ferredoxin-activated fructose diphosphatase of spinach ...ferredoxin-activated fructose...
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THE Joua~a~ OF BIOLOGICAL CHE~~~STHY Vol 24F, No. 19, Issue of October 10, pp. 5952-5959, 1971
Printed in U.S.A.
Ferredoxin-activated Fructose Diphosphatase of Spinach Chloroplasts
RESOLUTION OF THE SYSTEM, PROPERTIES OF THE ALKALINE FRUCTOSE DIPHOSPHATASE COM- PONENT, AND PHYSIOLOGICAL SIGNIFICANCE OF THE FERREDOXIN-LINKED ACTIVATIOK*
(Received for publication, March 22, 1971)
BOB B. BUCHANAN, PETER SCH~~RMANN, AND PETER P. ~~~~~~~~~~
From the Department of Cell Physiology, University of California, Bedceley, California 94Y20
The hydrolytic cleavage of Pi from fructose diphosphate by the fructose diphosphatase (FDPase) system of spinach chloroplasts was found to require (a) an alkaline FDPase component insensitive to inhibition by AMP or fructose diphosphate; (b) reduced ferredoxin; (c) a protein factor; and (d) Mg++.
Reduced spinach ferredoxin could be replaced by reduced methyl viologen or dithiothreitol but not by reduced bacterial ferredoxin. Activation of the alkaline FDPase component by the nonphysiological substitutes required the protein factor. The alkaline FDPase component in isolated intact chloroplasts was also activated by light.
The protein factor component of the FDPase system has been partly purified and found to have a molecular weight of 40,000. The alkaline FDPase component, purified to homogeneity, had a molecular weight of 145,000 and contained 2 % carbohydrate. Except for a remarkably high half-cystine content (16% by weight as compared to 2 % for mammalian FDPases) which reflects an apparent abundance of cystine, the amino acid composition of the chloroplast alkaline FDPase component resembled that of the mammalian FDPases.
In the absence of reduced ferredoxin and protein factor, the chloroplast alkaline FDPase component can be activated by high concentrations of Mg+f. Like liver alkaline FDPase, the chloroplast enzyme component is also activated by cysta- mine or 5,5’-dithiobis(Z-nitrobenozic acid) (DTNB).
Ferredoxin-activated FDPase was inhibited completely by 5 x 10V4 M EDTA but was insensitive to KCN, cuprizone, and diethyldithiocarbamate (all at 1 X lop3 M).
The activity of chloroplast FDPase during photosynthesis appears to be controlled photochemically through reduced ferredoxin. What, if any, physiological role can be ascribed to the activation of the enzyme by high concentrations of Mg++ and disulfide reagents, independently of reduced ferredoxin, is not clear.
* This work was aided by Grant GB-23796 from the National Science Foundation.
$ Present address, Swiss Federal Research Station, Wadenswil, Switzerland.
Ferredoxins are iron-sulfur proteins noted for t,heir strongly electronegative oxidation-reduction potentials. Ferredoxins are widely distributed in nature and function as electron carriers in all photosynthetic cells and in certain anaerobic nonphotosyn- thetic bacteria (1). Work with isolated chloroplasts has shown that ferredoxin is the endogenous catalyst of cyclic and noncyclic photophosphorylation-the two processes that provide the as- similatory power, made up of ATP and NADPI-I, needed for carbon dioxide assimilation (1, 2).
An additional role of ferredoxin came to light when Buchanan, Kalberer, and Arnon (3) found that photochemically reduced ferredoxin activated the hydrolytic cleavage of fructose 1,6- diphosphate to fructose 6-phosphate and Pi by a fructose diphos- phatase present in an aqueous extract of chloroplasts (Equa- tion 1).
reduced ferredoxin Fructose 1,6-diphosphate + H20 >
chloroplast extract (1)
fructose g-phosphate + Pi
Since chloroplasts reduce ferredoxin only in the light, Buchanan et al. (3) suggested that a requirement of reduced ferredoxin for activation of the chloroplast FDPaser (4-7)-a key enzyme of the photosynthetic reductive pentose phosphate cycle @)-could serve as a light-actuated mechanism for regulation of carbon dioxide assimilation in photosynthesis. Kinetic evidence for a light activation of FDPase in whole algal cells was reported by Bassham, Kirk, and Jensen (9) and Pedersen, Kirk, and Bassham
(10). The fructose 1,6-diphosphatase reaction in chloroplasts was
previously considered to require a single enzyme, alkaline FDPase, analogous to mammalian alkaline FDPase-a “pace- maker” enzyme in gluconeogenesis (II). The present experi- ments show that the chloroplast FDPase is, however, more com- plex. It consists not only of the alkaline FDPase component but also of a protein factor and ferredoxin. Ferredoxin, in re- duced form, and the protein factor are needed to activat,e the alkaline FDPa.se component (Equation 2) prior to its catalysis
1 The abbreviations used are: FDPase, fructose 1,6-diphos- phatase; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid) ; HEPES, N-2-hydroxyethylpiperazine-iv’-2-ethanesulfonic acid; Tricine, N-tris(hydroxymethyl)methylglycine.
