles, with samples taken every 10s.

Assay of PRK. Cells were stored frozen in a pasteat minus 20°C. They were thawed and resuspendedin a standard buffer consisting of: 10 mM triethanol-amine (pH 7.5), 1.0 mM NaCl, and 0.2 mM dithio-threitol (DTT), or 0.2 mM 13-mercaptoethanol and 10mM MgCl2. Each subsequent step was done in thisbuffer. The cells were broken with a French pres-sure cell using 20,000 lb/in2; usually the cells had tobe passed several times through the cell to obtainmaximum breakage. Broken cells were centrifugedfor 15 min at 36,000 x g, and the supernatant frac-tion was saved and centrifuged a second time, 60min at 36,000 x g. The clear, dark blue-green super-natant fraction was used for the assay of phosphori-bulokinase (PRK) in cell-free extract. The reactionmixture for the enzyme consisted of the followingreagents in 1 ml: MgCl2, 10 mM; tris(hydroxy-methyl)aminomethane (pH 7.5), 20 mM; adeno-sine 5'-triphosphate (ATP), 0.5 mM; adenosine di-phosphate (ADP), 0 or 20 mM; ribulose 5-phosphate(Ru5P), 0 or 1.2 mM; DTT or ,B-mercaptoethanol, 0or 1.0 mM; NADPH, 0 or 1.0 mM; NADH, 0 or 1.0mM; purified spinach RuDP carboxylase (PRK-free),5 U; and NaH'4CO3, 20 mM (5.0 ,Ci/,umol). Thereaction was started with the addition of enoughcrude extract to catalyze the fixation of about 0.1,umol of 14C02/min in the coupled enzyme reaction.The crude enzyme preparation was kept unfrozen at4°C until used.The reactions were carried out in 10-ml vials, 1 ml

per vial. The enzymes were denatured by the addi-tion of 250 ,/d of 3 N trifluoracetic acid, and thecontents of each vial were evaporated to drynessunder a nitrogen stream. Five-tenths milliliter ofwater was added to each of the dried vials and the

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CO2 FIXATION IN APHANOCAPSA 6714 635

contents were resuspended; then 6.5 ml of Aquasol(New England Nuclear Corp.) was added. The vialswere capped and shaken to form a light blue-greensolution in which radioactivity in PGA was deter-mined (as a measure of PRK activity).

RESULTSInitial-velocity experiments: '4C and 33P.

When '4CO2 fixation rates were compared withADP and ATP levels during alternating 45-speriods of light and darkness (Fig. 1), no clearimmediate dependence of CO2 fixation on theratio of ATP/ADP was seen. Fixation of CO2began immediately and was approximately lin-ear during the light periods, although therewas a slow rate of uptake during the first cycle(probably owing to time required for equilibra-tion of already present CO2 with "4CO2 tracer).In contrast, ATP/ADP ratios were low duringthe dark periods (e.g., about 1:1) and rose rap-

cE

E0-0~,c 4._

0

120

EC-)

u) 80E2 0C7

40

0.0 1.5 3.0 4.5 6.0

Mi n utesFIG. 1. Adenylate concentrations (top) and net

CO2 fixation (bottom) in a flashing-light experiment.Samples were taken every 0.25 min (15 s); the lightand dark periods were 0.75 min (45 s) each.

idly in the initial seconds of each light period(about 5:1 within 15 s in the light).Except for the initial dark period, CO2 was

fixed more rapidly during the first 15 s of eachdark period than during the preceding lightperiod. Over the course of the entire experi-ment, CO2 fixation in the initial 15 s of the darkperiods accounted for nearly one-third of allCO2 fixed and occurred at a rate at least 1.5times the light fixation rate. Perhaps this accel-erated dark fixation rate (just after a light pe-riod) is similar to the burst of CO2 fixation inthe dark after photosynthesis reported byEmerson and Lewis (6), in studies with Cho-rella.

