U. S. Department ofAgriculture, Agricultural Research Service, and Departments ... United States Department of Agriculture and the North Carolina Agri-.
Plant Physiol. 1983 73, 989-994 0032-0889/83/73/0989/05/$00.50/0
Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH1 Received for publication June 15, 1983 and in revised form August 29, 1983
DOUGLAS C. DOEHLERT AND STEVEN C. HUBEl U. S. Department ofAgriculture, Agricultural Research Service, and Departments of Botany and Crop Science, North Carolina State University, Raleigh, North Carolina 27650 ABSTRACT
Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-aP was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, eg. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo.
Sucrose phosphate synthase (UDP-glucose: D-fructose-6-P-2glucosyl transferase, EC 2.4.1.14) is the enzyme believed to be of major importance in the pathway of sucrose biosynthesis (1). The enzyme is inhibited by UDP and sucrose-P, products of the reaction (2, 8). Inorganic phosphate has also been shown to be a potent inhibitor of spinach leaf SP-synthase2 (2, 8) as well as the wheat germ enzyme (12), and appears to be competitive with UDPG. Earlier we reported (6) that G6P activated spinach leaf SP-synthase by decreasing the Km (F6P) and increasing the Vma. The Km (UDPG) was not affected by G6P. Glucose-6-P activation of SP-synthase was antagonized by Pi. We suggested that the G6P/Pi ratio could provide a mechanism for the metabolic fine
control of SP-synthase activity and, hence, sucrose synthesis. The objectives of the present study were to characterize the regulation of SP-synthase activity by G6P and Pi under conditions of limiting substrate and variable pH. In addition, we describe the procedure used to stabilize SP-synthase activity during purification and for separating SP-synthase from interfering enzymes such as PG-isomerase (a prerequisite for studying G6P activation). MATERIALS AND METHODS Plant Materials. Spinach (Spinacia oleracea L.) was either bought at a local market, or was grown (var Dark Green Bloomsdale) in controlled environment growth chambers with 12-h light/dark cycles with 22°C day and 19°C night temperatures. Light intensity was 215 uE m2-s'. Plants were watered daily and fertilized with a commercial slow release 12-12-12 fertilizer every 2 weeks. Leaves were harvested about 95 d after planting. Enzyme Extraction and Partial Purification. Chopped and deveined spinach leaves were ground (100 g/400 ml) in ice cold extraction buffer (50 mM Hepes-NaOH [pH 7.0], 5 mm MgCl, 2.5 mm DTT, 0.5 mm EDTA) with a Brinkman Polytron3 homogenizer. After centrifugation at 10,000g for 10 min, the crude supematant was applied to an w-aminohexyl-agarose column (bed volume 6 ml) that had been equilibrated with the extraction buffer. The column was washed with 20 ml of extraction buffer followed by 25 ml of extraction buffer plus 0.1 M KCI. Sucrose phosphate synthase was then eluted with extraction buffer plus 0.5 M KCI. Fractions containing high SP-synthase activity were pooled and 6 ml was applied to a 2 x 120 cm Ultrogel AcA 34 (LKB, Bromma Sweden) column equilibrated with extraction buffer plus 20% (v/v) ethylene glycol and 0.2 M KCI. Fractions that contained SP-synthase and no trace of PGisomerase activity determined by the method described previously (6) were pooled and stored at -80°C. Protein concentration at different stages of purification was determined by the procedure of Bradford (3). Molecular weight was estimated by gel filtration on a calibrated I x 60 cm Ultrogel AcA 34 column, calibrated with the following proteins: thyroglobulin (mol wt 669,000), ferritin (mol wt 440,000), catalase (mol wt 232,000), and y-globulin (mol wt 152,000). The column was calibrated with the same buffer used in the enzyme preparation. SP-Synthase Assays. Sucrose phosphate synthase was assayed by measuring sucrose phosphate production by a modification of the procedure of Cardini et al. (4) or by monitoring UDP
' Cooperative investigations of the Agricultural Research Service, United States Department of Agriculture and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 8907 of the Journal series of the North Carolina Agricultural Research Service Raleigh, NC. I 2 Abbreviations: SP-synthase, sucrose phosphate synthase; F6P, frucMention of a trademark or proprietary product does not constitute tose-6-P; UDPG, uridinediphosphate glucose; G6P, glucose-6-P; PG- a guarantee or warranty of the product by the United States Department isomerase, phosphoglucoisomerase; A5o (G6P), concentration of G6P of Agriculture or the North Carolina Agricultural Research Service and necessary for half-maximal activation; 150 (Pi), concentration of Pi nec- does not imply its approval to the exclusion of other products that may also be suitable. essary for half-maximal inhibition. 989
990
DOEHLERT AND HUBER
Plant Physiol. Vol. 73, 1983
280
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FRACTION NUMBER FIG. 1. Elution profiles of SP-synthase and PG-isomerase activities and protein, from a I x 60 Ultrogel AcA 34 column. Fraction volume was
0.75 ml.