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Issue of October 10, 1971 B. B. Buchanan, P. Schiimann, and P. P. Kalberer 5953
of t,hc hydrolytic cleavage of fructose I ,6-diphosphate (Equa- t,ion 3).
reduced ferredoxin FDPase;n3ctivc > FDPasesotivo (2)
active FDPase Fructose 1,6-diphosphate + Hz0 f
Fructose 6-phosphate + Pi
The alkaline FDPase component was earlier extensively purified from Fpinach leaves by Racker and Schroeder (12) and Preiss, Biggs, and Greenberg (13); its crystallization has recently been reported by El-Badry and Bassham (14).
The chloroplast alkaline FDPase component resembles the alkaline FDPase from mammalian cells (15) in showing stimu- lation by cyst,amine and DTNB. However, unlike the mam- malian FDPase (11, 16-al), the alkaline FDPase component of t,he chloroplast FDPase system is not inhibited by AMP or fructose diphosphate (13).
A preliminary report of some of these findings has been pub- lished (22).
Rabbit muscle aldolase, liver catalase, pig heart malic dehydro- gennsc, horse heart cytochrome c, ovalbumin, and bovine serum albumin were purchased from Sigma. Homogeneous alkaline FDl’ase from rabbit liver and plastocyanin from spinach leaves were gifts of Dr. 13. Horecker (Albert I<Znstcin College of Medi- cine) and Mr. R. Chain of this Department., respectively. A sample of homogeneous alkaline FDPase from spinach leaves was an earlier gift of Dr. J. Preiss (University of California at Davis).
Protoil was estimated by a modified phenol reagent procedure (23). Chlorophyll was determined as described by Arnon (24). The carbohydrate content of chloroplast FDl’ase was determined by a naphthalene procedure (25). Zinc was determined by atomic absorption spectrophotomet.rg.
Amino acid analyses on 22- ancl 72.hour acid hydrolysates (6 s HCl, 105“) were carried out by Dr. II. Matsubara with the method of Spackman, Stein, and Xoore (26), modified as de- scribed previously (27, 28). Total half-cystine (cysteine + cystine) residues were determined as cysteic acid after performic acid osidation of the FDPase. Tryptophan was determined spectrophotometrically (29) and by the thioglgcolate procedure (30). The amino acid content of FDPxsc was based on an average of two analyses; the composition shown was calculated on the basis of 12 histidine residues per mole of enzyme.
Fcrredoxin with A.izs:A276 of 0.46 (or higher) was isolated from spinach leaves by the method of Tagawa and Arnon (as given in the review by Buchanan and rirnon (31)). Intact chloroplasts were isolated in isotonic sucrose (not sorbitol which interfered with l’i determination) as described by Kalberer, Buchanan, and Arnon (32). Chloroplast fragments (l’&) were prepared from whole chloroplast,s (isolated either in isotonic NaCl as described by Whatley and Arnon (33) or in isotonic sucrose (32)) by suspending in a 1 to 10 dilution of the preparative solu- t,ion, collecting by centrifugation (5 min, 35,000 x g), and re- suspending in the dilute preparative solut.ion.
Assay and PwiJkation of Alkaline FDI’ase Component of Chloroplast FDPase System
For routine assay of the alkaline FDl’:~sc component, the reaction mixture contained (in micromoles) aside from the en-
zyme: Tris-HCl buffer, ~1-1 8.5, 100; MgClz, 16; sodium EDTA, 0.1; and sodium fructose 1 ,6-diphosphate, 6, in a final volume of 1.0 ml. The assay, carried out in test tubes at room tempera- ture for 10 min, was started by adding fructose diphosphate and stopped by 0.5 ml of 10% trichloroacetic acid. The Pi released was determined calorimetrically by a modified Fiske-SubbaRow procedure (34). One unit of enzyme activity is defined as that amount of enzyme which catalyzes the release of 1 pmole of l’i per min under the assay conditions.
Unless indicated otherwise, all purification steps of the FDPase component given below were carried out ai. 4”.
