Chloroplastic 3-Phosphoglycerate Kinase from Spinach Leaves - NCBI

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Div., New Hartford, CT). The homogenate was pressed through two layers of cheesecloth and the filtrate was centrifuged for 45 minat 13,680g in a GS3 rotor of a.
Received for publication August 29, 1989 and in revised form December 5, 1989

Plant Physiol. (1990) 93, 40-47

0032-0889/90/93/0040/08/$01 .00/0

Isolation and Characterization of the Cytosolic and Chloroplastic 3-Phosphoglycerate Kinase from Spinach Leaves Evelyn Kopke-Secundo, Imre Molnar', and Claus Schnarrenberger* Institut fur Pflanzenphysiologie und Mikrobiologie, Freie Universitat Berlin, Konigin-Luise-Strasse 12-16a, D-1000 Berlin 33 (West), Federal Republic of Germany ABSTRACT

reported here to purify the cytosolic and chloroplastic 3-PGK from spinach leaves and to determine some of the differences or homologies in the structure and the kinetic properties of the two enzymes.

The cytosol and chloroplast 3-phosphoglycerate kinases (3PGK) from spinach (Spinacia oleracea L.) were purified to apparent homogeneity. The procedure included a conventional anionexchange chromatography on DEAE-cellulose and mainly a series of HPLC columns. The charge differences of the two isoenzymes were so small that separation was only successful by anionexchange chromatography on a HPLC SynChropak AX 300 column. The portion of the two isoenzmyes in leaf tissue was estimated as 5% and 95%. The major 3-PGK was associated with isolated chloroplasts while the other 3-PGK was only found in the soluble cell fraction. The specific activity of the purified enzymes were in the order of 800 units (per milligram of protein). The molecular weight for the two 3-PGKs under nondenaturing (size exclusion chromatography) and denaturing (SDS-PAGE) conditions were in the order of 40 kilodaltons, with the cytosolic 3-PGK being slightly smaller than the chloroplastic 3-PGK. An antiserum against the chloroplastic 3-PGK showed only 4.6% cross-reaction of the chloroplastic 3-PGK with the cytosolic 3-PGK. The kinetics for glycerate-3-phosphate and MgATP2- were biphasic. The presence of Na2SO4changed the MgATP2- dependence to linearity but not the glycerate-3-phosphate dependence.

MATERIALS AND METHODS Materials

Spinach (Spinacia oleracea L., Sorte Norveto) was grown in the green houses of the Institute. Chemicals were obtained either from Merck or Sigma and enzymes from Boehringer Mannheim. Cibacron blue (AffiGel Blue) and hydroxylapatite (chromatographic grade) were purchased from Bio-Rad (Munchen/FRG), DEAE-cellulose and P-cellulose from Whatman. Sephadex G 75 and the FPLC Mono Q column were obtained from Pharmacia. All HPLC equipment and columns were purchased through Molnar (Berlin/FRG). Separation of the 3-PGK Isoenzymes The two isoenzymes of 3-PGK were separated by anionexchange chromatography on a HPLC SynChropak AX 300 column. For this purpose the proteins from a 0 to 80% ammonium sulfate fraction of a crude extract were dialyzed against buffer A (10 mM K2HPO4/K.H2P04 [pH 7.5], 10 mM 2-mercaptoethanol, 0.05% NaN3) and loaded onto a 16 x 250 mm HPLC SynChropak AX 300 column (Molnar, Berlin/FRG) equilibrated with buffer A. The column was washed with buffer A until the absorbance at 280 nm had reached the baseline again. Proteins were eluted through a 60 min linear gradient of 0 to 300 mm KCl in buffer A at a flow rate of 6.5 mL min-'. Fractions were tested for 3-PGK activity.

3-PGK2 (ATP: D-3-phosphoglycerate l-phosphotransferase, EC 2.7.2.3), which is associated with glycolysis, gluconeogenesis, and the reductive pentose phosphate cycle, catalyses the reversible interconversion of 3-PGA and DPGA with concomitant utilization or generation of ATP. In pea shoots, a cytosolic and chloroplastic form of 3-PGK has been separated by isoelectric focusing (2) and been characterized (19). One 3PGK (presumably the chloroplastic enzyme) has been purified from spinach (7, 9, 13), spinach chloroplasts (20), and Beta vulgaris (6). In addition, McMorrow and Bradbeer (17) reported the successful purification of the two barley leaf 3PGKs, although no electrophoretic display or other properties (except data on immunological cross-reactions) of the pure enzymes were presented. It was the purpose in the work

Subcellular Localization Previous reports have established that 3-PGK isoenzymes in plants occur in the cytosol and in the chloroplasts, respectively (2). To identify the subcellular compartment of the respective 3-PGK isoenzymes of spinach, intact chloroplasts were isolated from spinach leaves by differential centrifugation (8) and the 3-PGK isoenzyme pattern from such a preparation was examined by HPLC on a SynChropak AX 300 as described above.

