Chloroplast and Cytoplasmic Enzymes - NCBI

Plant Physiol. (1975) 55, 168-171

Chloroplast and Cytoplasmic Enzymes VI. PEA LEAF 3-PHOSPHOGLYCERATE KINASES' Received for publication June 10, 1974 and in revised form August 1, 1974 IVAN PACOLD AND LOUISE E. ANDERSON2

Department of Biological Scien7ces, University of Illinois ABSTRACT Pea (Pisum sativum) leaf chloroplastic and cytoplasmic 3-phosphoglycerate kinases (ATP: D-3-phosphoglycerate 1phosphotransferase, EC 2.7.2.3) have similar Michaelis constants for ATP, 0.7 and 0.55 mM, for ADP, 0.18 and 0.22, and for 3-P-glycerate, 0.59 and 0.54 mM at low substrate concentrations, and 1.6 and 1.25 mM at high substrate concentrations. Both enzymes are inhibited by ADP and AMP in the ATP-utilizing direction and by ATP and AMP in the ATPgenerating direction and are controlled by energy charge. Apparently, whether the cytoplasmic and chloroplastic kinases in the plant cell will participate in the reductive pentose phosphate cycle and gluconeogenesis or in glycolysis will be determined by the environment in the cell compartment and not by the differential properties of the enzymes themselves.

3-Phosphoglycerate kinase (ATP: D-3-phosphoglycerate 1phosphotransferase, EC 2.7.2.3) catalyzes the reversible interconversion of 3-P-glycerate and 1,3-diP-glycerate with the concomitant utilization or generation of ATP. This enzyme participates in the reductive pentose phosphate cycle and in glycolysis and gluconeogenesis in green plants. Although the chloroplastic and cytoplasmic forms of the enzyme have been separated by isoelectric focusing (4), further characterization of the two forms has been lacking. The purpose of the experiments described here was to determine and compare the properties of the two forms of 3-P-glycerate kinase. The pea (Pisum sativum L.) chloroplastic and cytoplasmic 3-P-glycerate kinases have identical pH optima, with Pglycerate as substrate, and similar or identical Michaelis constants for ATP, ADP, and 3-P-glycerate. Both forms are inhibited by AMP and ADP and the Ki values differ by a factor of 1.5. The only naturally occurring nucleoside triphosphate which serves as a phosphate donor is ATP. The pea leaf 3-P-glycerate kinases are apparently very similar enzymes and also resemble in many, but not all, respects the enzymes from pea seeds (8), yeast (13-15), rabbit muscle (13), and the chemosynthetic bacterium Hydrogenomonas facilis

(19). 'This research was supported by grants from the National Science Foundation (GB 28160) and University of Illinois, Chicago Circle, Research Board to L. E. A. and Sigma Xi to I. P. This study is included in a thesis submitted by Ivan Pacold to the Graduate School, University of Illinois at Chicago Circle, in partial fulfillment of the requirements for the degree of Master of Science. 2To whom reprint requests should be sent.

at

Chicago Circle, Chicago, Illinois 60680

We have previously reported that the pea leaf P-glycerate kinases are under energy charge control (21).

