somne properties of partially purified. pyruvate kinase ... · somne properties of partially...

Plant Physiol. (1974) 54, 617-623

Somne Properties of Partially Purified. Pyruvate Kinase fromEuglena gracilis Klebs var. bacillaris1

Received for publication December 26, 1973 and in revised form June 25, 1974

DENNIS VACCARO2 AND MICHAEL H. ZELDIN3Department of Biology, Tufts University, Medford, Massachusetts 02155

ABSTRACT

A method of purification of pyruvate kinase (EC 2.7.1.40)from light-grown Euglena gracilis var. bacillaris was developedwhich yielded an enzyme preparation purified 115-fold overcrude extracts. During organelle formation, levels of pyruvatekinase in extracts prepared from cells engaged in light-inducedchloroplast development do not change significantly. The en-zyme has a molecular weight of approximately 240,000 and arequirement for both K+ and Mg'+. Fructose 1,6-diphosphateactivates the enzyme when the concentration of phosphoenol-pyruvate is limiting; it does not activate when the concentra-tion of ADP is limiting. ATP, citrate, and Ca2+ are inhibitorsof the enzyme and inhibit the fructose 1,6-diphosphate stimu-lation of the enzyme activity. ATP inhibition is only partiallyreversed by high concentrations of fructose 1,6-diphosphate.Further reversal of inhibition can be achieved by dialysis. Ca'-dependent inhibition can be reversed by a chelating agent butnot by increased concentrations of Mg' .The significance of the properties of pyruvate kinase in the

regulation of photosynthetic carbohydrate metabolism, espe-cially in connection with the inability of fructose 1, 6-diphos-phate to reverse Ca2+ and ATP inhibitions, is emphasized.

The formation of pyruvate and ATP from P-enolpyruvateand ADP is now recognized as a controlling step in the regula-tion of glycolysis in many organisms. During the last severalyears the enzyme pyruvate kinase, which catalyzes this conver-sion, has been isolated and purified from many sources and itsproperties described in some detail (4-7, 12, 14. 18-20, 24).

Although information concerning the properties of pyruvatekinase in heterotrophic organisms is available, the situation inautotrophic organisms has not been examined extensively (8,23, 26-28). Because photosynthetic carbon metabolism is oper-ative, as well as the metabolic conversions found in hetero-tropic organisms, the role of glycolysis in metabolic regulationwould be expected to be very important; in turn the control ofthe reaction catalyzed by pyruvate kinase would be central tosuch regulation.

'This work was supported in part by National Science Founda-tion Grant GB-27502 and by Tufts University Faculty ResearchFund.

2Present address: Department of Physiology, Harvard MedicalSchool, Boston, Mass.

'Present address: Biological Laboratories, Harvard University,Cambridge, Mass.

Pyruvate kinase has been isolated and partially characterizedfrom Euglena and several higher plant tissues. Using extractsof Euglena gracilis Z strain, Ohmann (19) purified pyruvatekinase about 20-fold and found that the enzyme is stimulatedby fructose 1, 6-DiP,' but is not inhibited by ATP. Miller andEvans (18) described a very strict requirement for mono anddivalent cations, such as K+ and Mg2+ for pyruvate kinase ac-tivity in various higher plant tissues. Jacob and D'Auzac (14)have isolated pyruvate kinase from serum of latex and foundthat the enzyme is inhibited by citrate and Ca'+. Duggleby andDennis (6, 7) have reported the properties of pyruvate kinaseisolated from cotton seed. The enzyme from this plant sourceis reported to be inhibited by malate, citrate, UTP, and ATPand activated by AMP, GMP, and fumarate.

In this paper we report a method for obtaining partiallypurified pyruvate kinase from light-grown Euglenia gracilis var.bacillaris and describe some kinetic and physical properties ofthis purified enzyme. In addition, the levels of pyruvate kinaseactivity were measured during chloroplast development.

MATERIALS AND METHODS

Disodium ADP, tricyclohexylammonium P-enolpyruvate, di-sodium fructose-1, 6-diP, disodium NADH, and rabbit musclelactate dehydrogenase Type II were purchased from SigmaChemical Company. Acrylamide and DEAE-cellulose werefrom Biorad.

