Proc. Natd Acad. Sci. USA Vol. 79; pp. 4322-4326, July 1982 Botany

A special bisphosphate functions as a. cytoplasmic regulatory metabolite in green leaves (fructose 2,6-bisphosphate/sucrose synthesis/fructose bisphosphatase//) CSABA CStKE*, NORMAN F. WEEDEN*, BOB B. BUCHANAN*, AND KOSAKU UYEDAt *Division of Molecular Plant Biology, University of California, Berkeley, California 94720; and tDepartment of Biochemistry, Veterans Administration Hospital, University of Texas Health Science Center, Dallas, Texas 75235 Communicated by Daniel I. Arnon, April 19, 1982 ABSTRACT Fructose 2,6-bisphosphate (Fru-2,6-P2), a regu- have therefore conducted a study aimed at answering these latory metabolite discovered in animal cells and recently reported questions and now report evidence that Fru-2,6-P2 is both pres- to occur in etiolated seedlings, was found to be present in the cy- ent and functional in leaves of C3 plants. (C3 plants produce a toplasmic fraction ofleaves ofspinach and peas (typical C3 plants, 3-carbon carboxylic acid as a major early product of photosyn- in which a three-carbon carboxylic acid is a major early photo- thesis). At concentrations commensurate with those occurring synthetic product). At concentrations approximating those calcu- in vivo, Fru-2,6-P2 inhibited the cytoplasmic form of Fru-P2ase lated to occur physiologically, Fru-2,6-P2 modulated two (15, 16), a key in the synthesis of sugars, including sur of the leafcytoplasm: (i) Fructose-1,6-bisphosphatase (EC 3.1.3.11), crose, the most abundant sugar ofgreen plants (17, 18). By con- a key enzyme of sugar synthesis, was competitively inhibited by trast, PFP, a cytoplasmic enzyme that now appears to play an Fru-2,6-P2, and (ii) -linked phosphofructokinase by Fru-2,6-P2. The (inorganic pyrophosphate-D-fructose-6-phosphate 1-phospho- important role in glycolysis, was activated , EC 2.7.1.90), a cytoplasmic enzyme that now seems results are consistent with the view that Fru-2,6-P2 functions important in glycolysis of C3 plants, was activated by Fru-2,6-P2. as a cytoplasmic regulatory metabolite in leaves of C3 plants. There was no indication ofa role for Fru-2,6-P2 in photosynthesis ofeither chloroplasts or oxygenic prokaryotes. The results suggest MATERIALS AND METHODS that Fru-2,6-P2 functions in the regulation of glycolysis and glu- Plant and Microbial Material. Spinach plants (Spinacea oler- coneogenesis (carbohydrate synthesis) in the cytoplasm of leaves acea, var. Hipack, Asgrow Seed, Tracy, CA) were grown in a of C3 plants. nutrient solution in a greenhouse as described (19). Green peas (Pisum sativum, var. Progress no. 9, Ferry Morse, Mountain Fructose 2,6-bisphosphate (Fru-2,6-P2) is a recently discovered View, CA) were grown in vermiculite under natural lighting metabolite that functions in the regulation ofglycolysis and glu- conditions. Nostoc muscorum was grown on N2 as described by coneogenesis in mammalian tissues (1-7). The regulation ofeach Arnon et al. (20). Prochloron was collected in June 1981 near of these pathways by Fru-2,6-P2 is achieved through the mod- Palau, Western Caroline Islands, and kindly provided by R. ulation ofa target enzyme: an activation ofphosphofructokinase Lewin (University of California, San Diego). (PFK; EC 2.7.1.11) in the case of glycolysis (Eq. 1) and an in- Reagents. Except for fructose 6-phosphate (Fru-6-P), which hibition of fructose-1,6-bisphosphatase (Fru-P2ase; EC 3.1.3.11) was obtained from Boehringer Mannheim, biochemicals and in the case of gluconeogenesis (Eq. 2). the "coupling enzymes" for assay of PFF, PFK, and Fru-P2ase were obtained from Sigma (PFP and PFK: aldolase, a-glycer- PFK ophosphate dehydrogenase, triose phosphate , all Fructose 6-phosphate + ATP - from rabbit muscle; Fru-P2ase: yeast phosphoglucose isomer- Fructose 1,6-bisphosphate + ADP [1] ase, baker's yeast glucose-6-phosphate dehydrogenase). Fru- Fru-P2ase 2,6-P2 was synthesized as described (4). Other reagents were Fructose 1,6-bisphosphate + H20 - purchased from commercial sources and were of the highest quality available. Unless indicated otherwise, buffers were ad- Fructose 6-phosphate + Pi. [2] justed to the indicated pH at 20'C. Recently, Fru-2,6-P2 was reported to occur in plant tissues Enzyme Assays. PFP was routinely assayed by measuring (nonphotosynthetic) and to function therein in the activation of change in A340 in a reaction mixture containing, in a 1-ml final a pyrophosphate-linked phosphofructokinase (8, 9), a sacchar- volume, the following (mM): Hepes buffer, pH 7.3, 50; MgCl2, olytic enzyme of certain microbial (10-13) and plant (14) cells. 