Table I. Stoichoimetry of UDP and Sucrose Phosphate Production by Spinach LeafSP-Synthase at Different Substrate Levels with and without G6P Activation Each value is the mean of four determinations. Rate of Formation Assay Condition F6P 7.5 7.5 1.0 1.0 7.5 7.5
UDPG mM 7.5 7.5 7.5 7.5 1.0 1.0
G6P
UDP
0 5 0 5 0 5
2.74 4.08 0.66 2.04 0.82 1.79
Sucrose-P nmol/min 2.43 4.03 0.44 1.99 0.75 1.95
production (10). Unless otherwise noted, the reaction mixtures contained 50 mM Hepes-NaOH (pH 7.0), 5 mM MgCl2, 8 mM F6P, 8 mm UDPG, and enzyme. Reactions were typically run for 10 min at 30'C and terminated in boiling water (for UDP production) or by addition of I volume at I N NaOH (for sucrose phosphate determination). Color blanks were developed using the complete assay plus denatured enzyme. For the determination of pH effects, the enzyme was dialyzed into elution buffer that contained 10 mM Hepes-NaOH (pH 7.0). For Pi inhibition studies, reaction mixtures contained 90 mM Hepes-NaOH (pH 6.5 or 7.5). For the determination of the pH dependence of SPsynthase, a mixed buffer (64 mm Mes, 64 mm Hepes, 64 mM Tricine, and 64 mm 2-[N-cyclohexylaminoJethanesulfonic acid, adjusted with NaOH to the indicated pH) was used in the reaction mixture. For the effector experiments, all compounds tested were dissolved in 0.5 M Hepes-NaOH (pH 7.0) at 30X final concentration to eliminate pH changes in the final reaction mixture.
RESULTS Enzyme Stabilization and Partial Purification. The two major problems encountered in the preparation of SP-synthase for kinetic analysis were the elimination of interfering enzyme activities, especially PG-isomerase, and the stabilization of activity. The step elution of SP-synthase from w-aminohexyl-agarose produced an 8.5-fold increase in specific activity and a 24.5-fold concentration of activity with 60% yield, but did not eliminate PG-isomerase activity. Elution of the w-aminohexyl-agarose column with a salt gradient did not separate SP-synthase and PGisomerase activities, so was not used as a standard procedure. However, gel filtration on Ultrogel AcA 34 produced a partial separation between SP-synthase and PG-isomerase (Fig. 1). It was necessary to add 20% (v/v) ethylene glycol and 200 mM KCI to the Ultrogel elution buffer during this purification step. The absence of these reagents resulted in the complete loss of SPsynthase activity during gel filtration. Only those fractions that contained SP-synthase activity and no PG-isomerase activity were pooled for kinetic experiments. The final SP-synthase preparation also contained no activity of phosphoglucomutase, nonspecific phosphatase (for F6P or G6P), or UDPG-epimerase (6). The observed peak of SP-synthase (Fig. 1) corresponded to a mol wt of about 460,000 D. Previously, Harbron et al. (8) reported a mol wt of 260,000 for spinach leaf SP-synthase determined by gel filtration. A total purification of 15-fold was achieved with a yield of 14%. F6P Saturation Kinetics. Fructose-6-P saturation kinetics were strictly hyperbolic (Hill coefficient of 1.0). The Km (F6P) as estimated from 4 separate experiments was 3.2 + 0.8 mm in the absence of G6P and was reduced to 0.7 ± 0.1 mm in the presence of 5 mM G6P. The Km (F6P) decreased hyperbolically with increasing G6P concentration (data not shown). A problem encountered in determining the Km (F6P) was that G6P is a contaminant of F6P preparations. We have found no commercial F6P preparations that contained less than 0.5% G6P. This may
SPINACH LEAF SUCROSE PHOSPHATE SYNTHASE
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[G6 P] mM FIG. 2. The effect of F6P concentration on G6P activation of SP-synthase. All assays contained 8 mMi UDPG. Inset, The effect of F6P concentration on A50 (G6P) and relative activation. A50 (G6P) was determined by the abscissa intercept of a Woolf plot of [G6P]/( V - l"O) versus [G6PJ, where V is the SP-synthase activity at a given F6P and G6P concentration and V0 is the SP-synthase activity at the same F6P concentration with no G6P. The ordinate intercept of the Woolf plot is then A50 (G6P)/( V",= - l-'). The relative activation by G6P is V",,,XVO, using calculated V,,,.x values.