Cells of Euglena gracilis var. bacillaris Pringsheim weregrown and maintained under resting conditions as describedelsewhere (25). When grown in the light, cells were illuminatedby cool white and red fluorescent lights at an intensity of 3.4 X10' ergs cm-2 sec-'. Chloroplast development by dark-grownnongrowing cells was induced at the same light intensity. Ell-glena gracilis Z strain was grown and maintained under thesame conditions. Cells were counted in a hemocytometer afterthey were killed with a drop of saturated HgCl. solution. Atleast four separate counts were made for each determinationand an average was calculated from these counts.

Except where noted, all isolations and purification proce-dures were carried out in an 0.04 M IA buffer, containing 1mM KCl; 1 mm MgSO,; 0.3 mm EDTA; 1 mm 2-mercaptoethanol; 8.5% sucrose; the pH was adjusted to 6.8with concentrated acetic acid. This is defined here as IA buffer.

Pyruvate kinase activity was assayed according to themethod of Bucher and Pfleiderer (2). Measurements were takenat 30 C on a Gilford model 2000 recording spectrophotometerat 340 nm. Assays were carried out in either 40 mm MESbuffer, adjusted to pH 6.2 with KOH or 40 mm IA buffer, pH

'Abbreviations: Fructose 1,6-diP: fructose 1,6-diphosphate;EGTA: ethylene glycol-bis (aminoethylether) N,N' tetraacetate;DEAE: diethylaminoethyl; IA buffer: imidazole-acetate buffer.

617

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VACCARO AND ZELDIN

Table I. Purificationi of Euglelia Pyruvate KinaseOne unit is equal to 1 ,umole of NADH oxidized/min

TotalStep Protein

mng

Crude extract 720DEAE I 275Dialysis 79.6

25.31 .333

Specific TotalActivity Units

units 'mgproteins

0.722 5201.75 480I 11 A5.53

15.482.7

440390110

Yield

92%

85%,;

75'%21 (CC

12.5% trichloroacetic acid for 3 hr, and left to stand for 3 hrin a 0.05% solution of Coomassie blue in 12.5% trichloroaceticacid. Excess stain was removed by placing gels in 12.5% trichlo-

urification roacetic acid for about 24 hr.Factor Unstained gels were extracted for pyruvate kinase after elec-

trophoresis by cutting the gel into 2-mm discs, each of whichwas placed into 1 ml of IA buffer containing 1 mg of BSA.The gels were then homogenized by hand and allowed to stand

2.4 at room temperature for 2 days. A maximum of 10% of the7.7 total activity placed onto a gel was recovered in a single peak

21 fraction.115

RESULTS

6.8. Both reaction mixtures contained the following: 0.1 M KC1;8 mm MgSO,; 1 mm mercaptoethanol; 0.3 mm EDTA; 3.47mM ADP; 8.5% sucrose; 1 mg/ml of BSA, and P-enolpy-ruvate as described in the text; 87 fcM NADH; 0.37 unit of lac-tate dehydrogenase. The mixture was allowed to incubate for6 min. at 30 C. the enzyme was added to give a final volume of1 .4 ml in the cuvette to start the assay.During the first 3 min after the addition of the enzyme, the

average rate of oxidation of NADH was 10% slower than dur-ing the subsequent 4- to 6-min period. The latter period was

used to obtain the maximal average velocities. Protein was de-termined according to Lowry et al. (16).

Cells were prepared for extraction for purification steps as

follows: 12 X 109 cells were harvested by centrifugation,washed in succession with 1 liter of IA buffer, and then with120 ml of IA buffer at room temperature. The resultant cellpellet was quick-frozen by immersion of the tubes containingcells in a dry ice-ethanol mixture. Cells were stored at - 20 C.

Extracts of cells engaged in chloroplast development were

prepared as follows: 4 to 7 X 10* cells were divided into twoequal portions. Each portion was washed with 100 ml of IAbuffer and the cells were resuspended in 10 ml of IA buffer.The cell suspension was passed through a French pressure cellat 2000 p.s.i. at room temperature. The resultant homogenatewas then centrifuged at 18,000 rpm for 1 hr at 14 C. Thesupernatant contained all pyruvate kinase activity.