MATERIALS AND METHODS Plant Material. Pea plants (Pisum sativum, var. Little Marvel) were grown in a mixture of vermiculite and soil in a growth room for 10 to 20 days under 16 hr of artificial light and 8 hr of dark. P-glycerate Kinase Assay. A number of different assay methods were used. Unless otherwise specified the enzyme was assayed using the coupled assay method of Buicher (9). Each cuvette contained 10 umoles of ATP, 10 /moles of MgC12, 9 /,moles of 3-P-glycerate, 4.15 ,umoles of NADH, 50 jumoles of tris, HCl, pH 7.5, 100 /tg of glyceraldehyde-3-P dehydrogenase, and enzyme in a total volume of 1 ml. The rate of NADH disappearance was followed at 340 nm using a Gilford 2400 recording spectrophotometer. Temperature was 25 C. One enzyme unit represents disappearance of 1 ,tmole NADH per min. Specific activity is defined as enzyme units per mg protein. Protein Determination. Protein was determined by the biuret method modified by acetone precipitation (see Ref. 4) or directly by the spectrophotometric method of Warburg and Christian as described by Layne (18). Purification of 3-P-glycerate Kinase. Chloroplasts were prepared as described previously (4) except that the procedure was scaled up for kg quantities of tissue and the entire shoot of the pea plant was used. The fractionation and purification procedures were performed at 4 C. The chloroplasts were resuspended in 10 mm potassium phosphate buffer, pH 7.5, and disrupted by release from a French pressure cell at 10,000 p.s.i. The extract was brought to pH 7.5 with NaOH, 1 g PVPP3 added for each 10 ml of extract and the solution was centrifuged at 40,000g for 30 min. The supernatant was used in the ammonium sulfate precipitation (see below). The supernatant solution from the chloroplast isolation constituted the cytoplasmic fraction. It was brought to pH 7.5 with NaOH and treated with 1 g of PVPP per 10 ml of the extract. Debris and PVPP were removed by centrifugation at 20,000g for 20 min. The same procedure was used for further purification of the chloroplastic and cytoplasmic kinases. The chloroplast or cytoplasmic extracts were treated with 0.313 g of ammonium sulfate per ml of extract to obtain 50% ammonium sulfate saturation. After 15 min the precipitate was removed by 15 min centrifugation at 20,000g. The supernatant solution was treated with 0.137 g of ammonium sulfate per ml to obtain 70% saturation. After 15 min the precipitate was collected by 3

168

Abbreviation: PVPP: polyvinylpolypyrollidone (polyclar).