Leaf %&act-Fresh market spinach leaves, 5 kg, were washed and frozen overnight at -20”. The frozen leaves (in l-kg batches) were blended in 1,400 ml of water for 3 min in a Waring Blendor (gallon size, model CR-5). Ten milliliters of 1 11 K&IP04 (sufficient to give a final pH of 7) were added prior to blending. The homogenate was filtered through four layers of cheesecloth and the residue was discarded. The remaining particulate material was sedimented by centrifugation at 13,000 x g for 5 min and was also discarded.
pH 4.5 Precipitation-The green supernatant fraction (10,700 ml) was adjusted to pH 4.5 with 1 N formic acid and centrifuged (5 min, 12,000 x g). The yellow supernatant fraction obtained in this step contained the bulk of the protein factor needed for activation of alkaline FDPase by reduced ferredoxin; the frac- tion was neutralized with 1 M Tris and fractionated as described below. The green precipitate containing the alkaline FDPase component was suspended in a HEPES-EDTA solution (0.025 hI HEPES-NaOH buffer, pH 7.6, containing 2.5 X lop4 M EDTA- Na) and then brought to a volume of 1,000 ml. The pH was adjusted to 6.5 with 0.1 N NH,OH. The suspension was cen- trifuged for 10 min at 35,000 x g and the residue was discarded. Final volume of the supernat,ant fraction containing the FDPase was 700 ml.
~lmmonium Sulfate I+actionation-Solid ammonium sulfate was added to the redissolved ~1-1 4.5 precipitate fraction to give 50% saturat’ion. The solution was centrifuged (10 min, 13,000 x g) and the precipit,ate was discarded. Ammonium sulfate
was added to the supernatant fraction for 90% saturation. The solution was centrifuged as above, the supernatant fraction was discarded, and the precipitate (containing the bulk of the FDPase activity) was dissolved in 0.03 v Tricine buffer, pH 8.0, to a final volume of 80 ml. This slightly turbid solution was centri- fuged (15 min, 35,000 x g) to clarify.
Xephadex G-100 Chromatography-The 50 to 90% ammonium sulfate fraction was applied to a Sephadex G-100 column (5 x 90 cm) equilibrated beforehand and developed with 0.03 RI Tricine, pI-I 8.0. Fractions of 10 ml were collected with a frac- tion collector. FDl’ase chromatographed just behind the ex- cluded volume and was eluted before the bulk of applied protein. The slightly yellow fractions containing FDPase were pooled; and sufficient HEPES-NaOH buffer, pH 7.6, and NaCl were added to give a final concentration of 0.05 &I HEPES, pH 7.6, and 0.2 RI NaCl.
DEAE-Cellulose Chromatography-The combined FDPase fractions were applied to a DEAE-cellulose column (5 x 45 cm) equilibrated beforehand with a solution containing 0.05 M HEPES-NaOH buffer, pH 7.6, and 0.15 M NaCl. The column was mashed with a solution containing 0.05 hI HEPES-NaOH buffer, pH 7.6, and 0.3 M NaCl until the eluate was free of pro- tein, attached to a fraction collector, and eluted in lo-ml frac- tions with a solution containing 0.05 M HEPES-NaOH buffer,
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5954 Ferredoxin-activated Fructose Diphosphatase Vol. 246, No. 19
TABLE I Summary of purijication of chloroplast alkaline FDPase
component from spinach leaves
mg Leaf extract. ................ 48,100 pH 4.5 precipitate. .......... 8,400 50-90s (NH&Sod. .......... 1,760 Sephadex G-100. ............ 384 DEAE-cellulose. ............ 84
3,200 2,720 2,160 2,050
-~ % units/mg 100 0.06 110 0.38
94 1.54 75 5.62 71 24.4
pH 7.6, and 0.5 M N&I. FDPase was eluted just behind the solvent front; the active fractions were pooled and concentrated by vacuum dialysis.
The FDPase component purified by this procedure was homo- geneous. The enzyme was relatively stable and could be stored in HEPES-EDTA buffer at -20” for several weeks with little loss of activity. Repeated freezing and thawing caused a slow loss of activity.
Puri$cation of Protein Factor Component of Chloroplast FDPase System
The activity of the protein factor was determined by the increase of Pi release from the fructose diphosphate in the pres- ence of photoreduced ferredoxin and homogeneous FDPase component (cf. Table IV).
The neutralized pH 4.5 supernatant fraction from Step 2 of the alkaline FDPase procedure was used as a source of the pro- tein factor. Acetone, cooled to -2O”, was added with constant stirring to a final concentration of 750/,; the solution was left 1 hour at -20” to allow the precipitate to settle. The supernatant fraction was decanted and discarded; the precipitate (containing the protein factor) was collected by centrifugation (3 min, 1000 x g) and suspended in 0.03 M Tris-HCl buffer, pH 8.0. The turbid solution was then dialyzed 24 hours against 0.03 M Tris- HCl buffer, pH 8.0.
Denatured protein was centrifuged off (15 min, 13,000 x g), and solid ammonium sulfate was added to the supernatant frac- tion to 50% saturation. The heavy precipitate was sedimented by centrifugation as before and discarded. The ammonium sul- fate concentration in the supernatant solution was increased to 90% saturation and the precipitate (containing the protein fac- tor) was collected by centrifugation as above and dissolved in 100 ml of 0.02 M Tris-HCl buffer, pH 8.0. The slightly turbid solution was centrifuged (5 min, 35,000 x g) to clarify.