DEAE-cellulose was washed as described in the literature(11), resuspended in IA buffer, and pH was adjusted to 6.8 withacetic acid. The DEAE was then thoroughly washed and equili-brated with IA buffer.

Sedimentation velocity of pyruvate kinase was determinedin a 5% to 20% sucrose gradient. Approximately 12 ,ug ofprotein representing 0.52 unit of purified Euglena pyruvatekinase was layered on top of the sucrose gradient. The gra-dients, having a total volume of 5 ml were centrifuged for 13hr at 40,000 rpm in an SW-65 Rotor (Beckman Instruments)at 4 C. Alkaline phosphatase and catalase were included tocalibrate the gradient. Fractions of 200 ,ul each were taken foras-ay after centrifugation.

Estimation of the mol wt by gel filtration was carried out on

a Sephadex G-200 column (0.9 X 55 cm) according to themethod of Andrews (1). Pyruvate kinase was layered onto thecolumn with beef heart Cyt c, BSA, aldolase, and catalase, andelution was carried out with 40 mm IA buffer, pH 6.8 at 4 C,containing 0.1 M KCI, 8 mm MgSO4, 0.3 mm EDTA, 1 mM2-mercaptoethanol, 8.5% sucrose. Fractions of 0.66 ml eachwere collected.

Polyacrylamide gel electrophoresis was carried out on gels(1O cm) prepared according to the method of Davis (3). Elec-trophoresis of the enzyme (in 8.5% sucrose and IA buffer) wascarried out at 2 mamp per tube. 200-300 v, for 2.5 to 3 hr ina jacketed bath maintained at 4 C. Each gel was then fixed in

The following five-step purification method for pyruvatekinase provides an enzyme preparation of higher purity thanhas been described previously when photosynthetic organismsserve as the source.

Step 1: Preparation of the Crude Extract. All operationswere carried out at room temperature (20-25 C), unless other-wise noted. The frozen cells, about 12 X IO" total, obtained as

described in "Materials and Methods," were rapidly thawedby the addition of 120 ml of IA buffer. The mixture was placedin a 45 C water bath and vigorously swirled until the cells were

brought into suspension. The cells were then passed through a

French pressure cell at 2000 p.s.i.The resultant homogenate was centrifuged for 10 min at

20,000g to remove debris. The supernatant was decanted andcentrifuged for 2 hr at 100,000g in a Beckman L-2 65. Thepellet from this centrifugation lacked pyruvate kinase activity.The supernatant was designated the crude extract (Table 1).

Step 2: DEAE-Cellulose Column. The crude extract was

loaded onto a DEAE column, 2.4 x 15 cm. The pyruvate ki-nase did not adsorb to the DEAE-cellulose at this stage and was

collected in the wash and designated DEAE I. This stepyielded a 2-fold purification without significant loss in totalpyruvate kinase activity (Table I).

Step 3: Ammonium Sulfate Precipitation. Crystalline ammo-nium sulfate was added with stirring to DEAE I to 45% satu-ration over a period of 5 min. Stirring was continued for an-

other 10 min. The slurry was centrifuged for 20 min at 10,000rpm. Most of the pyruvate kinase activity was found in thepellet which was resuspended in 100 ml of IA buffer. Occasion-ally, the ammonium-sulfate-treated DEAE I was cloudy aftercentrifugation. Activity in this suspension could be removedby further centrifugation at 10,000 rpm. The total resuspendedmaterial was dialyzed for 8 hr against 30 volumes of IA buffer.Longer periods of dialysis resulted in an increasing loss of en-

zyme activity. This preparation, the dialyzed enzyme, was 2- to3-fold purified over DEAE I with a yield of 70 to 100% (Ta-ble I).