' Present address: Blucherstrasse 22, D-1000 Berlin 61 (West), FRG. 2 Abbreviations: 3-PGK, 3-phosphoglycerate kinase; DPGA, 1,3diphosphoglycerate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPHT, high-performance hydroxylapatite; 3PGA, 3-phosphoglycerate; TPI, triosephosphate isomerase.

40

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3-PHOSPHOGLYCERATE KINASES FROM SPINACH LEAVES

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2

1

3

4

120 a D

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94

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9

10

11

12 min

13

14

15

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Figure 2. Silver stained SDS polyacrylamide gel with purified 3-PGK isoenzymes (see Fig. 1; Tables I and 11) and with purfied 3-PGK from isolated chloroplasts. Lane 1, mol wt markers; lane 2, cytosolic 3PGK of peak I (1.2 Ag); lane 3, chloroplastic 3-PGK of peak 11 (0.6 ,qg); lane 4, 3-PGK isolated from chloroplasts (0.5 Ag). 600

Figure 1. Elution profile of 3-PGK after HPLC on a SynChropak AX 300 anion-exchange column. Proteins of a dialyzed 0 to 80% ammonium sulfate fraction from a crude or chloroplast extract were loaded and eluted with a linear gradient of 0 to 300 mm KCI in buffer A. Top, Separation of proteins from a crude extract; bottom, separation of proteins from isolated chloroplasts.

Purification of the 3-PGK Isoenzymes All steps were carried out at 4°C except HPLC runs which were performed at 22C. HPLC experiments were performed with a Knauer HPLC System (Knauer, Berlin/FRG). During HPLC runs, proteins were monitored at 280 nm and recorded on a compensating strip chart recorder. After loading of the sample onto a HPLC column, the column was washed with the respective equilibration buffer (until zero absorbance at 280 nm was reached again) before applying a salt gradient. All enzyme preparations were dialyzed before chromatography with the equilibration buffer of the subsequent column. About 500 g of fresh spinach leaves were homogenized in three volumes of buffer A (see above) in a Waring Blendor (Waring Prod. Div., New Hartford, CT). The homogenate was pressed through two layers of cheesecloth and the filtrate was centrifuged for 45 min at 13,680g in a GS 3 rotor of a

E P.

460

I

0-L.

A. I-

*

8

260

26

8 :w

1

is

25

20

30

35

man

Figure 3. Elution profile of cytosolic (small peak) and chloroplastic (large peak) 3-PGK and TPI after HPLC on a SynChropak AX 300 anion-exchange column. Proteins of a 3-PGK preparation after conventional chromatography on DEAE cellulose were applied.

Sorvall RC 2B centrifuge. The supenatant was collected and solid ammonium sulfate was slowly added up to 35% saturation under constant stirring and maintaining the pH at 7.5. After 15 min, the solution was centrifuged for 45 min at 13,680g. The ammonium sulfate concentration of the supernatant was increased up to 80% and, after 15 min of stirring,

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KOPKE-SECUNDO ET AL

9)4

j.-.

T~x,

I_

*14

_

--E

Figure 4. Silver stained SDS polyacrylamide gel with purified cytosolic 3-PGK from spinach leaves. Lane 1, mol wt markers; lane 2, cytosolic 3-PGK (1.2 ,ag).

the proteins were centrifuged for 45 min at 13,680g. The pellet was resuspended in a minimal volume of buffer A and dialyzed twice for at least 8 h each time against buffer A. Proteins were then loaded onto a 4.5 x 32 cm DEAE-cellulose column (DE 52, Whatman, Springfield Mill, Maidstone, Kent/England) equilibrated with buffer A. The column was washed with two column volumes of the same buffer and the sample was eluted with 2 L of a 0 to 300 mm KCl gradient in the same buffer. Both 3-PGKs eluted as one peak. For the separation of the isoenzymes, the fractions were pooled, dialyzed, loaded onto a 16 x 250 mm HPLC SynChropak AX 300 column equilibrated with buffer A, and chromatographed as described for the 'separation of the 3-PGK isoenzymes' (see above). For further purification, the 3-PGK isoenzymes were processed separately.