The 50 to 90% ammonium sulfate fraction was applied to a Sephadex G-100 column (5 x 90 cm) equilibrated beforehand and developed with 0.03 M Tris-HCl buffer, pH 8.0. Fractions (13 ml) containing protein factor activity were pooled and stored at -20”. The protein factor was stable for several months at -20".
Ultracentrifugation was carried out in a Spinco model E ultra- centrifuge. Sedimentation velocity experiments were done in a 12-mm aluminum, l-sector cell with quartz windows. The high speed sedimentation equilibrium experiments were done accord- ing to Yphantis (35) with 3-mm liquid columns in a g-channel cell. The time required to reach equilibrium (24 hours) was
determined by measuring fringe displacement until it became constant. Fringe and schlieren boundary displacements were measured with a Nikon a-coordinate comparator. Partial speci- fic volume was determined from sedimentation equilibrium cen- trifugation in DzO and Hz0 as described by Edelstein and Schach- man (36) and from amino acid composition as described by Schachman (37).
The molecular weight of chloroplast alkaline FDPase was estimated by gel filtration as described by Andrews (38). A Sephadex G-200 column (1.5 x 80 cm, equilibrated beforehand with a solution containing 0.1 M potassium phosphate buffer, pH 7.3, and 0.1 M NaCl) was calibrated with liver catalase, pig heart malic dehydrogenase, horse heart cytochrome c, ovalbumin, bovine serum albumin, and plastocyanin as standards. Chlo- roplast alkaline FDPase was applied to the calibrated column and was eluted between aldolase and bovine serum albumin, corresponding to a molecular weight of approximately 130,000.
The procedure of Davis (39) was used in acrylamide gel elec- trophoresis with 7.5% gels and Tris-glycine, pH 8.3, as electrode buffer. A current of 2 to 3 ma per gel was applied for 2 hours at room temperature. Gels were stained in Coomassie blue (0.5% solution in 12.5% trichloroacetic acid) (40).
The reaction of FDPase with p-mercuribenzoate was followed spectrophotometrically as described by Boyer (41). p-Mer- curibenzoate (10u3 M) was added in 5+1 amounts to a cuvette containing 0.3 mg of FDPase in 3 ml of 0.1 M Tris-HCl buffer, pH 8.0. After 5 min, absorbance at 250 nm was measured against a cuvette containing FDPase but no p-mercuribenzoate. Additional incubation did not increase the amount of p-mercuri- benzoate reacting with the FDPase.
The reaction of FDPase with DTNB was carried out spectro- photometrically as described by Ellman (42). A cuvette con- tained 0.3 mg of FDPase in 3 ml of 0.1 M Tris-HCl buffer, pH 7.3 (in the presence or absence of 8 M urea) and 20 ~1 of a 0.4% DTNB solution. Absorbance at 412 nm was read immediately after mixing and again after 12 hours at room temperature, when the reaction was complete.
RESULTS AND DISCUSSION
Chemical and Physical Properties of Alkaline FDPase Component of Chloroplast FDPase Xystem
Alkaline FDPase, protein factor, and ferredoxin are the three protein components of the FDPase system of chloroplasts. Early in this investigation the FDPase and protein factor com- ponents were obtained from the aqueous extract of isolated chloroplasts. However, for preparation on a large scale, pro- cedures were devised for purification of the FDPase and protein factor from spinach leaves. Table I shows a summary of the purification and yield of the alkaline FDPase component (from leaves) in each of the purification steps described under “Meth- ods.”
The freshly purified FDPase component appeared to be homo- geneous. The purified protein showed a single peak in the ultracentrifuge (Fig. 1,lcwer patterns in A and B) and in Sephadex G-200 chromatography. The FDPase component traveled in polyacrylamide gel as one main band trailed by diffuse protein material (Fig. 2). This slow moving material, present in all preparations, was probably due to change in the homogeneous enzyme during electrophoresis-an interpretation supported by the observation that under certain conditions (particularly after
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Issue of October 10, 1971 B. B. Buchanan, P. Schiimzann, and P. P. Kalberer
the enzyme was concentrated by vacuum or pressure dialysis) the FDPase component dissociated into subunits.
The subunits formed during concentration dialysis were sep- arated from the parent alkaline FDPase component of the FDPase system by Sephadex G-200 chromatography. The sub- unit fraction showed a single peak in the ultracentrifuge (Fig. 1, upper pelteras in A and B) and, based on both gel filtration and ultracentrifugation (see below), had a molecular weight half that of the original enzyme. This finding indicates that the active alkaline FDPase component is composed of two subunits of about equal molecular weight; the isolated subunit fraction had no FDPase activity.
