Plant Physiol. (1985) 79, 242-2470032-0889/85/79/0242/06/$01.00/0

Pyruvate Decarboxylase from Zea mays L.'I. PURIFICATION AND PARTIAL CHARACTERIZATION FROM MATURE KERNELS ANDANAEROBICALLY TREATED ROOTS

Received for publication February 7, 1985 and in revised form April 25, 1985

THOMAS C. LEE AND PAT J. LANGSTON-UNKEFER*2Department ofAgronomy, University of Wisconsin, and United States Department ofAgriculture,Agricultural Research Service, Madison, Wisconsin 53706

ABSTRACT

Pyruvate decarboxylase (PDC) was purified from mature, dry maizekernels and from roots of anaerobically treated maize seedlings andpartially characterzed. PDC was purified to a specific activity of 96 unitsper milligram protein from kernels and to 41 units per milligram proteinfrom root. The subunit molecular masses were estimated to be 61,000and 60,000 for kernel PDC and 59,000 and 58,000 for root PDC. ThepH optimum for each enzyme was 5.8. Since the pH optimum is nearlyone pH unit below the value reported for the cytoplasm of anaerobicallymetabolizing maize roots (pH 6.7 1 0.2), we investigated the effects ofpH 5.8 and 6.6 on the cooperative kinetics observed for PDC from eachsource. The maximum Hill coefficients (nH) were much greater at eachpH for the kernel PDC (pH 5.8, nH = 2.5 and pH 6.6, nH = 3.2) than forthe root PDC (pH 5.8, nH = 14A and pH 6.6, nH = 1.8). The cooperativekinetics observed with respect to pyruvate were asymmetric. Potassiuminhibited maize PDC and was competitive with pyruvate (root PDC K,=16 millimolar and kernel PDC KJ= 10 millimolar).

Pyruvate decarboxylase, PDC3 (EC 4.1.1.1) catalyzes the de-carboxylation of pyruvate to acetaldehyde; ADH (EC 1.1.1.1)then catalyzes the reduction ofthe acetaldehydeto ethanol. Thesetwo enzymes thus catalyze a pathway in which NAD+ is regen-erated under anaerobic conditions. Pyruvate, the first substrateof this pathway, occurs at a major branch point in glycolysisbetween anaerobic and aerobic metabolism, and the regulationbetween these two metabolic routes is likely to occur both at thelevel of PDC synthesis and of PDC activity. The syntheses ofPDC and ADH are specifically enhanced under anaerobic con-ditions (9, 18), and significant increases in the activities of theseenzymes are observed (9, 18). One functional mechanism pro-posed to control this anaerobic pathway is the regulating effectof cytoplasmic pH on PDC activity (7). PDC is inactive at thepH ofaerobically metabolizing tissues. However, the cytoplasmic

' Supported by United States Department of Agriculture CRG 83-CRCR-1-1201, the University of Wisconsin, and United States Depart-ment of Energy. The investigations reported were included in the disser-tation submitted by T. C. L. to the Graduate College, University ofWisconsin, Madison, in partial fulfillment of the requirements for thePh.D. degree.

2Present address: INC4, MS C345, Los Alamos National Laboratory,Los Alamos, NM 87545.

3Abbreviations:PDC, pyruvate decarboxylase (EC 4.1.1.1); ADH, al-cohol dehydrogenase (EC 1.1.1.1); EDTA-Na, ethylenediaminetetraace-tic acid, sodium salt; TPP, thiamine pyrophosphate.

pH of anaerobically metabolizing tissues is significantly lowerthan the pH of aerobically functioning tissues and this decreaseallows PDC to function. The PDC's from sweet potato root (14,15), wheat (7, 22), and brewer's yeast (3, 24) bind pyruvatecooperatively; thus, pyruvate levels have also been proposed asa regulatory mechanism. The effect(s) that changing the pH mayhave on these cooperative kinetics has not been investigatedusing a plant PDC. An additional mechanism to regulate thisanaerobic pathway in plants is the inhibition of PDC by phos-phate, a mechanism similar to the phosphate inhibition of yeastPDC (3). A complete study of the possible effects of phosphatesalts on plant PDC activity has not been done.