Step 4: DEAE-Cellulose Column Chromatography. Immedi-ately after step 3, dialyzed enzyme was subjected to chromatog-raphy on a DEAE-cellulose column, 2.4 X 10 cm, preparedas described for step 2. The enzyme activity was retained on

the column and materials which had not adsorbed were re-

moved by washing with 50 ml of IA buffer. Pyruvate kinasewas eluted from the column with a 10 mM to 160 mm KCl gra-dient. The enzyme was eluted from the column at approxi-mately 30 mm KCl. All fractions with enzyme activity were

combined and dialyzed as described above. This preparationdesignated DEAE II was 3-fold purified over the dialyzed en-

zyme (step 3) with an 80 to 100% yield of activity.Step 5: Second DEAE Column Chromatography. The DEAE

II was divided into two portions, each containing 12 mg ofprotein. Each portion was applied to a second DEAE-cellulose

DEAE IIDEAE Ill

618 Plant Physiol. Vol. 54, 1974



PYRUVATE KINASE OF EUGLENA

column (1.4 X 10 cm), and the pyruvate kinase was elutedwith 40 mm KCl. The fractions with the highest specific activ-ity were combined and a 5-fold purification over DEAE IIwas obtained. This over-all procedure results in a greater than100-fold purification from crude extracts with 21% over-allrecovery of activity (Table I). Electrophoresis of DEAE IIand DEAE III on polyacrylamide gels shows a decline in thenumber of components in going from step 4 to step 5. Thereare, however, approximately 8 visible bands for DEAE III.Only one region, associated with a band, contained detectablepyruvate kinase activity after electrophoresis.

Tests for aldolase activity and triose phosphate dehydrogen-ase were negative in the pH range 6 to 7. A P-enolpyruvatephosphatase activity was readily detectable at pH 5.0 andbelow.

Freezing and thawing of cells prior to breakage retarded theappearance of a gummy precipitate during early steps of puri-fication. In addition, the operations up to the completion ofStep 4 had to be carried out in less than 48 hr to avoid theappearance of the same gummy precipitate. Once this precipi-tate formed, further purification methods did not work. DEAEII could be stored at 4 C for many weeks with only slight lossesin activity.

Properties of Euglena Pyruvate Kinase. The enzyme has anoptimum in the range from pH 6.2 to 6.5, but is quite active inthe pH range 5.4 to 7.2 (Fig. 1).

Euglena pyruvate kinase has an absolute requirement forboth Mg2" and K+, as has been found for pyruvate kinases fromother sources (8, 14, 24). The concentration required for maxi-mum activity was found to be 8 mM for Mg2, (as Mg SO4) and10 mm K+ (as KCl) at pH 6.2. If either one of these cations areeliminated from the reaction mixture, the activity of the en-zyme is negligible.The average sedimentation velocity of partially purified py-

ruvate kinase was 8.8 S as determined on sucrose gradientsusing catalase and alkaline phosphatase to calibrate the gradi-ent. No more than one peak of activity can be found in theenzyme preparations by this method.

rhe mol wt of the partially purified enzyme as determinedusing Sephadex G-200 was approximately 240,000 (Fig. 2).Some Kinetic Properties of Pyruvate Kinase. The effect of

increasing concentrations of P-enolpyruvate in the presence ofsaturating levels of ADP on the velocity of the reaction at pH6.8 is shown in Figure 3. The sigmoidal character of suchcurves decreases with decreasing pH. The S0<. for P-enolpyru-vate has been determined from Hill plots (when log v/V-v =0); the S0.5 does decrease from 2 mm at 6.8 to 1.2 mm at pH 6.2.The SQ,0 for ADP was determined by holding the P-enolpy-

ruvate concentration at saturating levels while varying the ADPconcentration at pH 6.2. The S,.., for ADP, as determined fromHill plots, was 0.48 mM. CDP, UDP, and GDP were also usedin place of ADP each at a concentration of 3.9 mm and noneof these gave more than 15% of the activity found with ADP.

Fructose 1,6-diP is a strong activator of Euglena pyruvatekinase (Fig. 3). Maximum stimulation of enzyme activity wasobtained with 2.6 mM fructose-1,6-diP. The SO.- values at pH6.2, 6.5, and 6.8 were approximately 0.2 mm P-enolpyruvatefor the stimulated enzyme.