Figures 2 through 5 show the fate of CO2fixed during the experiment of Fig. 1. Eachfigure is in two parts: top panels show ratios of14C/32P, a measure of specific activity (see Mate-rials and Methods); the bottom panels show thecontent of 14C in the metabolite pools of thereductive pentose cycle.The concentration of "4C in PGA increased

and decreased in a cyclic manner (bottom, Fig.2). Noteworthy were the rapid increases of 14C

4003 - Phosphoglycerate 0.

30~~~~~~~~~

0-0/

120

10 f 0

30

20-

1010

0. L5 3.0 4.5 6.0Minutes

FIG. 2. 14C/32P ratios (top) and concentration of14C in PGA (bottom) during the flashing-light experi-ment of Fig. 1.

0 lZ

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30

20

0.0 L5 3.0Minutes

FIG. 3. 14C/32P ratios (top) and co

14C in FDP + SDP (bottom). Data fronFig. 1.

0L

cn

E

E0

cn

10

10

0.0

in this metabolite in the first 15 s of the dark

periods (cycles to IV). The total "4CO2 fixed

into PGA in these initial seconds of darknesswas nearly equal to the total "4CO2 fixed in thedark: i.e., approximately 40 of 48 ng-atoms of'4C per mg of algae. Thus, fixation into PGA

|- is the route of uptake during the first 15 s of0o / darkness.

0-0-0 This conclusion is also supported by the factthat the specific activity of "4C in PGA in-creased markedly in the initial seconds of thedark periods. As is especially apparent in cycles

___ II to IV, ratios of '4C/32P increased at about thesame rate in the first 15 s of darkness as in thelight. Since the specific activities of the otherintermediates were uniformly lower than PGA,

-\/ rearrangement of previously (light) fixed car-

bon could not have accounted for the increasedspecific activities in PGA, in the dark. Only a

more highly labeled source, i.e., "4CO2 from thegas phase, would have increased the 14C/32Pratio in PGA. The slight decrease in 14C/32P inPGA during the latter part of the dark periodsprobably indicates a flow of unlabeled carboninto PGA, coming from the oxidative pentosecycle.

This experiment also makes possible the esti-4.5 6.0 mation of relative rates of carbon flow through

certain parts of the reductive pentose cycle, inthe light and in the dark. In the light periods,

)ncentration of newly fixed carbon was observed almost imme-n experiment of diately in FDP + SDP and corresponded to a

decrease in "4C content in PGA (cycles III to IV;

1.5 3.0 4.5 6.0

MinutesFIG. 4. '4C/32P ratios (top) and concentration of 14C in G6P and S7P (bottom); data from experiment ofFig.

1.

II

Fructose Diphosphate plusSedoheptulose Diphosphate

_ L

/0-0- 0-0-/

c-

10

0

40

30

vr

E

E

20

10

0

I~~ ~~~~e~~~~~0-'Glucose-6-Phosphate plus l-

Sedoheptulose-7-Phosphate 0

I 090 I 0 0

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VOL. 128, 1976

CL

1-T

E-

grO

E0

c

10 -

10 L

0.0

C02 FIXATION IN APHANOCAPSA 6714 637

1.5 3.0 4.5 6.0

MinutesFIG. 5. 14C/32P ratios (top) and concentration of 14C in F6P (bottom); from experiment of Fig. 1.

Fig. 2 and 3). It should be noted that rates ofC02 fixation quickly reached steady-state val-ues in these light periods (shown in Fig. 1),showing that carbon flow into PGA was occur-ring at the maximal rate for this experiment.Thus, fixed carbon in PGA was very quicklyconverted to FDP + SDP in the light afterdarkness at a rate in excess of its maximumrates of formation by RuDP carboxylation (i.e.,equivalent to the rate of C02 fixation). Fromthese observations we conclude that the en-zyme sequence PGA-kinase, triose phosphatedehydrogenase, and aldolase synthesized FDPand exchanged label into SDP (via erythrose-4-phosphate) from PGA in the light faster than itwas replaced by C02 fixation. Since NADPH isrequired for these conversions (i.e., in the re-duction of 1,3-phosphoglycerate to glyceralde-hyde-3-phosphate), it can be concluded that theconcentration of reduced pyridine nucleotidewas sufficient not to limit the flow of carbon incells transferred from dark to light.Although the conversion of PGA to FDP and

SDP was rapid in the initial seconds of a lightperiod, further conversion of these metabolitesto the corresponding sugar monophosphateswas slow. The 14C labeling of GOP, S7P, andfructose-6-phosphate (F6P) was very slow rela-tive to that in FDP and SDP (Fig. 4 to 5). Thisindicates that the reactions mediated byFDPase and SDPase are inactivated in the darkand do not rapidly become activated during the45-s light period.