affect results, particularly at high F6P concentration with no added G6P. The response of SP-synthase to F6P concentration in the complete absence of G6P is, as yet, unknown. The plant material used for these studies was either obtained from a local market or grown in growth chambers and used fresh. No differences in F6P saturation kinetics or G6P activation kinetics were found in SP-synthase obtained from these different sources (data not shown). G6P Activation. Experiments were performed to determine the effects of G6P activation on the stoichiometry of UDP and sucrose phosphate production (Table I). Both products were formed in nearly a 1:1 ratio as expected and this ratio remained constant with G6P activation. This demonstrated that G6P activation affects UDP production identically with sucrose phos-
phate production. By measuring Pi production simultaneously with sucrose during an SP-synthase assay, we estimated that 10% of the sucrose phosphate produced was converted to sucrose by residual sucrose phosphate phosphatase activity (not shown). It is important to note that G6P could not replace F6P as a substrate (6). This can only be demonstrated when the SP-synthase preparations are completely free of PG-isomerase activity. The effect of F6P concentration on G6P activation was determined (Fig. 2). Glucose-6-P caused a 4. 1-fold increase in activity with 1 mm F6P (and 8 mm UDPG). As the concentration of F6P was increased (UDPG held constant), a progressive decrease in the relative activation (V,,,j/V.) was observed so that at 8 mm F6P, activation by G6P was only 1.5-fold (Fig. 2, inset). The A-v (G6P) was also affected by F6P concentration. At 8 mm F6P, the A5o (G6P) was 0.55 mm and at 1 mm F6P, the A5O (G6P) was increased to 1.05 mm (Fig. 2, inset). Hence, G6P affected F6P binding and vice versa. The effect of Pi on G6P activation at a limiting F6P concen-
tration (1 mM) was investigated (Fig. 3). Phosphate antagonized the activation by G6P and changed the shape of the G6P response curve from hyperbolic to sigmoidal (Fig. 3A). Maximal velocities (V,,,") were determined experimentally by adding excess G6P and these decreased as the Pi concentration was increased (Fig. 3B). At very high concentrations of G6P (over 10 mM), activity decreased. The A50 (G6P) increased from 0.85 mm with no Pi to 9.90 mm with 20 mm Pi (Fig. 3B). Replots of the data in Fig. 3 by the Hill equation yielded a series of parallel lines, with slopes or Hill coefficients of 1.62 ± 0.14. Pi Inhibition. Inhibition of SP-synthase by Pi was hyperbolic and partial (Fig. 4A). However, inhibition was more pronounced at pH 7.5 than at pH 6.5. Woolf plots of the data (Pi concentration/% inhibition versus Pi concentration) showed that the maximal inhibition by Pi remained at about 65% at either pH, but the 1-v (Pi) decreased from 3.45 mM at pH 6.5 to 1.15 mm at pH 7.5. Glucose-6-P also affected Pi inhibition (Fig. 4B). In the presence of 5 mm G6P, phosphate inhibition was sigmoidal (Hill coefficient of 1.3) at pH 6.5, whereas at pH 7.5 (with 5 mm G6P) it was hyperbolic (Hill coefficient of 1.0). Woolf plots (not shown) were linear at pH 7.5 and the I50 (Pi) was calculated to be 6.59 