The sedimentation velocity pattern (determined with schlieren optics) of the fresh alkaline FDPase component (lower patterns) and the isolated subunits (upper patlerns) in Fig. 1 were meas- ured 11 min (A) and 27 min (B) after the centrifuge reached full speed. The log of the displacement of the boundary of each protein under these conditions was linear with time. The data obtained with four different levels of both FDPase and subunit fraction showed that the sedimentation coefficient was dependent on the protein concentration; extrapolation to zero protein gave an $20,~ of 8.3 for the FDPase component (and of 4.2 for the subunit fraction).
The sediment,ation equilibrium technique showed a pattern typical of a homogeneous preparation and gave a molecular weight of 145,000 for the active undissociated FDPase component (Fig. 3) based on an average partial specific volume of 0.700. (The partial specific volume was determined experiment.ally by sedimentation equilibrium in DzO and Hz0 and found to be 0.697; a value of 0.706 was calculated from amino acid composi- tion.) The molecular weight of the subunit fraction would there- fore be 73,000. Values of 130,000 and 139,000 for the active FDPase component were obtained by the Sephadex G-200 chro- matography technique and by amino acid analysis. A molecular weight of 145,000 is substantially lower than 195,000 obtained by Preiss (quoted by Preiss and Kosuge (43)) for his alkaline FDPase preparation.
Table II shows the amino acid composition of the alkaline FDPase component from chloroplasts in comparison with the enzyme from rabbit muscle and liver. Each of the three pro- teins is characterized by a high content of glutamate, aspartate, and glycine, a low content of histidine, and the absence of tryp- tophan. A striking feature of the chloroplast component (which distinguishes it from the mammalian enzymes) is the unusually high half-cystine content (210 residues per mole of enzyme, accounting for 16% of the enzyme by weight as compared to 2% for the mammalian enzymes). This feature is especially note- worthy when considered in the light of sulfhydryl group titration data. The chloroplast alkaline FDPase component contained only 10 p-mercuribenzoate-reactive -SH groups per mole (a value not increased by treating the enzyme with 8 M urea) and three to four DTNB-reactive -SH groups per mole (a value increased to 10 by treating the enzyme with 8 M urea). The data point to an unusually large cystine content of the alkaline FDPase component.
Homogeneous preparations of the chloroplast alkaline FDPase component were characterized by a single peak in the ultraviolet at 278 nm and showed no absorption in the visible region. An absorbance at 278 nm of 1.0 (l-cm light path) was equal to 1.3 mg of protein determined by the phenol reagent method.
Three different homogeneous preparations of the alkaline
FIG. 1 (lop). Sedimentation velocity pattern of chloroplast alka- line FDPase component and the FDPasc subunit fraction. Pat- terns were determined (in a solution of 0.1 M potassium phosphate buffer, pH 7.3, and 0.1 M NaCI) with 0.3ml samples containing 2.9 mg per ml of the FDPase (lower pattern) and 0.8 mg per ml of the subunit fraction (upper partlern). Measurements were made at a rotor speed of 59,780 rpm; average temperature, 20’. Photo- graphs were taken 11 min (A) and 27 min (B) after reaching full speed.
Fro. 2 (bottom). Electrophoresis of chloroplast alkaline FDPase component in polyacrylamide gel.
2.001 I I I I I
50.4 50.6 50.8 51.0 51.2 51.4
fmdiu*) 2, cm 2 FIG. 3. Sedimentation equilibrium centrifugation of chloroplast
alkaline FDPase component. Protein concentration was 0.48 mg per ml in a solution containing 0.1 nf potassium phosphate buffer, pH 7.3, and 0.1 M NaCl. Centrifugation speed was 16,200 at an average temperature of 20”; time, 24 hours.
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5956 Ferredoxin-activated Fructose Diphosphatase Vol. 246, No. 19
Number of residues per mole in FDPase of:
Lysine. ...................... Histidine. ................... Arginine. .................... Aspartic acid. .............. Threonine .................. Serine ...................... Glutamic acid ............... Proline. .................... Glycine ..................... Alanine ..................... Half-cystine ................ Valine ...................... Methionine ................. Isoleucine ................... Leucine. .................... Tyrosine .................... Phenylalanine. .............. Tryp tophan. ................
65 89 12 10 30 43 98 103 28 78 78 76
216 118 43 48
166 104 64 111
210 21 67 104 14 21 57 58 73 119 36 63 25 32
117 14 34
128 70 79 81 52
34 70 94 52 40
Amino acid composition of chloroplast alkaline FDPase comp0nen.l in comparison with FDPases -from liver and muscle
Amino acid residue
0 Data of Fernando, Pontremoli, and Horecker (44).
FIG. 4. Effect of AMP on alkaline FDPase component from spinach chloroplasts in comparison with the enzyme from rabbit liver. Except-for adding A&IP, as indicated, t&e enzymes were assaved in 12 mM M&l% at nH 8.0 as described under “Methods.” 0ne”hundred per cent &ti;ity corresponds to 1.8 and 2.1 pmoles of Pi released by 0.05 mg of the chloroplast and liver enzymes, respectively.