Another experiment was designed to testwhether conversion of G6P to S7P and F6Pdepends on light or darkness. In this experi-ment, [U-_4C]glucose was used as the source oflabel. In both light and dark periods, carbonpassed quickly into GOP, S7P, and then fromthe sugar monophosphates into PGA, FDP, andSDP (Fig. 6 to 8). Thus, during a flashing-lightexperiment, all of the steps leading from thesugar monophosphates to sugar diphosphatesand PGA are fast.That slow hydrolysis of FDP and SDP was

not due to factors other than those produced byflashing the light is shown by the data of Fig. 9.This experiment was carried out with continu-ous illumination of the cells; otherwise, theconditions were the same as described above.The '4C/32P ratio showed that carbon fixed from14CO moved quickly throughout the metabolitepools of the reductive pentose cycle, approach-ing steady-state concentrations by 6 min. Moreimportantly, the specific activities of the mono-phosphate sugars (made by estimating thenumber of phosphorous and carbon atoms in agiven compound; see Materials and Methods)were approximately equal to those for PGA,FDP, and SDP (after correcting for the numberof phosphorous atoms per metabolite). Theseresults should be compared with the compara-ble data of Fig. 1 to 5 for the flashing-lightexperiments.

Steady-state experiment: 14C and 32p. In thisexperiment, both tracers were administered


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al-

50

E

50

~ ~ ~ A A AL

A

0.0 L5 3.0 4.5 6.5Minutes

FIG. 6. '4C/32P ratios (top) and 14C concentration(bottom) in G6P and S7P using [14C]glucose as thesubstrate.

long enough to achieve steady-state (satura-tion) labeling of the metabolite pools before thealternating light-dark periods were begun. Ascan be seen, G6P, F6P, and S7P decreased inconcentration fairly rapidly during the darkperiods (Fig. 10, top). When cells were reillumi-nated, approximately 20 s passed before carbonbegan to flow into these monophosphate pools.It should be noted that in the reductive pentosecycle, the FDP + SDPase reactions must func-tion for carbon to pass from FDP and SDP to theG6P, F6P, and S7P. Thus, the approximately20-s lag time before the latter compounds were

restored to photosynthetic levels was indicativeof the time required for reactivation of the FDP+ SDPase reactions. This estimate is also ingood agreement with initial-velocity data (Fig.3), which showed accumulation of carbon inFDP and SDP for 20 to 30 s after cells were

transferred from dark to light.

The decrease in the concentration of themonophosphate sugars in the dark periods(Fig. 10, top) could be interpreted in a similarway: dark inactivation of FDP + SDPase oc-curred in approximately 10 s, thus cutting the

75

50

25

0

50

E

C, 25

0.0 1.5 3.0 4.5 6.0

Minutes

FIG. 7. I4C/312P ratios (top) and 14C concentration(bottom) in PGA. Data from experiment ofFig. 6.

40

20

0

80

60C7

E 40

c

20

O I

0.0 1.5 3.0 4.5 6.0

Minutes

FIG. 8. '4C/32P ratios (top) and 14C concentration(bottom) in FDP and SDP. Data from experiment ofFig. 6.

I0

/.I\Y I0\0 e*

- .-0 "

I0\ e

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40

30

-% 20

10

30

E

20E

10

0.0 1.5 3.0 4.5 6.0

Minutes

FIG. 9. 14CI32P ratios (top) and 14C concentration(bottom) in a labeling experiment using "CO2 andcontinuous light.

major flow of carbon into the monophosphatepools. However, since CO2 fixation was stillactive (i.e., 15 to 20 s into the dark: Fig. 1 and2), the level of carbon in the sugar monophos-phates fell as a result of CO2 fixation and theassociated conversion of sugar monophosphatesto PGA, via RuDP.