Since maize seedlings must carry out anaerobic metabolism tosurvive growth in anaerobic conditions such as in water-loggedsoils, the regulation ofPDC is important. Therefore, we purifiedand partially characterized PDC from anaerobically treatedmaize roots and from maize kernels. The effects of pH and ofpyruvate concentration on the cooperative kinetics were inves-tigated for the enzyme from each of these two sources. Thepossible inhibition of maize PDC by phosphate and potassiumwas also studied.

MATERIALS AND METHODS

Reagents and Buffers. Sepharose 6B was purchased from Phar-macia Inc.4 The mol wt standards and biochemical reagents werefrom Sigma. All other chemicals were of reagent grade. Buffer Acontained 100 mM K-phosphate (pH 6.5), 50 mm TPP, 5 mmMgCl2, and 5 mm 2-mercaptoethanol. Buffer B contained 25 mMimidazole-HCI (pH 6.8), 5 mM MgC92, 50 zlM TPP, and 5 mm 2-mercaptoethanol.

Purification of Pyruvate Decarboxylase from Kernels. Mature,dry maize (Zea mays L., W438 x A632) kernels (500 g) wereground and the flour was homogenized with 1 L of buffer A ina Waring Blendor (medium speed for 1 min). All purificationsteps were at 4C. The homogenate was filtered through cheese-cloth and centrifuged (10,000g, 25 min). Protamine sulfate (2%w/v) was added to the supernatant with approximately 1 ml ofprotamine sulfate solution added per 20 ml of supernatant. Thesolution was stirred for 10 min, clarified by centrifugation(15,000g, 20 min) and pH adjusted to 7.5 with I M K2HPO4.The solution was brought to 25% saturation with solid(NH4L)SO4, sfirred for 45 min, and centrifuged (20,000g, 20min). The supernatant was brought to 37% saturation with(NH4)2SO4, stirred for 45 min, and centrifuged (30,000g, 20

4The mention of firm names or trade products does not imply thatthey are endorsed or recommended by the United States Department ofAgriculture or by the University ofWisconsin over other firms or similarproducts not mentioned.

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min). This last pellet was resuspended in buffer B (-30 ml). Thesuspension was clarified by centrifugation (20,000g, 20 min) anddialyzed (6 h) against buffer B (2 x 21). Precipitated protein wasremoved by centrifugation (25,000g, 20 min), and the samplewas applied to a column of DEAE-cellulose (2.2 x 17 cm) whichhad been equilibrated in buffer B. The resin was washed withapproximately 1 bed volume of buffer B, and the enzyme waseluted with a linear gradient of KCG (0-0.5 M) in 250 ml ofbufferB. The fractions containing enzyme activity (20-25 ml) werepooled and concentrated in dialysis tubing surrounded by PEG20,000 to a final volume of 4 to 5 ml. An aliquot (2 ml) wasapplied to a column of Sepharose 6B (1.85 x 110 cm) equili-brated in buffer A which here contained 0.75 mm TPP, andethylene glycol (15% v/v). The enzyme was eluted with thismodified buffer A at a flow rate of 7 ml h-'. The fractionscontaining PDC activity were pooled and the enzyme was storedat 4C.

Purification of Pyruvate Decarboxylase from Root Tissue.Kernels (W438 x A632) were surface sterilized with 1% NaOClfor 10 min, allowed to imbibe with aerated distilled H20 over-night, and then rolled between sheets of presterilized germinationpaper which had been moistened with 2 M autoclaved CaSO4.The rolls of kernels were placed vertically in containers whichhad been soaked in 50% NaOCG to minimize fungal growth andwere fitted to allow for a continuous flow of N2 through them.The seedlings were grown in the dark with air flowing throughthe container until the primary roots were 6 cm long (3 d), andthe plants were then grown (48 h) with N2 flowing through thecontainers.