5; tetrasodium EDTA, 1; NADH, 0.1; Fru-6-P, 1; inorganic [This enzyme, officially named pyrophosphate-D-fructose-6- pyrophosphate, 0.2; and coupling enzymes: 100 tug of aldolase phosphate 1- (EC 2.7.1.90), is here desig- (1 unit); 8 Ag ofa-glycerophosphate dehydrogenase (1 unit); and nated PFP (Eq. 3).] 6 jug of triose phosphate isomerase (10 units). For kinetic mea- surements, the concentrations of Fru-6-P, pyrophosphate, and PFP MgCl2 were varied as indicated. Unless indicated otherwise, Fructose 6-phosphate + pyrophosphate - PFP was activated with 1 p.M Fru-2,6-P2. Similar activation was + observed when Fru-2,6-P2 was added to the reaction mixture Fructose 1,6-bisphosphate Pi. [3] either prior to or after the addition of PFP. In most cases, the It is not known whether Fru-2,6-P2 occurs in green tissues extent of activation of PFP was determined in a single assay by or, importantly, whether it plays a role in photosynthesis. We Abbreviations: Fru-1,6-P2, fructose 1,6-bisphosphate; Fru-2,6-P2, fruc- The publication costs ofthis article were defrayed in part by page charge tose 2,6-bisphosphate; Fru-P2ase, fructose-1,6-bisphosphatase; PFK, payment. This article must therefore be hereby marked "advertise- phosphofructokinase; PFP, inorganic pyrophosphate-D-fructose-6- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. phosphate 1-phosphotransferase; Fru-6-P, fructose 6-phosphate. 4322 Downloaded by guest on September 25, 2021 Botany: Cseke et al. Proc. NatL Acad. Sci. USA 79 (1982) 4323

adding Fru-2,6-P2 to the reaction mixture 3 min after starting and 28 mM 2-mercaptoethanol, and then applied to a 2 x 5 cm the reaction with PFP. DE 52 column that had been equilibrated with'the same buffer PFK was assayed asgiven for PFP except that the pH ofthe and was eluted with 40 ml of a pH 7.5 sodium phosphate gra- Hepes buffer was 7.0 and 0.2 mM ATP was added in place of dient (10-100 mM). Fractions showing AMP-sensitive Fru- inorganic pyrophosphate. Pyrophosphatase activity was assayed P2ase activity were pooled and concentrated by.dialysis against in a reaction mixture containing, in a 0.-5-ml final volume, the solid sucrose. The concentrated-fraction, which was free ofchlo- following (mM): Hepes, pH 7.3, 50; MgCl2, 5; pyrophosphate, roplast Fru-P2ase, was used as a source of cytosolic Fru-P2ase. 2. After 15-min incubation at 20TC, the reaction was stopped The preparation in 50% sucrose could be stored at 5C for sev- by adding 2.0 ml of the mixture used for Pi analysis, and the eral weeks without appreciable loss ofactivity. For kinetic stud- samples were allowed to stand for 10 min before A6w was mea- ies, the preparation was diluted in a solution containing 50 mM sured (19). sodium phosphate buffer at pH 7.5, 5% sucrose, and 14 mM Cytoplasmic Fru-P2ase was also assayed spectrophotometri- 2-mercaptoethanol. cally by coupling the reaction to the reduction of NADP with Preparation of Fractions for Fru-2,6-P2 Analysis. Leaf ex- phosphoglucose isomerase and glucose-6-phosphate dehydro- tract. Washed spinach leaves (25 g) were homogenized for 10 genase and measuring the change in Aw. The reaction mixture sec in a Waring Blendor containing 60 ml of 0.5 M Tris1HCl contained, in a 0.5-ml final volume, the following (mM): imidaz- buffer, pH 8.5, supplemented with 5 mM ethylene glycol bis(/- ole HCl buffer, pH 7.5, 50; MgSO4, 1; NADP, 1; and the cou- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The ho- pling enzymes: 0.3 ,ug of glucose-6-phosphate dehydrogenase mogenate was filtered through four layers of cheesecloth, and (0.1 unit) and 0.4 ,ug of phosphoglucose isomerase (0.2 unit). the residue was discarded. NaOH was added to the filtrate im- The reaction was started by addition of 0.02 mM