Plant Physiol. Vol. 93, 1990

After precipitation with 35 to 80% ammonium sulfate and dialysis, the cytosolic 3-PGK was applied to a 2 x 90 cm Sephadex G-75 column equilibrated with buffer B (50 mM Tricin [pH 7.5], 10 mm 2-mercaptoethanol, 0.05% NaN3). Proteins were eluted with buffer B at a flow rate of 1.2 mL min-'. Next, fractions with cytosolic 3-PGK activity eluting from the Sephadex G-75 column, were pooled, dialyzed, and applied to a 5.8 x 100 mm HPLC hydroxylapatite column (Bio-Gel HPHT, Bio-Rad, Richmond, CA) equilibrated with buffer B. 3-PGK was eluted with a linear, 60-min gradient of 0 to 350 mm KH2PO4/K2HPO4 (pH 7.5) in buffer B at a flow rate of 0.4 mL min-'. In the two final purification steps, chromatography on a 5 x 50 mm FPLC Mono Q column was performed. The pooled fractions of the HPHT column were dialyzed and loaded onto a FPLC Mono Q column. The cytosolic 3-PGK was eluted with a 30-min linear gradient of 0 to 300 mM KCI in buffer B at a flow rate of 2 mL min-'. After dialysis, fractions with 3-PGK activity were rechromatographed on a Mono Q column as before. The purification of the chloroplastic 3-PGK was initiated by ammonium sulfate precipitation from 35 to 80% saturation and chromatography on DEAE-cellulose as described for the cytosolic 3-PGK. After dialysis, 3-PGK was applied to a 3 x 16 cm hydroxylapatite column equilibrated with buffer B. The column was washed with 2 volumes of buffer B, one volume of 1 M NaCl in buffer B, and one volume of 1 mM K2HPO4/KH2PO4in buffer B. 3-PGK was eluted with a linear gradient (400 mL) of 1 to 200 mm K2HPO4/KH2PO4 in buffer B. After dialysis, 3-PGK was chromatographed on a 8 x 250 mm HPLC SynChropak AX 300 column equlibrated with buffer A. After loading, the column was washed with one volume of buffer A and proteins were eluted with a 30 min gradient of 0 to 200 mm KCl in buffer A at a flow rate of 3 mL min-'. Since separation of isoenzymes was unsatisfatory because of a too high protein load, all fractions with 3-PGK activity were pooled, dialyzed, and rechromatographed on the SynChropak AX 300 column under the same conditions, yielding good separation of the 3-PGK isoenzymes. To remove TPI it was necessary to include next an affinity chromatography on 2 x 20 cm P-cellulose P 11 (Whatman) column equilibrated with buffer C (20 mM Tes [pH 7.5], 10 mM 2-mercaptoethanol, 0.05% NaN3). After loading the dialyzed 3-PGK, the column was washed with two column volumes of buffer C and the chloroplastic 3-PGK was eluted with 260 mL of a linear gradient of 0 to 100 mM 3-PGA in

Table I. Purification Scheme of the Cytosolic 3-PGK from Spinach Leaves Activity units

Crude extract DEAE-cellulose

26847 18813 709 12311 788 682 400

Protein mg

5490.0 762.0 5.4 SynChropak AXP 300a 38.3 SynChropak AXP 300b 4.0 Sephadex G75 HPHT 2.3 1 st Mono Q 1.3 2nd MonoQ 75 0.094 b a Cytosolic 3-PGK. Chloroplastic 3-PGK.

Specific Activity units mg-1

Purification

5 25 130 321 199 297 308 798

1 5 26 64 40 48 62 160

-fold

Yield %

100.0 70.0 2.6 46.0 3.0 2.5 1.5 0.3

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3-PHOSPHOGLYCERATE KINASES FROM SPINACH LEAVES

Table II. Purification Scheme of the Chloroplastic 3-PGK from Spinach Leaves Activity units

Protein mg

5 400 20 333 Crude extract 14 972 543 DEAE-cellulose 364 13 056 Hydroxylapatite 7 500 31 1 st SynChropak AX 300 4.5 325 2nd SynChropak AX 300a 8.6 3367 2nd SynChropak AX 300b 2.4 1228 P-cellulose 2.4 1289 3rd SynChropak AX 300 1.3 813 TSK G 2000 SW 0.9 737 4th SynChropak AX 300 b a Chloroplastic 3-PGK. Cytosolic 3-PGK.