Roots (300 g from 1000 g kernels) were homogenized in aWaring Blendor with buffer A (900 ml) which contained poly-vinyl polypyrolidone (5% w/v) as an aid in the removal ofphenolics. This step and all others were at 4C. The homogenatewas filtered through cheesecloth and centrifuged (10,000g, 25min). Protamine sulfate (2% w/v) was added to the supernatant(-1 ml/20 ml) and the preparation was stirred for 10 min andcentrifuged (15,000g, 25 min). The pH of the supernatant was

adjusted to 7.5 with 1 M K2HPO4. Solid (NH4)2SO4 was added(35% saturation) and the preparation was stirred for 1 h andcentrifuged (20,000g, 30 min). The resulting pellet was resus-

pended in 8 to 10 ml of buffer A with 0.25 mM TPP and ethyleneglycol (15% v/v) added. The sample was centrifuged (25,000g,20 min) and concentrated to a volume of 4 ml in dialysis tubingsurrounded by PEG-20,000. An aliquot (2 ml) was applied to acolumn of Sepharose 6B (1.85 x 110 cm) which had beenequilibrated in buffer A with 0.25 mm TPP and ethylene glycol(15% v/v) added. The PDC activity was eluted with this bufferusing a flow rate of 7 ml h-'. The fractions containing theenzyme activity were pooled and stored at 4C.Enzyme Activity and Protein Assays. PDC activity was meas-

ured by the assay of Oba and Uritani (15) in which the acetal-dehyde produced is reduced by ADH. The coupled oxidation ofNADH was monitored at 340 nm at 25°C. The reaction mixtureused to monitor only the purification contained 100 mM Mes-KOH (pH 5.8), 20 mm sodium pyruvate, 0.26 mM NADH, 1

mM TPP, 1 mM MgCl2, and 10 units of yeast ADH per ml ofassay solution. The procedure ofMcClure (1 1) was used to ensurethat appropriate quantities of the ADH were used in the coupledassay. We also added additional coupling enzyme to test assaysand found no increase in detectable PDC activity. One unit ofPDC activity catalyzed the oxidation of 1 umol of NADH/min.A Cary 219 spectrophotometer was used. Full scale deflectionsof 2, 1, 0.5, and 0.1 O.D. were used as needed. Protein wasdetermined by the method of Bradford (4) using BSA as astandard.

Kinetic Procedures. PDC used for all kinetic studies was di-alyzed against 500 volumes of 50 mM His-HCl (pH 6.5), 5 mM

MgCl2, 0.25 mm TPP, and 5 mM 2-mercaptoethanol to removeall potassium. The kinetic studies utilized purified enzyme prein-cubated (30 min, 25C) in 50 mM His-HG (pH 6.5), 30 mmTPP, 30 mm MgCl2, and 2 mM DTT; 100 mM His-HC replacedthe Mes-KOH in the assay buffer described above. Any changesin these procedures are given in the figure legends All enzymevelocities were determined in the linear portion of the reaction.At low concentrations ofpyruvate up to 1 h was needed to reachthe linear portion of the reaction. The data were analyzed usingthe method of least squares to compute the line of best fit.SDS Electrophoresis. SDS denaturing PAGE was done using

a modification of the procedure of Laemmli (8) in which the geland electrode buffers were made 1 mM EDTA-Na. Gels werestained for protein using a solution of0.05% Coomassie brilliantblue R-250 in 10% acetic acid and 50% methanol in water. Gelswere destained using a solution of 10% acetic acid and 20%methanol in water.