mM and maximal inhibition was 83%. At pH 6.5 Woolf plots curved upward, which is characteristic of a sigmoidal response. When the concentration of dibasic Pi was calculated, it was found that the per cent inhibition of SP-synthase increased hyperbolically with dibasic Pi concentrations independent of pH (Fig. 5). This suggested that pH effects on Pi inhibition were caused by increased dibasic Pi concentration and that dibasic Pi, and not monobasic Pi, is responsible for inhibition of SP-synthase. An alternative explanation would be that pH affected the Km (UDPG), but we have found little or no change in the Km
Plant Physiol. Vol. 73, 1983
DOEHLERT AND HUBER
992
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FIG. 3. A, The effects of Pi on G6P activation of SP-synthase with limiting F6P concentration at pH 7.0. All assays contained 1 mm F6P and 8 mm UDPG. B, The effects of Pi concentration on A"0 (G6P) and V,,,¢. The V,,,, was determined experimentally as the maximal SPsynthase activity obtained with increasing G6P concentration. A50 (G6P) was obtained graphically, by determining the G6P concentration where (V~ V) = (V..x- Vo)/2. -
(UDPG) from pH 6.5 to 7.5 (data not shown). The 15o for dibasic Pi as derived from Woolf plots was 0.74 mm in the absence of G6P and 4.45 mm in the presence of 5 mm G6P. The maximal inhibition of SP-synthase by Pi, expressed as a percentage of the uninhibited rate (VO), increased from 65% to 85% with the addition of 5 mm G6P. However, because Vo was doubled by 5 mm G6P, the activity of SP-synthase that remained in the presence of a saturating Pi concentration was about the same with or without G6P. This indicated that Pi eliminated all of the activating effect of G6P, but did not eliminate a certain basal activity. pH Profile. The effect of pH on SP-synthase activity in the absence and presence of effectors is shown in Figure 6. In the absence of effectors (control) enzyme activity exhibited a pH optimum at pH 7.5, but retained over 50% of maximum activity from pH 6.5 to 8.0. In the presence of 10 mm Pi, the pH optimum was pH 7.0. Phosphate inhibition (as a per cent of control) was absent at pH 5.5, but increased with increasing pH. This corresponded to the change in dibasic Pi concentration caused by the pH change. Glucose-6-P stimulated SP-synthase activity over the entire pH range tested. Additional Effectors. A number of compounds were tested for possible effects on SP-synthase activity and these effects are tabulated in Table II. Glucose-l-P and fructose-l-P stimulated activity slightly and appeared to be weak activators. Fructose-
[Pi
mM
FIG. 4. The effect of pH on Pi inhibition of SP-synthase in the absence (A) and presence (B) of 5 mM G6P. All assays contained 8 mM F6P and 8 mm UDPG. Per cent inhibition of SP-synthase activity was calculated as ( VWV) x 100, where V. is the SP-synthase activity with no Pi, and V is the SP-synthase activity at the indicate Pi concentration. In the absence of G6P, V. at pH 6.5 was 86.3 nmol sucrose-P/min/ml1' and at pH 7.5 was 74.3 nmol sucrose-P/min/ml-'. In the presence of G6P, V. at pH 6.5 was 126.6 nmol sucrose-P/min/ml-' and at pH 7.5 was 140.5 nmol sucrose-P/min/ml-'.