FDPase component contained 2 To carbohydrate (with glucose as a standard). As determined by atomic absorption analysis, these same preparations contained less than 0.4 eq of zinc per mole of enzyme.
E,fect of AMP, Mg*, awl Light on Alkaline PDPase Component of Chbropkzst FDPase System
In 1954, Krebs (45) discussed the importance of FDPase in regulating gluconeogenesis in animal cells. Nine years later, three laboratories (16-B) independently made the important observation that relatively low concentrations of AMP inhibit
TABLE III E$ect of light on a&dine FDPase componed of intact chloroplasts
The reaction was carried out in Warburg vessels fitted with double side arms and center wells. The complete system con- tained (in one side arm) 0.25 ml of intact chloroplasts equivalent to one mg of chlorophyll; (in the second side arm) sodium fructose 1,6-diphosphate, 6 pmoles; and (in the main compartment) Tris- HCl buffer, pH 8.0, 100 pmoles; neutralized reduced glutathione, 5 pmoles; MgC12, 12 pmoles; and EDTA, 0.1 pmole. Vessels also contained, in center wells, 0.1 ml of 7 N KOH. Final volume, 3.0 ml; temperature, 20”; and gas phase, argon. After gassing, ves- sels were incubated, as indicated, 10 min in the light (10,000 lux) to activate the FDPase. The light was turned off, the chloro- plasts and fructose diphosphate were added from the side arms, and the FDPase was assayed in the dark in the osmotically broken chloroplast preparation. Reaction time, 30 min. Reaction was stopped and (after centrifuging off the precipitate) Pi was esti- mated by a modified Fiske-SubbaRow procedure (34).
Dark, control.................................. 0.5 Light, control.. . 0.9 Light, minus fructose diphosphate.. . 0.0
mammalian FDPase-a finding that has led to the conclusion (11, 19-21) that the activity of FDPase in gluconeogenesis is regulated by the level of AMP. Such a role for AMP in sugar formation in photosynthesis can, however, be excluded. Fig. 4 shows that the chloroplast alkaline FDPase component, unlike the liver enzyme, is not inhibited by AMP. Insensitivity of the chloroplast alkaline FDPase component to AMP was also ob- served by Preiss et al. (13).
Apart from differential sensitivity to AMP, plant and mam- malian (or microbial) FDPases differ in the level of Mg* required for maximal activity. The FDPases from rat liver (46), rabbit liver (47), and nonphotosynthetic microorganisms (48, 49) require, depending on the source, 0.7 to 5.0 mM Mg* for maximal activity, whereas the enzyme from photosynthetic cells requires about a lo-fold greater concentration of Mg*. Spinach leaf alkaline FDPase, dependent on pH, was reported to require 5 to 50 mM Mg* (12, 13), and the EugZena grucilis enzyme was reported to require 50 mM Mg* (50).
It is not known whether chloroplasts can maintain the high concentrations of Mgft reported to be required by the alkaline FDPase component. A possible explanation is that light induces an accumulation in chloroplasts of Mg* sufficient to activate the alkaline FDPase component. Such a dependence on light might also explain the photoactivation of the FDPase component observed in isolated intact chloroplasts (Table III); activity of the alkaline FDPase component was nearly doubled by illumi- nating the chloroplasts for 10 min prior to breaking osmotically and assaying the enzyme. The flux of Mg* required for acti- vation would, however, be in a direction opposite to that observed experimentally: recent studies from several laboratories have shown that Mg* is expelled from chloroplasts on illumination and is reabsorbed in the dark (51-53). We searched, therefore, for a mechanism for activation of the chloroplast alkaline FDPase other than a light-induced enhancement of the intrachloroplastic level of Mg++. This search was made at a low level of Mg* (0.67 mM)-which cannot itself activate the FDPase component.
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Issue of October lo,1971 B. B. Buchanan, P. Schiirwzann, and P. P. Kalberer 5957
TABLE IV Requirements for activation of chloroplast alkaline FDPase
component by jerredoxin
The complete system contained alkaline FDPase component, 0.04 mg; protein factor, 0.15 mg; spinach ferredoxin, 0.12 mg; spinach chloroplast fragments, PI&, equivalent to 0.1 mg of chlorophyll; and (in pmoles) Tris-HCl buffer, pH 8.0, 100; neutra- lized reduced glutathione, 5; sodium fructose 1,6-diphosphate, 6; sodium ascorbate, 10; 2,6-dichlorophenol indophenol, 0.1; EDTA- Na, 0.1; and MgC12, 1. Final volume, 1.5 ml; gas phase, argon. The reaction (carried out at 20” in Warburg flasks) was started by adding fructose diphosphate and chloroplast fragments from the side arm and was continued for 30 min under illumination (10,000 lux). The reaction was stopped and (after centrifuging off the precipitate) Pi was estimated-as in Table III.