It is significant that, in the dark periods ofthis flashing-light experiment, the change inRuDP concentration was small in comparisonto the rapid expansion of the PGA pool (Fig. 10,bottom). Since RuDP is the major precursor forPGA, it follows that the RuDP pool was turningover rapidly in the initial seconds of darkness.In fact, the RuDP pool must have been replen-ished several times to account for the totalamount of dark-synthesized PGA. Recall thatdata from one of the initial-velocity labelingexperiments (Fig. 1 and 2) showed that thepredominant route of PGA synthesis in theearly dark periods was via carboxylation ofRuDP. Thus, both phosphoribulokinase and thecarboxylase were active in the initial seconds ofthe dark periods.As seen in the initial-velocity experiments,

the overall effect of flashing the light was a

steady accumulation of carbon in FDP + SDP(Fig. 11).As a final point, it should be noted that light-

dark and dark-light changes in the adenylatepools required 15 s or less (Fig. 12), which isconsiderably faster than reactivation of FDP +SDPase or inactivation of CO2 fixation.Activity of PRK in cell-free extracts. With

0.5 mM ATP and 0.5 mM Ru5P, PRK was onlyslightly inhibited by 5 mM adenosine mono-phosphate (AMP) (Fig. 13). Five millimolarADP, a 10-fold higher concentration than thesubstrate ATP, gave approximately 50% inhibi-tion of the enzyme. From the data presented inFig. 1 and 12, it can be calculated that ATP/ADP ratios in vivo never fell below 0.8 in theflashing-light experiments.Figure 14 shows the activity of PRK as a

function ofRu5P concentration, at fixed ADP orfixed AMP concentrations. Inhibition by 2.0mM ADP or AMP did not exceed 20 to 30%.These comparatively weak inhibitions by AMPshould be contrasted with the strong AMP inhi-bition ofPRK from thiobacilli, hydrogen-oxidiz-ing bacteria (10, 11), and certain photosyntheticbacteria (20). On the other hand, the PRK ofAphanocapsa 6714 does not differ greatly fromthe chloroplast enzyme in its lack of response toinhibition by ADP and AMP (1, 10).PRK from Aphanocapsa 6714 requires a re-

15-

10

5

E

- 0

E 0 ~~~~~G6P+S7PA F6PO PGA

v0.00 0.66 1.33 2.00 2.66

Time (Minutes)

FIG. 10. 14C concentration in F6P, G6P, and S7P(top); PGA and RuDP (bottom) in a steady-stateexperiment with all pools saturated with respect totracer.

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0.4

14

a4~~~~~~Cl-i~~ ~ ~ ~ ~ IE(MNTS

FI.1 -4ocnrto nFP D,adRD;dt rmeprmn fFg 0

a-

E0m

-21CL

clim

G=1.,

0.0 0.66 1.33 2.00 2.66Minutes

FIG. 12. 32P concentration in ATP, ADP, andAMP; data from experiment of Fig. 10.

ducing agent for activity (Table 1). DTT was

the most effective reducing agent tested. Gluta-thione was about one-seventh as effective as

DTT; f8-mercaptoethanol did not stimulate en-

zyme activity at the concentrations used. Nei-ther NADPH nor NADH stimulated enzyme

activity. This should be contrasted with NADHstimulation of PRK from Hydrogenomonas fa-cilis and Rhodopeudomonas spheroides (11,20). On the other hand, stimulation of the chlo-roplast enzyme by DTT or by light-generatedreducing agents has been reported (9), and DTT

16.5I.-,

Phosphoribulokinase

E 5.5tviyE 5. 5 X ADP\

C: ATP - 5 x 10 4M\

0 1 2 5 10 20

Conc. x 10- 3M

FIG. 13. PRK activity as a function of AMP orADP concentration. NaH' 4CO3 specific activity, 5,utCilumol; 3 mg ofprotein/ml of reaction mix. Ru5Pconcentration was 5 x 10-4 M.

activation of PRK from the blue-green algaAnacystis nidulans has also been demonstrated(5).