1,6-bisP and fructose-2,6-bisP had little or no effect on SPsynthase activity. Pyrophosphate inhibited slightly, especially at low UDPG. Phosphoenolpyruvate also inhibited SP-synthase, but the concentrations required suggested that it is not of physiological significance. Uridine diphosphate was a strong inhibitor of SP-synthase which is consistent with earlier reports that it is competitive with UDPG (2, 8). Potassium fluoride at 20 mM was a strong inhibitor of SP-synthase. DISCUSSION The absence of sigmoidal substrate saturation kdnetics for F6P and UDPG (6, 8) suggests that cooperativity of substrate binding is not a regulatory mechanism for SP-synthase (but cf. Ref. 2). However, regulation by metabolic effectors may be important. Glucose-6-P was the first metabolic activator of SP-synthase to be identified (6). Glucose-6P is not a substrate and consequently appears to act on a site other than the catalytic site, e.g. an
SPINACH LEAF SUCROSE PHOSPHATE SYNTHASE
993
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allosteric or modifier site as described by Koshland (1 1). Binding of G6P may induce a conformational change in the enzyme that increases the affinity of SP-synthase for F6P as reflected by the corresponding decrease in the Km (F6P). It appears that SP-synthase is a regulatory enzyme and is affected by an activator, G6P, and in inhibitor, Pi. The activity 20~~ of the enzyme is determined by the interaction of these effectors and substrate concentration. Glucose-6P increases the V,,,,= and the Iso (Pi), and decreases the Km (F6P), whereas Pi decreases the
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Our observation that Pi is only a partial inhibitor of SPsynthase has not been reported previously. Earlier studies (2, 8) suggested that Pi was a competitive inhibitor with UDPG and assumed that it was acting on the catalytic site. However, partial competitive inhibitors can bind either at the active site or at a modifier site (5). The competitive interaction of Pi with G6P, as evidenced by the increase in A5o (G6P) with increasing Pi concentration (Fig. 3B), strongly suggests that Pi can also interact at the same site that binds G6P. Recently, additional kinetic evidence has been obtained that is consistent with interaction of effectors at a modifier site that is distinct from the active site (manuscript in preparation). The concentration curve for G6P activation was clearly sigmoidal (Hill coefficient of 1.6). Although this suggests the presence of multiple binding sites for G6P with positive cooperativity, the same phenomenon could be observed with one binding site if the binding constant for G6P was higher than that for the substrate (A. R. Main, personal communication). Detailed mechanistic analyses are necessary to resolve this. The interactions of G6P and Pi in SP-synthase regulation are similar to those of 3-PGA and Pi in the regulation of ADPglucose pyrophosphorylase (7). ADP-glucose pyrophosphorylase is activated by 3-PGA and inhibited by Pi. The saturation curve for 3-PGA is sigmoidal and the Hill coefficient is affected by pH and Pi. Although the saturation curve of SP-synthase for G6P is sigmoidal, the Hill coefficient is not affected by Pi. Metabolic Considerations. Previous schemes for the metabolic regulation of SP-synthase have emphasized the roles of UDP and fructose-1,6-bisP in modulating SP-synthase activity (2, 8). Whereas UDP is a potent inhibitor of SP-synthase, it is not known whether the UDP concentration in the cytosol varies
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Plant Physiol. Vol. 73, 1983
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Table II. Effect of Various Compounds on SP-Synthase Activity at Different Substrate Concentrations Each value is the mean of two determinations. Assay Conditions' Effector
Concentration
(A) 8 mM F6P 8 mM UDPG
(B) 8 mM F6P I mM UDPG
(C) I mM F6P 8 mM UDPG
% control 5 mM 120 120 132 Glucose l-P 5 mM 114 110 108 Fructose l-P 2.5 mM 99 103 Fructose 1,6-P2 103 82 92 Fructose 2,6-P2 10 AM 99 87 PPi 5 mM 73 78 5 mm 67 81 P-enolpyruvate 83 14 5 mM 30 UDP 23 41 KF 68 20 mM 43 'Control activities were 141, 49, and 50 nmol sucrose-P/min ml' for A, B, and C, respectively.
F6P and UDPG concentrations of 0.5 to 1.0 mM. At these substrate concentrations, which are similar to the Km values, SPsynthase activity would be most sensitive to changes in the concentration of effectors. It is not possible in studies with protoplasts (e.g. Ref. 13) to measure cytosolic Pi because of a large vacuolar Pi pool. Recently Stitt et al. ( 14) reported a close correlation between the G6P concentration in the cytosol and photosynthetic sucrose production in wheat protoplasts. They demonstrated large increases in G6P concentration and sucrose formation with increasing irradiance and CO2 concentration, while F6P and UDPG concentrations remained relatively constant. They also suggested that under conditions of limiting light or C02, the decrease in G6P concentration would be associated FIG. 7. Pathway ofcarbon flow for sucrose synthesis showing possible with an increase in the cytosolic Pi concentration of about 10 regulation of SP-synthase by G6P and Pi. 1, SP-synthase; 2, PG-isomer- mm. These results corroborate our hypothesis for the regulation ase; 3, phosphoglucomutase; 4, UDPG pyrophosphorylase; 5, sucrose-P of SP-synthase by G6P and Pi. phosphatase; 6, fructose-i1,6-bisphosphatase; 7, phosphate translocator shuttle; 8, aldolase; 9, NADP: Glucose-6-P dehydrogenase; 10, oxidative pentose phosphate shunt. TP, triose phosphate; FBP, fructose-1,6,-bisphosphate; G I P, glucose- I-phosphate.