buffer C. Minor impurities of such 3-PGK preparations were removed by chromatography on a 8 x 250 mm SynChropak AX 300 column, a 7.5 x 300 mm HPLC TSK G 2000 SW column, and again a 8 x 250 mm SynChropak AX 300 column. The TSK G 2000 SW column for size exclusion chromatography was equilibrated with buffer A, loaded with 400 ,L enzyme preparation, and developed with buffer A at a flow rate of 1 mL min-'. Subsequently, chromatography on a 8 x 250 mm SynChropak AX 300 column was performed as described above. The purity of the enzymes was checked by SDS-PAGE. Electrophoresis was carried out in 20% SDS polyacrylamide gels according to Laemmli (10). Silver staining was performed according to Merril et al. ( 18). Enzyme Assays The assay for the 3-P-glycerate kinase (EC 2.7.2.3) was carried out spectrophotometrically in the direction leading from 3-PGA to DPGA (4). The reaction mixture was composed of 100 mM Tricin/HCl (pH 7.0), 6 DTT, 4 mm ATP, 4 mM MgCl2, 10 mM 3-PGA, 0.2 mM NADH, 1 unit GAPDH (EC 1.2.1.12), and enzyme in a total volume of 1 mL. For the determination of triosephosphate isomerase (EC 5.3.1.1) the enzyme assay contained 200 mM triethanolamine (pH 7.6), 4 mM D,L-glyceraldehyde-3-P, 0.2 mM NADH, 2.6 units glycerol-3-P dehydrogenase (EC 1.1.1.8), and enzyme in a total volume of 1 mL. The rate of NADH disappearence was monitored at 365 nm using an Eppendorf 1101 M or a Kontron Uvicon 810 recording spectrophotometer. All measurements were performed at 22°C. One unit of activity represents the conversion of 1 mol NADH per min. The specific activity is defined as units per mg protein. For Km studies the activity of 3-PGK was measured at 8 or 10 different concentrations of one substrate. For all kinetic analyses a standard deviation was estimated out of three independent measurements. The results were presented as Eadie-Hofstee plots. Protein was determined with Coomassie brilliant blue G-250 as described by Bradford (5) with bovine serum albumin as standard. Mol Wt Determination The mol wt of the native 3-PGK isoenzymes was determined by HPLC gel filtration. Twenty ,L of a 3-PGK sample

Specific Activity units mg-'

4 28 36 240 73 392 511 537 568 800

Purification -fold

0 7

9 60 18 98 128 134 142 200

Yield %

100 73 64 37 2 17 6 6 4 4

was applied onto a 7.5 x 300 mm Bio Sil TSK 250 column (Bio-Rad, Richmond, CA) equilibrated with buffer D (50 mM Na2SO4, 20 mM NaH2PO4/Na2HPO4 [pH 7.5], 10 mM 2mercaptoethanol, 0.05% NaN3). Proteins were eluted with buffer D at a flow rate of 1 mL min-'. Markers used were

thyroglobulin (670,000), y-globulin (158,000), ovalbumin (44,000), myoglobin (17,000), and vitamin B-12 (1,350). The subunit mol wt of the 3-PGK isoenzymes was estimated by SDS-PAGE (10). Markers used were phosphorylase b (94,000), BSA (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (20,100), and a-lactalbumin (14,4000).

Immunochemical Methods Antiserum against the purified chloroplastic 3-PGK was raised in a rabbit. Approximately 100 ,g purified protein in Freund's complete adjuvant was injected into the leg muscle of an animal. After 3 weeks, boosts with 100 ,g protein in Freund's incomplete adjuvant were given three times at weekly intervals. Three d after the last boost blood was collected and, after 4 h at room temperature, serum was obtained through centrifugation. For immunotitration (8) of the 3-PGK, 10 L of a constant amount (6.25 ,ug protein) of 3-PGK and 10 uL of antiserum, or dilutions of it in one-tenth PBS, were incubated for 5 min. Then, 10 ,uL with of a 12% suspension of Protein A-coated Staphylococcus aureus Cowan I cells (Immuno-Precipitin, BRL, Gaithersburg, MD) were added. After incubation for 2 min, the suspension was centrifuged at 14,000g for 1 min in a Biofuge A (Heraeus Christ) and 20 MAL of the supernatant was used for an enzyme activity test. RESULTS Separation of the 3-PGK lsoenzymes The two 3-PGK isoenzymes from spinach could only be separated on a SynChropak AX 300 HPLC column into two peaks whereby the first peak (P I), which amounted to 5% of the total 3-PGK activity, eluted at 76 mM KCI and the second peak (P II), which amounted to 95% of the total 3-PGK activity, eluted at 95 mM KC1 (Fig. lA). When proteins from isolated chloroplasts were subjected to the same separation