fructose-1,6- mediately after filtration to a final concentration of 0.1 M; the bisphosphate (Fru-1,6-P2). Fru-2,6-P2 was added as indicated filtrate was then clarified by centrifugation (15 min at 27,000 prior to starting the reaction. X g). Purification of PFP. All steps were carried out at 4°C. Ciloroplasts. Intact spinach chloroplasts were prepared and Washed spinach leaves (60 g) were homogenized for 10 sec in washed once as described previously (21), except that the buffer a Waring Blendor containing 150 ml of 50 mM N-[tris(hy- used was 0.5 M Tris HC1, pH 8.5; the washed chloroplasts were droxymethyl)methyl]glycine (Tricine)/KOH buffer, pH 7.6, osmotically broken by suspension in a solution containing 0.5 supplemented with 14 mM 2-mercaptoethanol and 0.5 mM M Tris HCl buffer, pH 8.5, supplemented with 5 mM EGTA phenylmethylsulfonyl fluoride. The homogenate was filtered and 0.1 M NaOH. After centrifugation (10 min, 27,000 X g), through four layers ofcheesecloth, the residue was discarded, the membrane fraction was suspended in this same solution. and the filtrate was centrifuged for 15 min at 27,000 X g. The Mesophyll protoplasts. Spinach mesophyll protoplasts were supernatant fraction (155 ml) was clarified by centrifugation (30 prepared as described by Wirtz et at (22) except that the gra- min, 95,000 x g) and then applied to a DEAE-cellulose column dient centrifugation step was omitted. For isolation of meso- (Whatman DE 52), 2.6 x 15 cm, that had been equilibrated phyll cytoplasmic fraction, protoplasts that had been pelleted with 50 mM Tricine/KOH buffer, pH 7.6, containing 14 mM by centrifugation (5 min, 100 x g) were resuspended in 0.5 M 2-mercaptoethanol. The column was washed with 100 ml ofthe Tris-HCI buffer, pH 8.5, containing 1 mM CaCl2 and 0.375 M same buffer and then a linear 300-ml salt gradient was applied sucrose and then forced through a syringe fitted with nylon net (0-0.25 M NaCl in the buffer). Fractions (3 ml) were collected (20-Aum pore diameter). Chloroplasts and debris were re- and analyzed for PFP and PFK activities and protein content. moved by centrifugation (10 min, 27,000 X g), and NaOH to The fractions showing peak PFP activity were combined (6 ml) 0.1 M was added to the chlorophyll-free supernatant frac- and used as the source ofPFP. PFP purified by this procedure tion-i.e., the cytoplasmic fraction. Mesophyll cell extract was was enriched 20-fold over that in the initial leaf extract. The prepared in this same manner except that sucrose was omitted preparation was essentially free of PFK and pyrophosphatase from the protoplast resuspension buffer. activities. After addition of the appropriate alkaline buffer, each of'the Purification of Cytoplasmic Fru-P2ase. All steps were car- above fractions was frozen in liquid nitrogen immediately after ried out at 4°C. Washed spinach or pea leaves (100 g) were ho- centrifugationand stored at - 200C or lower. The fractions were mogenized in a Waring Blendor containing 200 ml of 0.1 M prepared in Berkeley and shipped to Dallas in dry ice for Fru- sodium phosphate buffer, pH 7.7, supplemented with 5% su- 2,6-P2 analysis. crose, 2% polyvinylpyrrolidone, 1 mM tetrasodium EDTA, 0.5 Other Preparative Procedures. Previously described pro- mM phenylmethylsulfonyl fluoride, and 28 mM 2-mercapto- cedures were used for the purification of spinach chloroplast ethanol. The homogenate was filtered and centrifuged as given PFK (21) and Fru-P2ase (see ref. 23). A published method was above for PFP. 7The supernatant fraction (220 ml) was subjected also used for preparing chloroplast extract for PFP localization to solid ammonium sulfate fractionation at 4°C, while the pH studies (21). was maintained at 7.2 by careful addition of0.2 M NaOH (cen- Analytical Procedures. Protein was estimated by a modifi- trifugation 15 min, 39,000 X g). The material precipitating be- cation of the Lowry procedure (24). Chlorophyll was deter- tween 0 and 40% saturation with ammonium sulfate was dis- mined as described by Arnon (25). Fru-2,6-P2 was analyzed by carded; the 40-65% ammonium sulfate fraction, which contained the rabbit muscle PFK procedure (26) and identified by its la- the bulk