The extent of stimulation by fructose 1 , 6-diP at low con-centrations of P-enolpyruvate (0.1 mm to 1.4 mM) was greatlyaffected by pH. For example, at a substrate concentration of0.4 mM P-enolpyruvate the activity was stimulated 10-fold byfructose 1 , 6-diP at pH 6.2; the activity was stimulated 68-foldat pH 6.8.When the concentration of ADP is varied and P-enolpyru-

vate kept at saturating levels, there is little stimulation in the

8

6

z

z 4

0

cr0

2

o -94.0 5.0 6.0 7.0 8.0 9.0

pH

FIG. 1. Effect of pH on reaction rate. Potassium acetate-aceticacid buffer was used between pH 4.0 to 5.4; MES buffer wasused in the range of pH 5.8 to 6.5; imidazole-acetate was used be-tween pH 6.8 to 7.6; and tris-acetate was used between pH 8.0 to9.0. Assay concentrations were 0.04 M for the buffer, 3.9 mM forADP, and 6.9 mm for P-enolpyruvate. All other conditions for as-say were described in "Materials and Methods."

presence of fructose 1,6-diP. The apparent S0., (ADP) in thepresence of fructose 1 ,6-diP is 0.4 mm, a small decline fromthe value obtained in the absence of fructose 1, 6-diP.

All the fructose 1, 6-diP was recoverable when incubatedwith the enzyme as determined by the method of Ling et al.(15). In addition, the sedimentation characteristic of the en-zyme determined on sucrose density gradients is not changedwhen the centrifugation is carried out in the presence of fruc-tose 1, 6-diP, or a combination of fructose 1, 6-diP, ADP, andP-enolpyruvate.Ammonium chloride, fructose 6-P, and AMP had no effect

on the kinetics of pyruvate kinase.The effects of ATP, citrate, and Ca2+ on the maximal velocity

of pyruvate kinase at pH 6.2 and at pH 6.8 were examined(Figs. 4, 5, and 6). In all cases, a maximum of 80% inhibitionwas obtained.

All three inhibitors are effective in inhibiting enzyme ac-tivity in the presence of fructose 1,6-diP at both pH 6.2 andpH 6.8 when one of the substrates is at less than saturatinglevels (Table II). Thus, Euiglena pyruvate kinase differs frompyruvate kinases isolated from other sources. The failure offructose 1,6-diP to reverse inhibition may be of considerableimportance in the in vivo regulation of pyruvate kinase andglycolysis. In view of the failure of fructose 1, 6-diP to reverse

Plant Physiol. Vol. 54, 1974619



VACCARO AND ZELDIN

35

Ul

zz0

IL

25

104 105 106

MOLECULAR WEIGHT ( DALTONS)

FIG. 2. Determination of mol wt of Euiglenia PK by gel filtration chromatography. X is the position of PK. The following proteins were usedto calibrate the column: 1: Cyt c; 2: BSA; 3: aldolase; and 4: catalase.

8

6-

z

0z

LU0

2

o w2 4 6 8

U MOLES PEP/ASSAY

FIG. 3. Effect of varying concentrations of P-enolpyruvate on the activity of Eiiglenia pyruvate kinase in the presence or absence of 2.64 mMfructose-1 ,6-dip at pH 6.8. Experimental conditions are described in "Materials and Methods" *-* no fructose-1 ,6-diP; 0--0 with fruc-tose-I, 6-diP.

inhibition of pyruvate kinase by Ca' and ATP, the extent ofreversibility of inhibition of the enzyme was studied.Almost complete recovery of activity from Ca2+ inhibition

was obtained by adding EGTA (Table III). Addition of Mg2+in the presence of Ca2+ results in some recovery.

Addition of both 7 mm ADP and 9.6 mm P-enolpyruvate didnot reverse the inhibition achieved with 3.6 mm Ca2 . Sincesuch a high concentration of substrate did not overcome the

inhibition, it is unlikely that a Ca2+-substrate complex is respon-sible for the inhibition.

Reversal of ATP-dependent inhibition is not complete whenMg2+, K+ or both are added regardless of the concentrations ofMg2+ and K+ employed (Table IV). Attempts to reverse com-pletely ATP inhibition with fructose 1, 6-diP were not success-ful. Almost complete recovery from ATP-dependent inhibitioncould be obtained by dialysis (Table IV).