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Plant Physiol. Vol. 93, 1990

Purification of the Cytosolic 3-PGK The first purification step for the cytosolic 3-PGK was a conventional chromatography on a large DEAE-cellulose column. After dialysis, the pooled 3-PGK fractions were applied directly to a preparative SynChropak AX 300 column for the separation of the two isoenzymes. Since the cytosolic 3-PGK was only slightly contaminated with TPI (Fig. 3), it was not necessary to perform a chromatography on P-cellulose or Cibacron blue for TPI removal. As a next purification step, the 3-PGK preparation was applied to a Sephadex G 75 column which was followed by FPLC on a HPHT column and two HPLCs on Mono Q columns. The specific activity of the purifled cytosolic 3-PGK was 798 units/mg and, according to the criteria of SDS-PAGE, the enzyme was considered pure with a minor comtamination (Fig. 4). Table I shows the purification scheme.

t: I-

c 9 co

rA

Antiserum

(el)

Purification of the Chloroplastic 3-PGK For the purification of the chloroplast 3-PGK, a conventional chromatography on a large DEAE-cellulose column was performed. This was followed by chromatography on a conventional hydroxylapatite column. Since, at this point, the 3-PGK preparation still contained 364 mg protein (Table II), the first HPLC on a SynChropak AX 300 column did not result in a separation of the 3-PGK isoenzymes due to the limited binding capacity of the column. However, separation was successful during rechromatography on the same column

c 40 0

es L.

0

0

(Fig. 3).

40 (U

1'01 ioo Antiscrum (el)

101

Figure 5. Immunotitration of the two purified 3-PGK isoenzymes from spinach leaves with a specific antiserum raised against the chloroplastic 3-PGK from spinach. The amount of antiserum yielding 50% precipitation of 3-PGK (see arrows) was used as a measure for the reaction of the antiserum with the respective 3-PGK. In all experments the same amount of enzyme was used. The bars represent the standard deviation of three measurements. A, Cytosolic 3-PGK; B, chloroplastic 3-PGK.

procedure, a single 3-PGK peak eluted at 93 mm KCI (Fig. 1 B). Therefore, the first peak with a crude extract represented the cytosolic and the second peak the chloroplastic 3-PGK. Further evidence for this conclusion was obtained from SDSPAGE of the two purified 3-PGK isoenzymes and the purified 3-PGK from isolated chloroplasts (Fig. 2): purified 3-PGK from peak P II (lane 3) and the purified 3-PGK from isolated chloroplasts (lane 4) showed the same migration pattern, whereas the purified 3-PGK of peak P I (lane 2) migrated slightly ahead of the other proteins.

An additional problem in the purification procedure was the presence of TPI activity which eluted together with the chloroplastic 3-PGK during anion exchange (Fig. 3), size exclusion, and hydroxylapatite chromatography. To remove TPI, it was necessary to perform a chromatography on a Pcellulose. 3-PGK could be eluted specifically with a linear gradient of 0 to 100 mm 3-PGA. Alternatively, TPI could be removed by chromatography on a Cibacron blue column. In this case TPI did not bind whereas 3-PGK did and, in turn, could be eluted in one step with 500 mM ammonium sulfate. For the final steps of purification, a third HPLC on a SynChropak AX 300 was performed which was followed by a size exclusion HPLC on a TSK G 2000 SW column and a fourth HPLC on a SynChropak AX 300 column. The chloroplastic 3-PGK was pure according to the criteria of SDSPAGE (Fig. 2) and had a specific activity of 800 units/mg. The purification scheme for the chloroplastic 3-PGK is shown in Table II. 3-PGK was also purified from isolated chloroplasts by chromatography on a preparative SynChropak AX 300 column. This procedure resulted in an almost pure form of the enzyme (Fig. 2), although the absolute amounts were small. Structural Properties

From size exclusion chromatography on a HPLC TSK G 2000 SW a mol wt of 39 kD was deduced for both the native cytosolic and chloroplastic 3-PGK (data not shown). During SDS-PAGE mol wt of 40.7 kD for the cytosolic 3-PGK and of 41 kD for the chloroplastic 3-PGK were obtained (Fig. 2).