Determination of Subunit Mr. The subunit mol wt of PDCfrom kernel and root tissues were determined using the methodof Weber and Osborn (25). A 12% acrylamide gel was used inthe SDS denaturing electrophoresis procedure described above.The mol wt standards used were BSA (Mr 66,200), ovalbumin(Mr 45,000), carbonic anhydrase (M, 31,000), soybean trypsininhibitor (Mr 21,500), and lysozyme (M, 14,400).

RESULTS

Purification of Pyruvate Decarboxylase. PDC was purified toapparent homogeneity from maize kernels to a specific activityof 96 units/mg protein by the steps summarized in Table I.Homogeneity was determined by SDS electrophoresis of purifiedPDC. PDC was also purified from anaerobically treated maizeroots to a specific activity of 41 units/mg protein by the proce-dure summarized in Table II. PDC obtained from each of thetissues was very stable and could be stored in 50% ethylene glycolfor 3 months at -20C with no loss of activity. A single speciesof PDC was observed from each of the sources when analyzed

Table I. Purification ofPDCfrom Maize Kernels

Steps Total Total SeiiSteps Protailn Enzyme Spctivity Purification YieldActivity

mg IU unitsl -fold %mgCrude extract 2048 512 0.25 1.0 100Protamine 1536 522 0.40 1.4 102Ammonium sulfate(25-37%) 220 200 1.10 4.4 39

Anion exchange-DEAE-Cellulose 19 145 7.70 31 29

Gel filtration-Sepha-rose-6B 0.85 82.5 96.4 385 15

Table II. Purification ofPDCfrom Anaerobically Treated Maize Roots

TtlTotal SpecificSteps Protein Enzyme Activity Purification YieldActivity

mg iU unitsl -fold %mgCrude extract 627 379 0.6 1.0 100Protamine sulfate 384 300 0.8 1.3 79Ammonium sulfate(0-35%) 23 227 10.0 17.0 60

Gel filtration-Sepha-rose-6B 3.1 129 41.6 68.5 34

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separately by gel filtration chromatography and by anion ex-change chromatography. PDC activity consistently eluted fromeach of the chromatography steps in a single, sharply definedand symmetrical region.

Subunit M, of Pyruvate Decarboxylase. The subunit mol wtofPDC from kernels were 61,000 and 60,000 (Fig. 1). The SDSelectrophoresis analysis of the purified PDC obtained from rootsresolved three proteins of Mr 59,000, 58,000, and 44,000 (Fig.1). The two major peptides of PDC from each of these sourceswere present in equal or nearly equal amounts. The third proteinspecies (Mr 44,000) in the root PDC was less than 10% of thetotal protein. The native mol wt of PDC was not estimated;however, it was clear that the protein was very large since all ofthe enzyme activity eluted from gel filtration chromatography ina single, sharply defined region immediately after the void vol-ume from a column of Sepharose CL-6B (fractionation range10,000-4,000,000).Investiption of the Kinetic Behavior of PDC. A lag phase was

observed for enzymic conversion after the substrate was addedto the resting enzyme. For this reason, all measured velocitiesare the steady state velocities, not time-zero velocities. The effectofpH on the kinetic behavior ofPDC from both kernel and roottissues was investigated. Each of the enzyme forms had a pHoptimum at 5.8 (Fig. 2). In order to determine the number ofionizable groups involved in the catalytic process, the data wereplotted as (log Vm=IVm,,, optimum) versus pH (15) (Fig. 2). Theslope of the limiting line on the acidic side is 1.15 and on theless acidic side is 2.35, suggesting one and two ionizable sidechains, respectively. This is consistent with the observation ofJordan et al. (6), who found apparent pKs of 5.3, 5.8, 5.95, and6.25 for yeast PDC. The slopes reported here account for onegroup with a pK more acidic than the pH optimum, one groupwith a pk at the pH optimum, and two groups with pKs lessacidic than the pH optimum.The cytoplasmic pH ofanaerobically metabolizing maize roots

was estimated to be 6.5 to 6.9 (17), and thus we chose toinvestigate the cooperative kinetics with respect to pyruvate at apH within this range. At the pH value 6.6, root PDC had 67%of its maximum activity and kernel PDC had 55% of its maxi-mum activity. The two PDCs exhibited markedly different de-grees of cooperativity in response to pH and to changing concen-