sufficiently to account for observed changes in sucrose synthesis. Harbron et al. (8) suggested that fructose- 1,6-bisP may modulate SP-synthase activity based on the observation that 2.5 mm fructose- 1,6-bisP caused a 70% inhibition of enzyme activity. We found little or no effect of 2.5 mm fructose- 1,6-bisP on SPsynthase activity (Table II). Regulation of SP-synthase by G6P and Pi provides a reasonable mechanism for the metabolic fine control of SP-synthase in relation to the rate of photosynthesis (Fig. 7). Inorganic phosphate is exchanged with the chloroplast for triose phosphates (9); thus, an accumulation of Pi in the cytosol could signal a reduction in the supply of carbon from the chloroplast. It is likely that the increase in Pi would correspond with decreased concentrations of P-esters such as G6P. Conversely, a high photosynthetic rate might be associated with decreased Pi and increased P-ester concentrations. Small reciprocal changes in Pi and G6P would have a large effect on SP-synthase activity (Fig. 3). Glucose-6-P is also an intermediate of the oxidative pentose phosphate pathway. In the dark, G6P concentration may be reduced as a result of cessation of photosynthesis and increased oxidative metabolism. An additional factor to consider is that Pi inhibition, in the presence or absence of G6P, is dependent on pH because dibasic Pi is the inhibitory form. At pH values below the pKa of Pi (7.2), Pi inhibition is reduced (Figs. 4-6). Consequently, conditions that generate high G6P concentration and either low Pi concentration or low pH would favor sucrose synthesis in spinach leaves. Stitt et al. (13) reported G6P concentrations of 1.7 to 2.5 mm in the extrachloroplastic compartment of spinach protoplasts with
LITERATURE CITED 1. AKAZAWA T, K OKAMOTO 1980 Biosynthesis and metabolism of sucrose. In J Preiss, ed, Biochemistry of Plants, Vol 3. Academic Press, New York, pp
199-220 2. AMIR J, J PREISS 1982 Kinetic characterization of spinach leaf sucrose phosphate synthase. Plant Physiol 69: 1027-1030 3. 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: 248-259 4. CARDINI CE, LF LELOIR, J CHIRIBOGA 1955 The biosynthesis of sucrose. J Biol Chem 214: 149-155 5. CLELAND WW 1963 The kinetics of enzyme catalyzed reactions with two or more substrates or products. II. Inhibition: Nomenclature and theory. Biochim Biophys Acta 67: 173-187 6. DOEHLERT DC, SC HUBER 1983 Spinach leaf sucrose phosphate synthase: Activation by glucose 6-phosphate and interaction with inorganic phosphate. FEBS Lett 153: 293-296 7. GosH HP, J PREISS 1966 Adenosine diphosphate glucose pyrophosphorylase. A regulatory enzyme in the biosynthesis of starch in spinach leaves. J Biol Chem 241: 4491-4504 8. HARBRON S, C FOYER, D WALKER 1981 The purification and properties of sucrose phosphate synthase from spinach leaves: The involvement of this enzyme and fructose bisphosphatase in the regulation of sucrose biosynthesis. Arch Biochem Biophys 212: 237-246 9. HEBER U, HW HELDT 1981 The chloroplast envelope: Structure, function and role in plant metabolism. Annu Rev Plant Physiol 32: 139-168 10. HUBER SC 1981 Interspecific variation in activity and regulation of leaf sucrose phosphate synthase. Z Pflanzenphysiol 102: 443-450 1 1. KOSHLAND DE JR 1970 The molecular basis for enzyme regulation. In P D Boyer, ed, The Enzymes. Academic Press, New York, pp 342-399 12. SALERNO GL, HG PONTIs 1978 Sucrose phosphate synthetase. Separation from sucrose synthetase and a study of its properties. Planta 142: 41-48 13. STrrT M, W WIRTZ, HW HELDT 1980 Metabolite levels during induction in the chloroplast and extrachloroplast compartments of spinach protoplasts. Biochim Biophys Acta 593: 85-102 14. STITT M, W WIRTZ, HW HELDT 1983 Regulation of sucrose synthesis by cytoplasmic fructose bisphosphatase and sucrose phosphate synthase during photosynthesis in varying light and carbon dioxide. Plant Physiol 72: 767774