3-PHOSPHOGLYCERATE KINASES FROM SPINACH LEAVES

45

310

30

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20

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0 0

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v/[MgATP2-] (U/mM)

20

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400

Figure 6. Eadie-Hofstee plot for MgATP2- and 3-PGK with the cytosolic and chloroplastic 3PGK.

300

s 200

= 200 0-

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v/[3-PGAJ (U/mM)

v/[MgATP2-J (U/mM)

Table Ill. Apparent Km Values for the 3-PGK Isoenzymes in the Absence and in the Presence of Na2SO4 If two values are given, the first value represents the apparent Km at low substrate concentrations and the second value the apparent Km at high substrate concentrations. Chloroplastic 3-PGK Cytosolic 3-PGK 3-PGA 3-PGA MgATP2 MgATP2mM

Without Na2SO4

Spinacha

Spinachb Peac

0.24/1.46 0.28/1.22 0.39/1.29 0.34/1.72 0.24 0.63 0.55 0.59/1.60 0.54/1.25 0.70

With Na2SO4

Spinach 5 mM 10 mm 20 mm 40 mm

0.34 0.44 0.37 0.38

0.26/1.36 0.24/1.08 0.35/0.98 0.43/0.92

Yeastd 40mM

0.55

1.00

0.47 0.50 0.43 0.44

a Present work. Larsson-Raznikiewicz (11). d Scopes (23). Anderson (19). b

400

0.34/1.53

0.33/1.38 0.34/1.75 0.50/1.90 c Pacold and

Therefore, the two 3-PGK isoenzymes are monomeric proteins. As illustrated in Figure 5, an immunotitration of the purified cytosolic and chloroplastic 3-PGK with an antiserum raised against purified chloroplastic 3-PGK showed a crossreactivity of 4.6% between the two 3-PGK isoenzymes when the same amount of 3-PGK protein was used. Thus, the two 3-PGK isoenzymes can be considered as structurally related but nonidentical polypeptides. Kinetic Characteristics of the 3-PGK lsoenzymes Both purified 3-PGK isoenzymes from spinach had a pH optimum at pH 7.5 with a strong drop of activity toward higher and lower pH values. They did not follow MichaelisMenten kinetics, but showed biphasic kinetics (Fig. 6). Both 3-PGK isoenzymes exhibited a 4 to 6 times higher affinity for their respective substrates in the lower substrate range than in the higher range (Table III). In addition, a substrate inhibition occurred in the high substrate range with respect to MgATP2-. To examine whether the Mg2" concentration had an effect on the nonlinear kinetics, kinetic measurements with the chloroplastic isoenzyme were performed at different Mg2` concentrations in the range of 2 to 8 mm Mg2+. The results indicated that the Mg2+ concentrations in this range had no influence on the nonlinearity of the 3-PGK kinetics (data not shown). The nonlinear kinetics of 3-PGK from yeast could be

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Plant Physiol. Vol. 93, 1990

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v/[MgATP2-1 (U/mNI) 50

ET AL

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Figure 7. Kinetics of the cytosolic and chloroplastic 3-PGK for MgATP2- and 3-PGA in the presence of 40 mm Na2SO4.

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converted to linear kinetics through addition of 40 mM Na2SO4 (23). To examine if this also applied to the 3-PGK isoenzymes from spinach, kinetic experiments were performed in the presence of 5, 10, 20, and 40 mm Na2SO4. All these Na2SO4 concentrations had no influence on the 3-PGK kinetics of the spinach leaf 3-PGKs with respect to the substrate 3-PGA, however, converted to linearity with respect to MgATP2-. Representative data for 40 mM Na2SO4 were documented in Figure 7.

DISCUSSION In the present study, we succeeded in purifying the cytosol and chloroplast 3-PGK to apparent homogeneity, with a specific activity of 800 units mg-' for both isoenzymes. The 3-PGK isoenzymes from spinach are monomers with a mol wt of approximately 40 kD, differing only by a few hundred daltons. These mol wt were somewhat smaller than the published values for animal and plant tissue 3-PGKs (7, 13, 20, 22). The chloroplastic isoenzyme amounted to 95% and the cytosolic component to only 5% of the total 3-PGK activity, which is in accordance with the findings for the 3-PGK isoenzymes from barley (17) but in contrast to findings of Anderson and Advani (2) who reported 90% 3-PGK activity for the cytosolic and 10% 3-PGK activity for the chloroplastic isoenzyme in pea leaves.