620 Plant Physiol. Vol. 54, 1974



Plant Physiol. Vol. 54, 1974 PYRUVATE KINASE OF EUGLENA 621

IC*4 tion employed for ATP, Ca2+, and citrate: AMP; adenosine;4 UDP; GDP; CDP; phenylalanine; alanine; NaCl; glucose-6-P,

ketoglutarate acid; 2-deoxy-D-glucose.Total Pyruvate Kinase Activity in Cells During Chloroplast

Development. Euglena gracilis Klebs var. bacillaris and Elu-glena gracilis Z strain were grown and brought to resting con-

\ ~~~~~~~~~ditionsas described in "Materials a;nd Methods." In each caseVI /v \xcultures were exposed to light and total pyruvate kinase levels

50Table II. Effect of Inihibitors oni Euglenia Pyruvate

Kinase in the Presence of Fructose-i,6-diPThe fructose-I ,6-diP concentration was 2.6 mm, ADP was 3.42

mM; and P-enolpyruvate was 0.39 mM.

i MOLES ATP / ASSAY

Concentration ofpH ~~Inhibitor ATP Citrate Ca-+

mM % inhibition

6.8 0.77 6 3 141.93 14 4 283.09 27 25 434.20 40 55 50

6.2 0.77 10 15 191.93 23 20 363.04 39 49 464.20 53 64 59VI/VXI

50Table III. Reversal of Calcium I,ihibitioni by EGTA anid Mg'Values in given %c inhibition. The ADP concentration was 3.47

mM; P-enolpyruvate, 5.4 mm for experiments 1, 2, and 4 and 6.94mm for experiments 3 and 5. All experiments were done at pH 6.8except experiment 5 which was done at pH 6.2. Ca2+ concentra-tions used to obtain desired inhibition are given in parenthesis.

10 _ _ EGTA concentration was 3.86 mM; Mg2+ concentration was 8.570l mM for each experiment.

p MOLES CITRATE / ASSAY

VI /Vo50 _

10

0 5 10p MOLES CA+/ASY

FIG. 4. Effects of ATP on pyruvate kinase activity at pH 6.2and pH 6.8. The concentration of ADP was 3.5 mM; the P-enol-pyruvate concentration was 7 mM; the inhibitor concentrations areshown. Experimental conditions are described in "Materials andMethods." *---, at pH 6.2; X--X at pH 6.8.

FIG. 5. Effects of citrate on pyruvate kinase activity at pH 6.2and 6.8. Details are as in Fig. 4.

FIG. 6. Effects of Ca'+ on pyruvate kinase activity at pH 6.2and pH 6.8. Details are as in Fig. 4.

The following compounds were without effect on pyruvatekinase activity, when tested at the same or greater concentra-

ExperimentReagent

1 2 3 4 5

% inhibition

Ca2+ 37 (2mM) 40(2mM) 52(3mM) 37(2mM) 67(4.3mM)Ca2G+A 2 3 1EGTA

Ca2+ + Mg2t 15 51

Table IV. Reversal of ATP Inzhibitioni by Mg2+,Fructose-1,6-diP, K+, anid Dialysis

The ADP concentration was 3.47 mM; P-enolpyruvate was 6.94mM; Mg2+ was 8.1 mm and ATP was 6.17 mM; K+ was 100 mM.Fructose-I ,6-diP concentration was 2.6 mm. Experiments 1, 2, and4 were carried out at pH 6.2; experiments 3, 5 and 6 were carriedout at pH 6.8.



622 VACCARO 0

estimated at various times during a 72-hr illumination period.During the 72-hr period that pyruvate kinase activity was esti-mated there was no marked change in the total activity on aper cell basis (data not shown).