5.0

0-j

FIG. 1. EstirpolyacrylamideMr standards us

(c) carbonic an

21,500; and (e)

1.0

0.8

-

I0

E0x

EcoE

0-j

0.6

0.4

oL I1I ½I14 S 6 7 8

pHFIG. 2. Influence of pH on activity and analysis of the number of

ionizable groups of purified PDC. (A), Kernel; (0), root PDC. Enzymewas first incubated in 50 mm His-HCI (pH 6.5), 30 mM TPP, 30 mmMgC92, and 2 mm DTT (30 min, 25C). The assay mixture (1 ml)contained 100 mm His-HCI, 20 mM sodium pyruvate, 0.26 mm NADH,I mM TPP, 1 mM MgCl2, and 10 units yeast ADH. The pH was adjustedafter addition of all reagents.

Table III. Comparison ofRoot and Kernel PDC and Effect ofpH onthe Hill Coefficients

Hill CoefficientspH S0.5 I.p.a

Minimum MaximummM nH

Root5.8 1.2 0.4 0.9 1.46.6 7.6 3.0 1.3 1.8

Kernel5.8 0.9 1.0 1.2 2.56.6 4.5 3.0 1.3 3.2

a Inflection point is the pyruvate concentration within the transitionbetween cooperative and Michaelis-Menten kinetics.

c trations of pyruvate (Table III; Fig. 3). PDC from root exhibitsvery little cooperativity with respect to pyruvate when analyzedusing a Hill plot of velocity data obtained at the pH optimum,

_dts 5.8. A Michaelis-Menten plot of these pH 5.8 data appearshyperbolic (not shown) and a Lineweaver-Burk plot (Fig. 4B) isapparently linear (r = 0.9878; app. Km= 0.5 mm pyruvate). At

e s pH 6.6, root PDC is cooperative with respect to pyruvate, andthe Hill coefficient increases with decreasing pyruvate concentra-

* |. s * | . , . tion. PDC from kernel is, however, clearly cooperative with0 0.2 0.4 0.6 0.8 1.0 respect to pyruvate at pH 5.8 and 6.6 (Table III; Fig. 3). Michae-

lis-Menten plots (not shown) of these data are sigmoidal, asRELATIVE MOBILITY expected since the maximum Hill coefficients are significantly

nation of subunit Mr of root and kernel PDC using SDS- greater than 1. As the pH was increased from 5.8 to 6.6, thegel electrophoresis; 12% polyacrylamide gels were used. maximum Hill coefficient increased (Table III).sed were: (a) BSA, Mr 66,200; (b) ovalbumin, Mr 45,000; Both PDCs exhibit positive cooperativity with pyruvate. Ithydrase, Mr 31,000; (d) soybean trypsin inhibitor, Mr must be emphasized that the sigmoidal saturation is not sym-lysozyme. Mr 14,400. metrical (Fig. 3) as with normal allosteric systems (21). The

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DECARBOXYLASE FROM ZEA MAYS L.

2.0

1.0

I

x

0

E

(D

o

-1.0

-2.0

-3.0'-2.4 - 1.0 0 1.0 2.0

LOG [Pyruvate, mM]

FIG. 3. Hill plot ofthe dependence ofsteady state rate on the pyruvateconcentration at pH 5.8 and pH 6.6 for kernel and root PDC. (K),Kernel PDC; (R), root PDC; (1), pH 6.6; (2), pH 5.8. Incubations andassays of PDC were as described in the legend of Figure 2.