Plant Physiol. Vol. 55, 1975

PEA LEAF 3-PHOSPHOGLYCERATE KINASES

Table 1. Purification of Chloroplastic and Cytoplasmic 3-P-glycerate Kinases

169

of NaCl, 100 ,ug of glyceraldehyde-3-P dehydrogenase, ATP varied in 12 even reciprocal intervals between 0.05 and 5.0 ,umoles, and 3-P-glyceric acid kinase in a total volume of 1.0 Puriml. Total Specific fication Activity Protein Activity Activity In the determination of Km for ADP, the cuvette contained 1 enzyme 0.4 /umole of NAD+, 1 pmole of glyceraldehyde-3-P, 50 j-moles enzyme mg/ml units/mg fold units/ml of potassium phosphate, pH 7.5, 50 ,ug of glyceraldehyde-3-P protein dehydrogenase, 10 umoles of MgC12, ADP varied in six even Cytoplasmic extract af4.1 0.16 25100 0.39 reciprocal intervals between 0.1 and 1.0,mole, and 3-P-glyter PVPP treatment' Ammonium sulfate frac8.7 22.4 3200 0.39 ceric acid kinase in a total volume of 1.0 ml. tionation In the determination of Ki for AMP or ADP, the cuvette DEAE-Sephadex 2.2 1.45 3.9 3580 1 .52 contained 0.1 ,umole of NADH, 50 pmoles of tris-HCl, pH 7.5, Sephadex G-50 8.8 1.0 23 3320 8.8 250 ptmoles of NaCl, 10 Mmoles of MgCl,, 5 MLmoles of 3-P-gly2.5 4.4 Chloroplastic extract af1440 0.57 ceric acid, 100 pg of glyceraldehyde-3-P dehydrogenase, 0.1 to ter,PVPP treatment' 2.5 Mumoles of AMP or ADP, ATP varied in six even reciprocal Ammonium sulfate frac- 67 10.5 11 1200 6.4 tionation intervals between 0.50 and 5.0 ,umoles, and 3-P-glyceric acid 1.8 40 0.08 400 > 22.5 DEAE-Sephadex kinase in a total volume of 1.0 ml. In the determintaion of Km for 3-P-glyceric acid, the cu1 2.5 kg of pea shoots were used in the preparation of the cytoplasmic enzyme vette contained 0.1 ,tmole of NADH, 10 Mmoles of MgCl,, 50 and 1.5 kg in the preparation of the chloroplastic enzyme. ,umoles of tris-HCl, pH 7.5, 250 Mmoles of NaCl, S or 1 jpmole of ATP, 100 trg of glceraldehyde-3-P dehydrogenase, 3-Pcentrifugation, resuspended in a small volume of 10 mm potassium phosphate buffer, pH 7.5, and dialyzed against a con- glyceric acid varied in 12 even reciprocal intervals between 0.1 tinuous flow of the phosphate buffer for 2 hr in a Bio-Rad and 10 Mmoles, and 3-P-glyceric acid kinase in a total volume dialyzer. The dialyzed fraction was adsorbed on DEAE A-50 of 1.0 ml. Determination of Nucleoside Triphosphate Specificity. VarSephadex (2.54 X 5 cm gel column) and eluted with 400 ml of a linear 0 to 0.3 M NaCl gradient in 10 mm potassium phos- ious ribonucleoside 5'-triphosphates were substituted for ATP phate, pH 7.5. The most active fractions were pooled and in the assay which contained 10 tumoles of 3-P-glyceric acid, stored at -20 C in the case of the chloroplastic enzyme, or, in 0.1 Mmole of NADH, 50 tris-HCl, pH 7.5, 10 Mmoles of the case of the cytoplasmic enzyme, brought to 70% ammo- MgCl1, 100 jug of glyceraldehyde-3-P dehydrogenase, 2.5 nium sulfate saturation. The precipitate from the cytoplasmic ,umoles of either ATP, GTP, CTP, ITP, TIP, or UTP, and preparation was collected by centrifugation, resuspended in a 3-P-glyceric acid kinase in a total volume of 1.0 ml. After small volume of phosphate buffer, and subjected to gel filtra- measuring the activity with substituted nucleotides, 2.5 ,umoles tion on a Sephadex G-50 column in 10 mm phosphate buffer of ATP (0.1 ml) were added into the reaction mixture to deter(3 X 40 cm). The most active fractions were collected and mine whether those nucleoside triphosphates had an inhibitory stored at -20 