DISCUSSION

In many systems pyruvate kinase activity has been reportedto consist of more than one active component (13, 17, 22).Using our methods of purification, analysis of the resultantenzyme preparation shows only one detectable component ofactive pyruvate kinase. This does not rule out the existence ofother pyruvate kinase isozymes in Euglena.Many of the physical and kinetic properties of the partially

purified pyruvate kinase from Euiglena are similar to those re-ported for pyruvate kinase purified from yeast and animalsources (4, 10, 12, 28). Activation by fructose-1 ,6-diP in com-bination with inhibition by ATP, citrate, and calcium have beenreported for the yeast and liver enzymes. We have also shownthat varying pH can greatly influence the extent of fructose-I,6-diP-dependent stimulation as has been described for liver(20). The enzyme from Euglena is rather specific in terms ofrequirements for the phosphoryl acceptor and the phosphorylactivator. ADP can serve as a substrate with P-enolpyruvate.Only fructose 1, 6-diP can stimulate the enzyme.

It is worth emphasizing that unlike pyruvate kinase isolatedfrom yeast and animal tissues, the Euglena enzyme remainssignificantly inhibited by Ca2 , citrate, or ATP in the presenceof fructose 1,6-diP and limiting concentrations of P-enolpyru-vate. In the case of ATP inhibition, raising the level of fruc-tose 1,6-diP did not overcome ATP inhibition. This suggeststhat Ca2", ATP, and citrate may play a role in inhibiting pyru-vate kinase activity even in the presence of high levels of fruc-tose 1, 6-diP.

Although extensive information concerning regulatory prop-erties for pyruvate kinase from plant systems is not yet avail-able, the studies which have been reported reveal some veryinteresting aspects about the enzyme from these sources. Inthe cases of Lemna and latex no stimulatory properties havebeen found (14, 19). An extensive study of the pyruvate kinasefrom cotton seed reveals that the enzyme from this plant tissuemost closely resembles the enzymes found in bacteria with re-spect to regulatory properties (6, 7). Euglena gracilis var. bacil-laris enzyme, described in this report, does not resemble anyplant enzyme yet reported with respect to regulatory propertiesbut more closely resembles yeast and animal pyruvate kinases.Since Euglena can grow both as a heterotroph and autotroph itis not surprising that it possesses a pyruvate kinase with regu-latory properties found for heterotrophy with features reflect-ing requirements for autotrophy.

In the absence of information concerning the concentrationand rate of flux of glycolytic intermediates in Euglena, thephysiological significance of some properties of Euglena pyru-vate kinase cannot be fully assessed. The ability of the inhibi-tors to block the fructose 1, 6-diP-stimulated enzyme, however,may be part of a regulatory system which can yield net synthe-sis of carbohydrates involving the substrates and the powerfulfeed-forward activator, fructose 1,6-diP, without permittingthe substrates and the modifier to increase the flux of metabo-lites in the direction of pyruvate formation. Thus, so long asinhibitors are available, increasing concentrations of fructose1,6-diP produced from photosynthesis, for example, wouldcontinue to serve as a reactant in the direction of photosyn-thetic gluconeogenesis instead of activating pyruvate kinase,thus increasing the rate of glycolysis.

ND ZELDIN Plant Physiol. Vol. 54, 1974

In higher plants, starch resulting from photosynthesis isstored within the chloroplast and its synthesis would be closelyassociated with carbon metabolism within the plastid (9). Incontrast, Euglena maintains paramylon depositions exterior tothe organelle and formation of this polysaccharide would beassociated with carbon metabolism from both the plastid andthe cytosol (21). If the regulation of carbohydrate metabolismis achieved as we have proposed, then the regulatory propertiesof Euglena pyruvate kinase would be very important in assur-ing formation of precursors of polysaccharide even in the ab-sence of the strict compartmentalization found for higherplants.Ohmann has reported a 3-fold increase in specific activity of

pyruvate kinase during chloroplast development by growingcells of Z strain Euglena (19). We observe no change in thespecific activity in resting clls. This is not surprising since inthe absence of carbon and nitrogen sources, control of pyru-vate kinase activity without synthesis of new enzyme may bethe most economical means of regulation. The mechanism ofcontrol of pyruvate kinase and the factors influencing thatcontrol during chloroplast development are unknown.

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16. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARRR, AND R. J. RANDALL. 1951.Protein measurenient with Folin phenol reagenit. J. Biol. Cllem. 193: 265-275.

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