transition from nH> 1 to nH = 1 (or Michaelis-Menten kinetics)occurs at or below the half-saturating pyruvate concentration.PDC was incubated in buffer containing 30 mM TPP and 30

mM MgCl2 to ensure that the enzyme was saturated with TPPand Mg before the kinetic analyses were done, and the assaymixtures contained I mM TPP. These cofactor concentrationswere greater and the length of the incubation was longer thanthe conditions required to ensure saturation of other Mg-TPPrequiring enzymes (1, 13). No difference in Hill coefficient wasobserved between enzyme preparations which had been incu-bated (30 min) with the high concentrations of TPP comparedwith enzyme which had not been incubated in this buffer. Theseresults suggest that the PDCs were saturated with TPP.

Effects of Phosphate and Potassium Ions on PDC Activity.The possible inhibition of maize kernel PDC by phosphate wasexamined (Table IV). Phosphate, added as phosphoric acid, didnot inhibit PDC activity at low concentrations and was a poorinhibitor at higher concentrations. However, potassium chlorideand monobasic potassium phosphate which were equally effec-tive inhibitors of PDC, were more effective inhibitors than wasphosphoric acid. Chlorides of potassium, lithium, ammonium,and sodium (25 mM) were tested as possible inhibitors of PDCactivity. Potassium chloride was the best inhibitor tested. Sodiumchloride did not inhibit PDC activity. The extent ofthe inhibitiondepended on the specific monovalent cation, suggesting that theinhibitor was the cation and that of the cations, potassium wasthe best inhibitor. Potassium, added as KCI, was an inhibitorcompetitive with pyruvate as shown by analysis of a Lineweaver-Burk plot and a Dixon plot (Fig. 4). A Ki value of 16 mm wasestimated from these plots for root PDC. Potassium inhibitionof root PDC was clearly linear at the six concentrations ofpyruvate we examined. The intersection ofthe lines in the Dixonplot is above the X-axis, which is expected for a linear, compet-itive inhibitor. A Kiof 10 mm for potassium was estimated fromthe Dixon plot for the kernel PDC studies. In contrast to theresults obtained with the root PDC, the inhibition by potassiumof the kernel PDC did not appear to not be linear as determinedby analysis of a Dixon plot (pH 5.8) (Fig. 5). However, kernel

20 40 60 80[POTASSIUM, mM]

0 100 200

"/PYRUVATE mM X 10'2FIG. 4. A, Dixon plot of the inhibition by KCI of the steady state

velocity of the root PDC at different, fixed, low concentrations ofpyruvate. B, Lineweaver-Burk plot of the data. Enzyme was first incu-bated in 50 mM His-HCI (pH 6.5), 30 mm TPP, 30 mm MgCI2, and 2mM DTT (30 min, 25'C). The assay (1 ml) contained 100 mM His-HCI(pH 5.8), 0.26 mM NADH, 1 mM Tpp, I mM MgC2, and 10 units yeastADH. A, Pyruvate concentrations used were: (A), 2 mM; (B), 1.5 mM;

(C), I mM; (D), 0.8 mM; (E), 0.6 mM; (F), 0.5 mM. B, The KCI concen-trations used were: (A), 0 mM; (B), 17 mM; (C), 50 mM; (D), 100 mM.

PDC exhibits positive cooperativity with respect to pyruvate inthe absence of potassium (Fig. 3; Table III). Plots of v versus[potassium] were indicative of cooperativity at 5 mM pyruvatebut did not indicate cooperativity at 2 and 1 mM pyruvate (Fig.6). This increasing cooperativity is expected as the substrateconcentration is increased for an enzyme assayed in the presenceof both a substrate (pyruvate) which binds cooperatively to theenzyme and a competitive inhibitor (potassium) which does notbind to the enzyme in a cooperative manner (20). This interpre-tation is consistent with the finding that potassium is a compet-itive inhibitor that does not bind cooperatively to root PDC.