An immunochemical comparison of the two 3-PGK isofrom spinach showed a cross-reaction of only 4.6%. This low degree of cross-reaction is in accordance with findings on other cytosol and chloroplast specific isoenzymes of the sugar phosphate metabolism (21); however, it is in contrast to the finding of McMorrow and Bradbeer (17) who found an almost 100% cross-reaction for the purified 3-PGK isoenzymes from barley. The two 3-PGK isoenzymes from spinach show biphasic kinetics with respect to their substrates MgATP2- and 3-PGA (Fig. 7). Both 3-PGK isoenzymes have an approximately 5 times higher affinity for their respective substrates in the lower substrate range than in the higher range (Table III). Nonlinear kinetics have also been reported for the 3-PGK from yeast (11, 23), spinach (12, 14), and erythrocytes (1). LarssonRaznikiewicz (1 1) reported that the nonlinear kinetics of yeast 3-PGK are dependent upon the ratio of Mg2" to ATP. However, our results on the 3-PGK kinetics of spinach showed no conversion to linearity in the Mg2" to ATP range reported by Larsson-Raznikiewicz for the yeast 3-PGK. Scopes (23) showed that, in the presence of 40 mm Na2SO4, the nonlinear kinetics of the yeast 3-PGK converted to linearity for both substrates. In our experiments it was demonstrated, that, with respect to the substrate MgATP2-, the kinetics of the two spinach leaf isoenzymes were converted to linearity, but that enzymes

3-PHOSPHOGLYCERATE KINASES FROM SPINACH LEAVES

Na2SO4 had no effect on the kinetics with respect to the substrate 3-PGA. Table III, which gives a summary of the apparent Km values for the 3-PGK of the present study as well as of published data from other sources, indicates that all Km values for the 3-PGK of spinach, pea, and yeast are in the same order. Lavergne et al. ( 14) reported biphasic kinetics for the chloroplastic 3-PGK from spinach but a higher affinity in the higher substrate range than in the lower one. This is in contrast to our results and the published data from the above mentioned sources. Furthermore, they calculated a Hill coefficient of 1.35 and postulated that the 3-PGK functions as an allosteric enzyme. Since the 3-PGK is a monomer, this interpretation seems to be unlikely. Through x-ray analysis of yeast and horse muscle 3-PGK, Banks et al. (3) illustrated the existence of only one binding site for each respective substrate. By means of NMR studies of the yeast 3-PGK, Transwell et al. (25) confirmed the results of Banks et al. (3) and, furthermore, showed that S042- binds competitively to the MgATP2- binding site. This competitive binding of S042- to the 3-PGK MgATP2- binding site may be an explanation for the conversion of the biphasic kinetics to linear kinetics for the 3-PGK of spinach with respect to the substrate MgATP2- as demonstrated in our studies. Many enzymes in the cell do not occur as separate entities, but as multienzyme complexes. Weber and Bernhard (26) showed through kinetic studies on the halibut muscle 3-PGK that the intermediate DPGA has a half-life of 30 min at physiological pH, in aqueous solution and at room temperature. Bound to 3-PGK under the same conditions, DPGA was stable for several days. If the enzyme-substrate complex 3-PGK-DPGA was looked upon as the substrate for the reaction with GAPDH, Michaelis-Menten kinetics were obtained with respect to this enzyme-substrate complex. These results were further confirmed by Srivastava and Bernhard (24) through NMR studies of the 3-PGK-DPGA complex of muscle tissue. Macioszek and Anderson (15) in their study on the photosynthetic induction of the 3-PGK/NADP-linked GAP DH from pea chloroplasts, also suggested that one DPGA is transferred directly from 3-PGK to GAPDH. In further studies by Malhotra et al. ( 16) it has been shown that one 3-PGK molecule binds to each of the four GAPDH subunits and that this complex kept all catalytic activities. Considering the possibility of a 3-PGK-GAPDH complex in an intact cell and the biphasic kinetics of isolated 3-PGK, one may suppose that the biphasic kinetics could be due to the formation of a heterologous 3-PGK-GAPDH complex since purified 3-PGK from diverse sources is usually coupled to commercially available muscle or yeast GAPDH. LITERATURE CITED 1. Ali M, Brownstone YS (1976) A study of phosphoglycerate kinase in human erythrocytes. II Kinetic properties. Biochim Biophys Acta 445: 89-103 2. Anderson LE, Advani VA (1970) Chloroplast and cytoplasmic

Three distinct isoenzymes associated with the reductive pentose phosphate cycle. Plant Physiol 45: 583-585 3. Banks RD, Blake CCF, Evans PR, Haser R, Rice DW, Hardy GW, Merrett M, Phillips AW (1979) Sequence, structure and activity of phosphoglycerate kinase: a possible hinge-bending enzyme. Nature 279: 773-777 enzymes.