Complete. . . Reduced glutathione omitted”. Ferredoxin omitted”. Alkaline FDPase component omitted. MgCl~omitted................................. Protein factor omitted. . . Fructose diphosphate omitted. Complete, ferredoxin not reduced (dark). .
4.0 1.4 0.1 0.1 0.1 0.0 0.0 0.1
a In a parallel experiment, the complete and the minus gluta- thione treatments showed, respectively, 2.0 and 0.4 rmoles of Pi released; 2-mercaptoethanol added in place of glutathione showed 3.3 rmoles of Pi released in a light-dependent reaction.
* Methyl viologen (0.01 pmole) and Glostridium pasteurianum ferredoxin (0.01 rmole) added in the absence of spinach ferredoxin showed, respectively, 2.5 and 0.0 pmoles of Pi released from fruc- tose diphosphate.
Activation of Chloroplast Alkaline FDPase Component by Photoreduced Ferredoxin
A possible mechanism for the activation of the alkaline FDPase component observed with intact chloroplasts is that light func- tions by way of a photochemical reductant. The finding that reduced ferredoxin sharply activated the homogeneous alkaline FDPase component (Table IV) constituted the first evidence that such a mechanism might be valid. Ferredoxin, photore- duced by chloroplasts, enhanced the release of Pi from fructose diphosphate 40-fold. The release of Pi required, in addition to reduced ferredoxin, MgCl,, fructose diphosphate, the alkaline FDPase component, and a new component, the protein factor. The reaction was stimulated by an -SH reagent (reduced glutathione or 2-mercaptoethanol) supplied in addition to re- duced ferredoxin. Native spinach ferredoxin could not be replaced by bacterial ferredoxin (from Clostridium pasteurianum) but could be partially replaced by the nonphysiological dye methyl viologen (Table IV). The FDPase component was shown earlier to be activated in the dark when ferredoxin was reduced with hydrogen gas in the presence of hydrogenase (3). Reduced NADP or NAD could not replace light or hydrogen gas.
The protein factor, a component of the soluble protein fraction of chloroplasts, has been partly purified. It is heat-sensitive (5 min, 80”) and, based on gel filtration, has a molecular weight of 40,000. Other properties are unknown.
A homogeneous chloroplast alkaline FDPase preparation (sup-
fructose diphosphofe, mhf
FIG. 5. Effect of fructose diphosphate concentration on fer- redoxin-activated FDPase. Except for varying the fructose diphosphate concentration, conditions were as described in Table IV.
plied by Dr. J. Preiss) behaved like our own preparation in the ferredoxin-dependent release of Pi from fructose diphosphate. A homogeneous preparation of the enzyme from rabbit liver, how- ever, showed no response to reduced ferredoxin under the con- ditions in Table IV.
Eg’,ct of Some Inhibitors on Ferredoxin-activated FDPase from Chloroplasts-Ferredoxin-activated FDPase was not affected by KCN or the copper chelating agents (54) diethyldithiocarbamate or cuprizone (at 1 X 10B3 M) but was 97% inhibited by 5 x 10m4 M EDTA. A lower level of EDTA (7 x 10-5 M) increased 2- to 4-fold the release of Pi from fructose diphosphate by certain preparations. Racker and Schroeder (12) and Preiss et al. (13) previously reported a stimulation of FDPase activity with EDTA.
Effect of Fructose Diphosphate Concentration on Ferredoxin- activated FDPase-A fructose diphosphate concentration greater than 0.1 mu (16, 18) to 1.0 mM (17) has been shown to inhibit mammalian FDPase-an effect believed to be important in regu- lation of that enzyme. The activity of the ferredoxin-activated FDPase of chloroplasts, by contrast, increased with fructose diphosphate concentration up to 8 mM (Fig. 5). The release of Pi from fructose diphosphate, in the absence of reduced ferre- doxin, was not appreciably affected by the fructose diphosphate conceL?tration.
Effect of Mg++ Concentration on Fcrredoxin-activated FDPase- Because chloroplast alkaline FDPase is activated by high con-
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5958 Ferredoxin-activated Fructose Diphosphatase Vol. 246, No. 19
FIG. 6. Effect of MgClz concentration on ferredoxin-activated FDPase. Except for varying the MgC12 concentration, condi- tions were as described in Table IV.
TABLE V Requirements fo? activation of chloroplast alkaline FDPase
component bg dithiothreitol
The complete system contained alkaline FDPase component, 0.04 mg; protein factor, 0.25 mg; and (in pmoles) Tris-HCl buffer, pH 30,100; MgCL, 1; EDTA-Na, 0.1; sodium fructose 1,6-diphos- phate, 6; dithiothreitol, 5; as indicated, 0.25 mg of bovine serum albumin was added. Final volume, 1.5 ml; gas phase, air. The reaction was carried out in test tubes at 22’ and was started by adding fructose diphosphate. Reaction time, 30 min. The reaction was stopped and Pi was determined as in Table III.