DISCUSSIONThe reductive pentose cycle in Aphanocapsa

6714 is blocked when cells are placed in thedark. This loss of function does not result froma reduced supply of cellular energy; it is morelikely the expression of a specific regulatorymechanism that is called into play when cellslose their capacity to carry out noncyclic photo-

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VOL. 128, 1976

E * vz0.6

.50

D } 13

o

1-

0 0.3 Phosphoribulokinase Activity

cm ~~~~~~*ControlAMP 2x 103M

A~~~~~II

0 4 8 12

RPx1-4m

FIG. 14. PRK activity as a function of Ru5P con-

centration at fixed AMP orADP concentrations. Con-ditions were those of Fig. 13.

TABLE 1. Phosphoribulokinase activity versusreducing agents and reduced pyridine metabolites a

tg-atoms of 14C/Compound Concn mg of protein per

10 min

DTT 1 x 10-4 M 8.73GSH 1 x 10-4 M 0.42Mercaptoethanol 1 x 10-4 M 0.08NADPH 1 x 10-3 M 0.15NADH 1 x 10-3 M 0.08DTT, 10-4 MNo ATP 0.15

DTT, 10-4No Ru5P 0.11

No addition 0.24a Conditions were those given in Fig. 13.

electron transport (18).Previous work showed that one consequence

of dark inactivation was the failure of dark cellsto fix CO2, a result probably attributable toinhibition of PRK. This conclusion was basedmainly on the observation that dark cells ofAphanocapsa 6714 failed to convert measurablequantities of highly labeled [14C]glucose intoRuDP (17). It should be noted that failure tosynthesize RuDP occurred even though darkcells were actively synthesizing the pentosemonophosphate sugars via the oxidative pen-tose (13, 14, 16). Thus, a supply of ribulose-5-phosphate was present (i.e., the substrate forRuDP synthesis). It is also possible that thecarboxylase reaction was inactivated in thedark. However, this result would not have beenresolvable in our experiments.The present work reveals three important

features of light-dark flashes on the reductivepentose cycle of Aphanocapsa 6714. (i) Dark


inactivation of FDP + SDPase was nearly com-plete, preventing almost all conversion of FDPand SDP to the corresponding monophosphatesugars. (ii) The times required for dark inacti-vation, and then the subsequent light reactiva-tion of the cycle, were significantly different forCO2 fixation and hydrolysis of FDP and SDP.(iii) Light-dark effects on the reductive pentosecycle are poorly correlated with changes in theconcentrations of the adenylates, arguingagainst adenylate (or energy charge) control ofthis pathway.The first point was established by measuring

the initial rates of 14CO2 fixation, or [14C]_glucose uptake, into soluble metabolites. Fromthe same experimental data, it was also deter-mined that CO2 fixation continued for some15 to 20 s into each of the dark periods of theflashing-light experiments. On the other hand,the FDP + SDPase reactions were rapidly in-activated in the dark. When cells were againplaced in the light, the reductive pentose cyclewas restored. However, the time required forreactivation of the FDP + SDPase reactionswas much longer than for renewal of CO2 fixa-tion (i.e., a delay of 20 to 30 s versus nearlyimmediate CO2 fixation in the light). As a con-sequence of these different time constants, CO2fixed during the flashing-light experiments be-came trapped in PGA, FDP, and SDP.Under conditions as they exist in nature, this

lack of coordination between these two sites ofthe reductive pentose cycle should not ad-versely affect the cell. Inactivation of the cyclewould occur once in the evening as cells shiftfrom growth to maintenance metabolism. Reac-tivation of the cycle would not begin until thefollowing day. Thus, the second point is reason-able in terms of a regulatory mechanism that isused intermittently to block completely or toreactivate photosynthetic metabolism, but notfor the fine control of carbon metabolism in thesteady state.The third point, which suggests that the level

of adenylates has little or no regulatory effecton the activity of the reductive pentose cycleduring light-dark transitions, is based on a gen-eral lack of correspondence between inactiva-tion or reactivation reactions and the intracel-lular concentration level of the adenylates.First, as noted in the initial-velocity experi-ments, CO2 fixation took place at an acceler-ated rate in the initial seconds of the darkperiods. At the same time, the concentration ofATP, the ratio of ATP/ADP, and also the en-ergy charge decreased sharply. This is not con-sistent with adenylate control over CO2 fixationby the inhibition of PRK, at least as it is