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4. Bergmeyer HU (1983) Methods of Enzymatic Analysis, Vol 2. Verlag Chemie, Weinheim 5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 6. Cavell S, Scopes RK (1976) Isolation and characterization of the "photosynthetic" phosphoglycerate kinase from Beta vulgaris. Eur J Biochem 63: 483-490 7. Fifis T, Scopes RK (1978) Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography. Biochem J 175: 311-319 8. Kruger I, Schnarrenberger C (1983) Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves. Eur J Biochem 136: 101-106 9. Kuntz GWK, Eber S, Kessler W, Krietsch H, Krietsch W (1978) Isolation of phosphoglycerate kinase by affinity chromatography. Eur J Biochem 85: 493-501 10. Laemmli MK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685 11. Larsson-Rainikiewicz M (1967) Kinetic studies on the reaction catalysed by phosphoglycerate kinase. II. The kinetic relationships between 3-phosphoglycerate, MgATP2- and activating metal ion. Biochim Biophys Acta 132: 33-40 12. Larsson-Rainikiewicz M (1983) Some comparative studies of phosphoglycerate kinase from spinach, wheat and yeast. Acta Chem Scand 37: 657-659 13. Lavergne D, Bismuth E (1973) Simultaneous purification of the two kinases from spinach leaves: ribulose-5-phosphate kinase and phosphoglycerate kinase. Plant Sci Lett 1: 229-236 14. Lavergne D, Bismuth E, Champigny ML (1974) Further studies of phosphoglycerate kinase and ribulose-5-phosphate kinase of the photosynthetic carbon reduction cycle: regulation of the enzymes by the adenine nucleotides. Plant Sci Lett 3: 391-397 15. Macioszek J, Anderson LE 1987 Changing kinetic properties of the two-enzyme phosphoglycerate kinase/NADP-linked glyceraldehyde-3-phophate dehydrogenase couple from pea chloroplasts during photosynthetic induction. Biochim Biophys Acta 892: 185-190 16. Malhotra OP, Kumar A, Tikoo K (1987) Isolation and quaternary structure of a complex of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Indian J Biochem Biophys 24: 16-20 17. McMorrow EM, Bradbeer JW (1987) The isolation and the immunological properties of chloroplast and cytoplasmic phosphoglycerate kinase from barley. In J Biggens, ed, Progress in Photosynthetic Research, Vol III. Martinus Nijhoff Publ., Dordrecht, pp 6.483-6.486 18. Merril CR, Goldman D, Sedman SA, Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211: 14371438 19. Pacold I, Anderson LE (1975) Chloroplast and cytoplasmic enzymes. VI. Pea leaf 3-phosphoglycerate kinases. Plant Physiol 55: 168-171 20. Persson L-O, Olde B (1988) Synthesis of ATP-polyethylene glycol and ATP-dextran and their use in the purification of phosphoglycerate kinase from spinach chloroplasts using affinity partitioning. J Chromatogr 457: 183-193 21. Schnarrenberger C (1987) Regulation and structure of isozymes of sugar phosphate metabolism in plants. Isozymes Curr Top Biol Med Res 16: 223-240 22. Scopes RK (1973) 3-Phosphoglycerate kinase. In PD Boyer, ed, The Enzymes, Vol 8. Academic Press, New York, pp 335-351 23. Scopes RK (1978) The steady-state kinetics of yeast phosphoglycerate kinase. Eur J Biochem 85: 503-516 24. Srivastava DK, Bernhard SA (1986) Metabolite transfer via enzyme-enzyme complexes. Science 234: 1081-1086 25. Tanswell P, Westhead EW, Williams JP (1976) Nuclear-magnetic-resonance study on the active site structure of yeast phosphoglycerate kinase. Eur J Biochem 63: 249-262 26. Weber JP, Bernhard SA (1982) Transfer of 1,3-diphosphoglycerate between glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase via an enzyme-substrate complex. Biochemistry 21: 4189-4194