DISCUSSION

PDC was purified from maize kernels to a higher specificactivity (96 units/mg) than the value reported by Singer (22) forthe enzyme from wheat germ (50 units/mg by our calculations).The specific activity of 96 units/mg protein compares favorablywith the 80 to 90 units/mg protein reported for highly purifiedPDC from yeast (5). The specific activity obtained for PDC ofmaize root was approximately 3-fold greater than the specificactivity reported for PDC of sweet potato root (14), the onlyother reported purification of PDC purified from root.

K I

R IK2R2

I 1

PYRUVATE 245

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LEE AND LANGSTON-UNKEFER

Table IV. Effect of Variving Concentrations ofKCI, H3PO4, andKH2PO4 on Kernel PDC Activity

Assay mixtures contained 100 mM His-HCI (pH 6.5), 0.26 mM NADH,1 mM TPP, I mM MgCI2, 20 mM pyruvic acid, and 2 mm DTT and 10units ADH in I ml.

Inhibitor PDC InhibitionActivitya nibto

mM m units %Control

0 8.6 0H3PO4

10 8.6 025 8.1 650 7.0 1 8

KH2PO410 7.1 1825 4.7 4650 3.0 76

KCI10 6.3 2725 4.1 5250 2.1 75

LiCI25 7.0 19

NH4CI25 7.0 19

NaCI25 8.3 4

a'Average of three values.

20 0 20 40 60

IKCI, mM)80 100

FIG. 5. Dixon plot ofthe inhibition by KCI ofthe steady state velocityof kernel PDC at different, fixed concentrations of pyruvate. Incubationand assays were as described in the legend of Figure 4. Pyruvate concen-trations used were: (A), 5 mM; (B), 2 mM; (C), 1 mM.

PDC of maize kernal and root were each present in a singleform as analyzed by anion exchange and gel filtration chroma-tography. This observation was consistent with the findings ofLaszlo and Lawrence (9) who analyzed the isozyme compositionof PDC from anaerobically and aerobically grown maize roots.The subunit mol wt were consistent with the values reported forPDC of sweet potato (14) and for PDC of yeast (19). Theobservation of two subunits of slightly differing mol wt for bothmaize PDCs was also consistent with the values reported foryeast PDC (23). This finding differed from the single mol wtspecies reported for PDC from sweet potato (14). The origin of

z

OLo 20 40 60 80 tOO

[POTASSIUM, mM]FIG. 6. Influence of KCI on the steady state velocity of kernel PDC

at different, fixed concentrations of pyruvate. Incubation and assays wereas described in the legend of Figure 4. Pyruvate concentrations usedwere: (A), I mM; (B), 2 mM; (C), 5 mM.

the small amount of the low mol wt species (44,000 Mr) in theroot PDC is unknown but is presumed to be a result ofproteolyticdegradation of the PDC during the purification since the relativeamount ofthis protein increased slightly after storing the purifiedpreparation for several weeks.The lag phase observed with maize PDC is also characteristic

ofPDC from yeast (23). We have investigated the lag phase, andthe results of this investigation will be reported elsewhere.PDC found in mature maize kernels will probably function in

anaerobic metabolism during seed germination. We comparedthe properties of the kernel PDC with those ofthe PDC obtainedfrom anaerobically treated roots and found significant kineticdifferences between these two enzymes. PDC from the roots ofanaerobically treated maize seedlings exhibited essentially Mi-chaelis-Menten kinetics at pH 5.8 with respect to pyruvate, whilethe enzyme from the kernel exhibited a high degree of coopera-tivity with pyruvate at pH 5.8. Both enzymes exhibited cooper-ative kinetics at pH 6.6 although the maximum Hill coefficientfor the kernel enzyme was much greater than the maximum Hillcoefficient for the root enzyme. Roberts et al. ( 17) estimated thecytoplasmic pH in aerobically and anaerobically grown maizeroots as 7.3 ± 0.2 and 6.7 ± 0.2, respectively. Thus, maize rootPDC with a pH optimum of 5.8, will probably be functioning invivo at pH values that are well above its optimum and which arein a range where PDC activity is strongly affected by cooperativebinding of pyruvate. This cooperative binding decreases as thepH decreases, and the enzyme is less dependent upon pyruvatefor activation.The saturation curves observed for maize PDC display a shift