C. They were stable for at least 2 months. The effect on the ATP-utilizing activity of 3-P-glyceric acid kinase. Reagents. Pea seeds were obtained from the Atlee Burpee results of a typical purification are given in Table I. When the most highly purified fractions were subjected to polyacryl- Company. Metal-free ammonium sulfate was obtained from amide gel electrophoresis six or seven protein bands were ob- Mann, Sephadex from Pharmacia, and biochemicals from served; the enzyme is apparently only partially purified. Addi- Sigma. All other reagents were the highest quality commercially available. tional purification was not attempted. Determination of pH Optima. pH optima were determined by assaying for 3-P-glyceric acid kinase activity in the ATP RESULTS utilizing direction in presence of acetate or tris-HCl buffers Chloroplastic and cytoplasmic 3-P-glycerate kinases have ranging in pH between 5 and 10. The cuvette contained 10 ,umoles of 3-P-glyceric acid, 5 ,umoles of ATP, 10 umoles of been purified 40- and 23-fold, respectively, from Pisum satiMgCl2, 0.1 ,umole of NADH, 100 ,ug of glyceraldehyde-3-P vum shoot tissue (Table I). The most highly purified fractions dehydrogenase desalted by passage through a Sephadex G-25 are contaminated with several other proteins. This enzyme has column, 100 ,tmoles of tris-HCl or sodium acetate, and 3-P- been purified to apparent homogeneity from spinach leaves by glyceric acid kinase in a total volume of 1 ml. The reaction Lavergne and Bismuth (17), but the chloroplastic and cytomixture was incubated for 15 min at 25 C prior to the initia- plasmic forms were not separated. tion of the reaction by addition of 3-P-glyceric acid kinase and With 3-P-glycerate as substrate the chloroplastic and cytothe coupling enzyme, glyceraldehyde-3-P dehydrogenase. The plasmic 3-P-glycerate kinases have similar broad pH optima activity was recorded, and pH of the assay mixture was mea- with a plateau between pH 6.75 and 9.25 (data not shown). sured at 20 C using a Radiometer pH meter 26. Comparably broad and flat pH optima have been earlier deDetermination of Kinetic Constants. The activity of a con- scribed for the pea seed kinase with a pH optimum between stant amount of 3-phosphoglyceric acid kinase was measured pH 6.7 and 9.7 (8) and for yeast and rabbit skeletal muscle at 6 or 12 different concentrations of the substrate in question. kinases with the optima between pH 6.0 and 9.2 (13). Each set of reaction rates and corresponding substrate conThe chloroplastic and cytoplasmic 3-P-glycerate kinases centrations was analyzed using the program of Hanson, Ling, have similar Km values for ATP, 0.7 and 0.55 mM, respecand Havir (11), and the IBM 360 computer at the University tively (Table II). Larsson-Raznikiewicz (14), working with of Illinois, Chicago Circle Computer Center. Mean Km and Ki yeast 3-P-glycerate kinase, obtained two Michaelis constants, values and standard error were estimated as described pre- 0.5 and 1 mM, in two ATP concentration ranges. In the presviously (5). ent experiments with pea leaf kinases, since the double reciproIn the determination of Km for ATP, the cuvette contained cal plots were linear only one constant was obtained for each 0.1 pmole of NADH, 10 t.moles of MgCI2, 5, 2.5, or 1 ,umoles enzyme. The Km (ATP) values for the pea leaf enzymes are of 3-P-glyceric acid, 50,Lmoles of tris-HCl, pH 7.5, 250 1umoles considerably lower than the value reported for the pea seed enI