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thought to occur in bacteria. In several auto-trophic, facultatively autotrophic, and photo-synthetic bacteria, AMP (or low energy charge)strongly inhibits the activity of this enzyme(7,10, 11). However, in cell extracts ofAphano-capsa 6714, the activity ofPRK was insensitiveto inhibition by AMP, or product inhibition byADP (although some inhibition was noted atvery high levels of these adenylates, but atATP/ADP ratios much lower than measured invivo).The evidence concerning control of the FDP

+ SDPase reactions also does not support a rolefor the adenylates in controlling this part of thereductive pentose cycle. When cells entered alight period (during the flashing-light experi-ments), CO2 fixation began almost immedi-ately, and the highest levels of ATP, ATP/ADP, and energy charge were restored within10 s. Moreover, the levels of NADPH and ATPwere sufficiently high to allow rapid conversionof PGA to FDP and SDP. This suggests thatneither energy nor reduced pyridine nucleo-tides were limiting in the environment of thereductive pentose cycle enzymes. Nevertheless,hydrolysis of FDP and SDP was delayed for 20to 30 s on entering a light period.In two other photosynthetic systems, Chro-

matium D (13) and spinach chloroplasts (12),the adenylate pools were measured under con-ditions preventing or limiting growth (in theformer case) or CO2 fixation (in the latter case).For both the bacterium and the chloroplast,energy charge was poorly correlated with me-tabolism-i.e., high ratios did not stimulatemetabolic activity (Chromatium) and low ra-tios did not inhibit activity (chloroplast).

Recently, Ihlenfeldt and Gibson (8) suggesteda regulatory role for NADPH, or the ratio ofNADPH/NADP, in controlling the rate of C02fixation by A. nidulans. They suggested that aprinciple site of metabolic regulation of C02fixation might be triose phosphate dehydrogen-ase. This was based partly on the rapid drop inNADPH concentration in cells placed in thedark. In earlier work from this laboratory (16,17), we obtained kinetic evidence based on in-corporation of 14CO2 and [140]glucose, also sug-gesting a restricted flow ofcarbon between hex-ose monophosphate sugars and PGA, whichwould be consistent with a dark inactivation oftriose phosphate dehydrogenase.However, in more recent work (18) we have

shown that NADPH levels in dark cells of glu-cose-fed Aphanocapsa 6714 are rapidly restoredto levels equal to or exceeding comparable pho-'tosynthetic values. It should be noted thatAna-cystis nidulans is impermeable or only poorly

J. BACTERIOL.

permeable to glucose (19); thus, similar resultswould not have been obtainable with the latterorganism.As shown in the present study, the rate-lim-

iting steps in the reductive pentose cycle of cellssubjected to stress by a flashing-light source arethe reactions specific to CO2 fixation (PRK orboth PRK and carboxylase) and the FDP +SDPase reactions. Thus, in Aphanocapsa 6714the light-dark regulation of the reductive pen-tose cycle is affected mainly at these two reac-tions via a mechanism that probably does notinvolve the concentration ofNADPH or ratio ofNADPH/NADP.One hypothesis to explain dark inactivation

of the reductive pentose cycle is that a reducingagent (or agents) specifically associated withphotosynthetic electron transport (but not in-cluding NADPH) regulates the activity of thispathway. It has been demonstrated that re-duced ferredoxin regulates the activity ofFDPase from spinach chloroplasts (4) and,more recently, that a number of reductive pen-tose cycle enzymes in cell extracts of A. nidu-lans are stimulated by reducing agents (5).Moreover, PRK from spinach chloroplasts isstimulated by DIT or by photosyntheticallyproduced reducing agents (9). These data sug-gest a similar pattern of metabolic regulationin these oxygen-evolving photosynthetic orga-nisms mediated by photochemically produced.reducing agents.

ACKNOWLEDGMENTSThis work was supported by the U.S. Energy Research

and Development Administration.