,Complete.......................................... 3.7 Protein factor omitted. . . . . . . . . . . 0.3 MgCla omitted.. . . . . . . . . . . . . . . . 0.1 Alkaline FDPase component omitted. . . . . 0.0 Dithiothreitol omitted.. . . . . . . . . . . . . . . . . . 0.0 Fructose diphosphate omitted. . . 0.0 Protein factor omitted, bovine serum albumin added. 0.3
centrations of Mgft (5 mu or greater), the extent of the ferre- doxin-linked activation is strictly dependent on the Mgft con- centration (Fig. 6). A consistent stimulation in Pi release was observed at all levels of MgClz tested but the effect of reduced ferredoxin was most pronounced at 0.67 to 2 mru MgClz.
Activation of Chloroplast Alkaline FDPase Component
by Dithiothreitol and --s--X-- Reagents
The nonphysiological -SH reagent dithiothreitol (55) could replace reduced ferredoxin in activation of the chloroplast alka- line FDPase component (Table V). Pi release from fructose diphosphate by the dithiothreitol-activated FDPase component occurred in the dark without chloroplasts and required MgCla and the protein factor. The requirement for the protein factor in activation of the FDPase component by dithiothreitol was specific-an equivalent amount of bovine serum albumin or of various protein fractions obtained in purification of the alkaline
FIG. 7. Effect of cystamine on chloroplast alkaline FDPase component. The control system contained 0.06 mg of alkaline FDPase component, 100 pmoles of Tris-HCl buffer (pH 8.0), 6 pmoles of fructose 1,6-diphosphate, and 1 pmole of MgC12. Cysta- mine (5 pmoles) was added as indicated. Final volume, 1.5 ml. Gas phase, argon. The reaction (carried out at 20” in Warburg flasks) was started by adding fructose diphosphate from the side arm and was stopped by adding 0.5 ml of 10% trichloroacetic acid. Pi released was estimated.
FDPase component was without effect. Cysteine, reduced glutathione, 2-mercaptoethanol, and sodium dithionite did not replace dithiothreitol in activation of the alkaline FDPase com- ponent. Dithiothreitol has been shown to activate other chlo- roplast enzymes: ribulose 5-phosphate kinase (56), NADP-linked glyceraldehyde 3-phosphate dehydrogenase (57), and pyruvate, Pi dikinase (58).
Cystamine and DTNB, S-S compounds found by Pontremoli et al. (15) to activate liver FDPase, also activated the homo- geneous chloroplast alkaline FDPase component. Fig. 7 shows that cystamine enhanced 50-fold the release of Pi from fructose diphosphate; the protein factor had no effect on the FDPase component under these conditions. When cystamine was re- placed by an equivalent amount of DTNB, a lo-fold increase in Pi release was observed. Activation by S-S reagents (via disulfide exchange) provides a model system for regulation of the FDPase reaction, first for mammalian cells (15) and now for chloroplasts. The physiological significance of this type of activation is, however, an open question.
In sum, the present experiments provide evidence that the FDPase system of chloroplasts is activated by light. Ferredoxin, a component of the system, is reduced photochemically and, in collaboration with another component, the protein factor, re- duced ferredoxin activates a third component, the alkaline FDPase. Once activated, alkaline FDPase catalyzes the forma- tion of fructose 6-phosphate (and Pi) from fructose diphos- phate-a key reaction in the reductive pentose phosphate cycle of photosynthesis.
The mechanism of activation of the alkaline FDPase com- ponent is not known, but it may involve a reduction by reduced ferredoxin of specific S-S groups that cannot be reduced by more electropositive agents such as reduced glutathione or 2- mercaptoethanol (Equations 4 and 5).
2 Ferredoxin.,, + Ha0 chloroplasts
> light (4)
2 ferredoxin,d f F, 0~
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Issue of October 10, 1971 B. B. Buchanan, P. Xchii~mann, and P. P. Kalberer 5959
protein factor FDPaseinactive + 2 ferredoxin,,d
(SS-1 (5) FDPaseactivc f ferredoxin,, (SH HS-)
The abundance of cysteine residues iu the alkaline FDPase and the replaceability of reduced ferredoxin by dithiothreitol or re- duced methyl viologen are in accord with such a mechanism.
Acknowledgment-We gratefully acknowledge the support and advice of Professor D. I. Rrnon throughout this investigation.
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Bob B. Buchanan, Peter Schürmann and Peter P. KalbererSIGNIFICANCE OF THE FERREDOXIN-LINKED ACTIVATION
FRUCTOSE DIPHOSPHATASE COMPONENT, AND PHYSIOLOGICALRESOLUTION OF THE SYSTEM, PROPERTIES OF THE ALKALINE
Ferredoxin-activated Fructose Diphosphatase of Spinach Chloroplasts:
1971, 246:5952-5959.J. Biol. Chem.
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