170

PACOLD AND ANDERSON

Plant Physiol. Vol. 55, 1975

Table II. Properties of 3-P-Glycerate Kintases

-~~~~~~~~~~~~~~~~~~~~~PaSe

Pea Leaf

Pea(Seed (81

abtMsl

Yat Rabbit -Muscle ~~Yeast13

Chloroplastic

Cytoplasmic

6.75-9.25

6.75-9.25

6.7-9.7

6.0-9.2 (13)

6.0-9.2

ATP

0.70 ±t 0.03

0.55 4 0.02

4.1

0.42

ADP

0.18 i 0.01

0.22 i 0.02

0.5,1.0 (14) 0.48 (13) 0.20 (13)

0.15

3-P-glycerate 0.1-0.3 mM 0.5-10 mN

0.59 i 0.06 1.60 + 0.11

0.54 4 0.03 1.25 i 0.07

0.69 (13) 1.28 (13)

0.42 1.37

pH optima, P-Glycerate, substrate

Bacterial (1's,,

Km (mM)

Ki (mM) competitive with ATP AMP ADP

pli

0.71 i 0.08 0.053 4 0.005 5.0 (4)

0.48 0.035

4 4

0.06 0.003

5.5 (4)

4.1 mM (8), but are similar to values obtained with bacterial (19), yeast (13, 14), and muscle (13) kinases (Table II). Two Kin values for 3-P-glycerate were obtained for each pea leaf kinase in two concentration ranges. Within each of these ranges, cytoplasmic and chloroplastic P-glycerate kinases have similar Km values (Table II). Yeast, muscle, and bacterial kinases have comparable Kml values, while the value reported for the pea seed enzyme is 10-fold higher than the Km determined at similar 3-P-glycerate concentrations for the pea leaf enzymes (Table II). The cytoplasmic and chloropalstic 3-P-glycerate kinases have similar Kin values for ADP, 0.22, and 0.18 mm, respectively (Table II). These values are very close to the Km values reported for yeast and muscle kinases (13). The inhibition of the pea leaf 3-P-glycerate kinases by adenosine phosphates is quite complex. When ATP is substrate, ADP and AMP inhibit. When ADP is substrate, ATP and AMP inhibit. In the ATP-utilizing reaction, both pea leaf kinases are competitively inhibited with respect to ATP by ADP and AMP. The Ki values for each particular inhibitor and cytoplasmic or chloroplastic kinase are similar, but ADP is about 10 times more effective as an inhibitor than is AMP (Table II). Essentially identical results have been reported for yeast 3-P-glycerate kinase (15). The effect of the inhibitors at low Mg2+ concentrations was not tested in the present experiments. In the ATP-generating direction the cytoplasmic and chloroplastic kinases are inhibited competitively, with respect to ADP, by AMP and ATP. In this case, the two nucleotides are about equally effective as inhibitors (Table II). The cytoplasmic and chloroplastic 3-P-glycerate kinases have similar specificities with respect to the nucleoside triphosphate utilized in the phosphorylation of 3-P-glycerate. ATP is the only effective substrate. UTP, TTP, CTP, ITP, and GTP are only 0.5 to 3% as effective as ATP. None of these compounds has an inhibitory effect on the utilization of ATP by the pea leaf kinases. Yeast and skeletal muscle kinases are much less specific with respect to the phosphate donor, with approximately 70% as much activity with ITP and 55% as much with GTP as with ATP (13). The following compounds were found to have no effect on the activity of cytoplasmic or chloroplast 3-phosphoglyceric acid kinase in the ATP-utilizing reaction: 10 mm fumarate, L-

zyme,

7.6

0.16

0.83

1.5 (15) 0.02 (15) 7.2 (13)

7.0

malate, acetate, succinate, glutamate, P-enolpyruvate, sucrose, pyruvate, ribose-5-P, fructose-6-P, glucose-6-P; 1 mm glyceraldehyde-3-P, ribulose-1,5-diP; 0.5 mm xylulose-5-P; 10 mM NH4Cl, and 50 mm potassium phosphate (pH 7.4). Citrate at 10 mm levels inhibited the activity of cytoplasmic and chloroplast 3-P-glyceric acid kinases 57% and 46%, respectively. Since citrate chelates Mg'+ ions required for the eenzymatic reaction the kinases are probably inhibited indirectly. DISCUSSION Those properties of pea leaf cytoplasmic and chloroplasmic 3-P-glycerate kinases which have been determined are very similar (Table II). In this respect the 3-P-glycerate kinases resemble the other chloroplastic and cytoplasmic isoenzymes which have been studied (1, 2, 6, 12, 20, 22, 23). The two kinases seem to be, from the kinetic point of view, essentially identical. Whether the cytoplasmic and chloroplastic kinases in the plant cell will utilize or generate ATP will be determined by the environment in the cell compartment and not by the differential properties of the enzymes themselves. Since the two kinases are not affected by most common metabolites but are affected by adenine nucleotides the major environmental effect will be mediated by differences in energy charge levels, in turn apparently mediated by light (see ref. 21). Although two other plant enzymes have recently been reported to be affected by energy charge levels (10, 25), 3-P-glycerate kinase is the only chloroplast enzyme, and the only higher plant reductive pentose phosphate pathway enzyme, which is known to be affected by energy charge. Pea leaf ribulose-5-P kinase, in contrast, has a much higher apparent affinity for ATP, but is not affected by ADP or AMP and is totally insensitive to energy charge (3). Ribulose-5-P kinase is activated by a light-dependent process (7, 16, 24). Light apparently controls the activity of both higher plant reductive pentose phosphate pathway kinases, directly in the case of ribulose-5-P kinase, and indirectly through energy charge, in the case of Pglycerate kinase. The pea leaf 3-P-glycerate kinases are very similar to the kinases from other organisms, differing mainly in nucleoside triphosphate specificity and pl' from the yeast and muscle enzymes (Table II).

Plant Physiol. Vol. 55, 1975

PEA LEAF 3-PHOSPHOGLYCERATE KINASES

Acknowledgments-We thank James 'McCorkle and staff at the Uni-ersity of Illinois, Chicago Circle, greenhouse for growing the pea plants used in this study. LITERATURE CITED

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