LITERATURE CITED1. Anderson, L. E. 1973. Regulation of pea leaf ribulose-5-

phosphate kinase. Biochim. Biophys. Acta 321:484-488.

2. Anderson, L. E., and M. Avron. 1967. Light modulationof enzyme activity in chloroplasts. Plant Physiol.57:209-213.

3. Avron, M., and M. Gibbs. 1974. Properties of phosphori-bulokinase of whole chloroplasts. Plant Physiol.53:136-139.

4. Buchanan, B. B., P. Shurmann, and P. Kalberer. 1971.Ferredoxin-activated fructose diphosphatase of spin-ach chloroplasts. J. Biol. Chem. 246:5952-5959.

5. Duggan, J. X., and L. E. Anderson. 1975. Light regula-tion ofenzyme activity in Anacystis nidulans. Planta122:293-297.

6. Emerson, R., and C. M. Lewis. 1941. Carbon dioxideexchange and the measurement of the quantum yieldof photosynthesis. Ann. J. Bot. 28:789-804.

7. Hart, B. A., and J. Gibson. 1971. Ribulose-5-phosphatekinase from Chromatium sp. strain D. Arch. Bio-chem. Biophys. 144:308-321.

8. Ihlenfeldt, M., and J. Gibson. 1975. CO2 fixation and itsregulation in Anacystis nidulans [Synechococcus].Arch. Microbiol. 102:13-22.

9. Latzko, E., R. V. Garnier, and M. Gibbs. 1970. Effectsof photosynthetic inhibitors and oxygen on the activ-


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VOL. 128, 1976 CO2 FIXATION IN APHANOCAPSA 6714 643

ity of ribulose-5-phosphate kinase. Biochem. Bio-phys. Res. Commun. 39:1140-1144.

10. MacElroy, R. D., E. J. Johnson, and M. K. Johnson.1968. Allosteric regulation of phosphoribulokinase ac-tivity. Biochem. Biophys. Res. Commun. 30:678-682.

11. MacEiroy, R. D., E. J. Johnson, and M. K. Johnson.1969. Control of ATP dependent CO2 fixation of Hy-drogenomonas facilis. Arch. Biochem. Biophys.131:272-275.

12. Miginiac-Maslow, M., and M. Champigny. 1974. Rela-tionship between the levels of adenine nucleotidesand the carboxylation activity of illuminated isolatedspinach chloroplasts. A study with Antimycin A.Plant Physiol. 53:856-862.

13. Miovic, M. L., and J. Gibson. 1973. Nucleotide poolsand adenylate change in balanced and imbalancedgrowth of Chromatium. J. Bacteriol. 114:86-95.

14. Moses, V., and K. K. Lonberg-Holm. 1963. A semiauto-matic device for measuring radioactivity on two di-mensional paper chromatograms. Anal. Biochem.5:11-17.

15. Pedersen, T. A., M. Kirk, and J. A. Bassham. 1966.Light-dark transients in levels of intermediary com-pounds during photosynthesis in air-adapted Chlo-rella. Physiol. Plant 19:219-231.

16. Pelroy, R. A., and J. A. Bassham. 1972. Photosyntheticand dark carbon metabolism in unicellular blue-green algae. Arch. Microbiol. 86:25-38.

17. Peiroy, R. A., and J. A. Bassham. 1973. Kinetics ofglucose metabolism in Aphanocapsa 6714. J. Bacte-riol. 115:943-948.

18. Pelroy, R. A., M. R. Kirk, and J. A. Bassham. 1976.Photosystem II regulation of macromolecule synthe-sis in the blue-green alga Aphanocapsa 6714. J.Bacteriol. 127:623-632.

19. Peiroy, R. A., R. Rippka, and R. Y. Stanier. 1972. Themetabolism of glucose by unicellular blue-green al-gae. Arch. Microbiol. 87:303-322.

20. Rindt, K. P., and E. Ohmann. 1969. NADH and AMPas allosteric effectors of ribulose-5-phosphate kinasein Rhodopseudomonas spheroides. Biochem. Biophys.Res. Commun. 36:357-364.


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