of the cooperative range to lower pyruvate concentrations tosuch an extent that the cooperative range appears below half-saturation (Fig. 3). This asymmetry is in contrast to normalallosteric systems which display saturation curves which seem tobe nearly symmetrical and in which the sigmoidal range is limitedon both sides by hyperbolic parts. Normally, the half-saturationpoint lies approximately in the middle of the sigmoidal range;and this is easily seen in Hill plots (1). Bisswanger (1) observedthat asymmetrical cooperativity can sometimes be explained onthe basis of the concerted model of Monod et al. ( 12) and then

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later developed the slow transition model (2) as another expla-nation of asymmetrical cooperativity. Such asymmetrical curveshave been observed for pyruvate dehydrogenase (1) and for PDCof sweet potato (14). The cooperative kinetics observed withrespect to pyruvate were the same for untreated PDC and forPDC preincubated in high concentrations of Mg2+-TPP.

Potassium was an effective, competitive inhibitor ofPDC frommaize while phosphate was not as effective as an inhibitor. Thesefindings were consistent with the results of Kenworthy andDavies (7) who also found that potassium but not phosphate wasan inhibitor of wheat PDC. PDC was inhibited by potassiumconcentrations of the same order of magnitude as reported to becontained in castor beans (10) and oat seeds (16). Thus, theinhibition ofPDC by potassium could be relevant in vivo.

In summary, the regulation of maize PDC activity in vivo isprobably due to a complex interaction of effectors, some ofwhich not only exert direct effects on PDC but also influenceeach other. The activity is directly affected by the pH and by thecooperativity with respect to pyruvate; these cooperative kineticsare in turn influenced by the pH.

LITERATURE CITED

1. BISSWANGER H 1974 Regulatory properties of the pyruvate-dehydrogenasecomplex from Escherichia coli. Eur J Biochem 48: 377-387

2. BISSWANGER H 1984 Cooperativity in highly aggregated enzyme systems. Aslow transition model for the pyruvate dehydrogenase complex from Esch-erichia coli. J Biol Chem 259: 2457-2465

3. BOITEAUX A, B HEss 1970 Allosteric properties ofyeast pyruvate decarboxylase.FEBS Lett 9: 293-296

4. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72: 248-254

5. GOUNARIS AD, I TURKENKOPF, LL CIVERCHIA, J GREENLIE 1975 Pyruvatedecarboxylase III. Specificity restrictions for thiamine pyrophosphate in theprotein association step, sub-unit structure. Biochim Biophys Acta 405: 492-499

6. JORDAN F, DJ Kuo, EU MONSE 1978 A pH-rate determination ofthe activity-pH profile of enzymes. Application to yeast pyruvate decarboxylase dem-onstrating the existence of multiple ionizable groups. Anal Biochem 86:298-302

7. KENWORTHY P, DD DAVIES 1976 Kinetic aspects of the regulation of pyruvicdecarboxylase. Phytochemistry 15: 279-282

8. LAEMMLI UK 1970 Cleavage ofstructural proteins during assembly ofthe headof bacteriophage T4. Nature 227: 680-685

9. LAZLO A, P ST LAWRENCE 1983 Parallel induction and synthesis ofPDC andADH in anoxic maize roots. Mol Gen Genet 192: 110-117

10. Lowr JNA, JS GREENWOOD, CM VOLLMER 1982 Mineral reserve in castorbeans: the dry seed. Plant Physiol 69: 829-833

1 1. MCCLUREWR 1969 A kinetic analysis ofcoupled enzyme assays. Biochemistry8: 2782-2786

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