ofthe AMP-sensitive Fru-P2ase activity, was saved and bility in acid solution. Accordingly, in the assay method for Fru- dissolved in 6 ml of50 mM sodium phosphate buffer, pH 7.7, 2,6-P2, cell-free extract equivalent to that being analyzed in containing 10% sucrose, 100 mM NaCl, and 28 mM 2-mercap- PFK activation was incubated in 0.1 M HC1 for 15 min at 30'C, toethanol. This fraction was applied to a 2.5 x 90 cm Sephacryl neutralized, and assayed as described (4). The Fru-2,6-P2 con- S-300 column (Pharmacia) that had been equilibrated and was centration was determined from the difference between acid- eluted with this same buffer. Fractions showing AMP-sensitive treated and untreated samples. Fru-P2ase activity were combined and concentrated to 5 ml by ultrafiltration with an Amicon PM 30 membrane filter. The con- RESULTS AND DISCUSSION centrate was dialyzed against 500 ml of 10 mM sodium phos- Effect of Fru-2,6-P2 on Spinach Chloroplast Fru-P2ase and phate buffer, pH 7.5, containing 15% sucrose, 2 mM EDTA, PFK. Fru-2,6-P2 is believed to function in the regulation ofglu- Downloaded by guest on September 25, 2021 4324 Botany: Cse-ke et d Proc. Nat Acad. Sci. USA 79 (1982) coneogenesis in mammalian cells through its capability -of in- hibiting Fru-P2ase. In view ofthe biochemical parallels between gluconeogenesis and photosynthesis, we decided to test the effect. of Fru-2,6-P2 on-the thioredoxin-linked Fru-P2ase that occurs in chloroplasts (23). This-experiment revealed that Fru- 2,6-P2 had no effect on chloroplast Fru-P2ase, in either the ab- sence or the presence of reduced thioredoxin, until the con- centration was raisedto about 100 times that reported to inhibit mammalian Fru-P2ase-i.e., 100 AM. Insensitivity to low con- centrations of Fru-2,6-P2 is thus an addition to a list of bio- chemical properties that distinguish chloroplast.Fru-P2ase from the mammalian equivalent (namely, capability for activation by reduced thioredoxin, low affinity for Fru-1,6-P2 substrate, and insensitivity to.AMP) (23, 27-29). PFK is generally recognized as being the metabolic antipode -0.2 -0.1 0.1 0.2 0.3 of Fru-P2ase in animal as well as plant cells. In the case of an- 1 imals, PFK is known to be activated by nanomolar concentra- Fru-1,6-P2, AM tions of Fru-2,6-P2 (3, 5, 26). We therefore tested the effect of from chloroplasts. When as- FIG. 2. Lineweaver-Burk analysis.of inhibition of cytoplasmic Fru-2,6-P2 on the PFK purified Fru-P2ase by Fru-2,6-P2. The x intercept of the line for no Fru-2,6-P2 sayed under a variety ofconditions, including those developed = 5 AM. for Fru-2,6-P2 was 0.13 ,uM. for mammalian systems (26), chloroplast PFK (21, 30, 31) indicates K. Ki showed no significant response to Fru-2,6-P2 up to concentra- those summarized above for spinach were obtained in the cur- tions of4 pAM. rent study for the cytoplasmic Fru-P2ase purified from pea Effect of Fru-2,6-P2 on Cytoplasmic Fru-P2ase from Spin- leaves by the above procedure (data not shown). ach Leaves. The absence ofa strong Fru-2,6-P2 effect on Fru- Effect of Fru-2,6-P2 on Cytoplasmic PFP from Spinach P2ase or PFK from chloroplasts prompted us to turn our atten- Leaves. PFP, an enzyme described for certain bacterial species tion to the cytoplasm and to test the possibility that Fru-2,6-P2 (10-13), was found by Carnal and Black (14) to be present in is involved in the regulation of leaf enzymes in that compart- leaves from pineapple, a plant showing crassulacean acid me- ment. The first to be studied was Fru-P2ase, an enzyme that tabolism. PFP activity was recently demonstrated in dark- is known to resemble mammalian Fru-P2ase in a number of grown (etiolated) mung bean seedlings and shown to be acti- properties, including inhibition by AMP, lack of a response to vated therein by micromolar concentrations of Fru-2,6-P2 (8, reduced thioredoxin, and strong affinity for Fru-1,6-P2 sub- 9). To our knowledge, PFP has not been reported to occur in strate (Km = 5 AM determined in this study) (refs. 15 and 16; photosynthetically competent leaves of C3 plants such as unpublished data). The results ofthese experiments were pos- spinach. itive: Fru-2,6-P2 caused a strong inhibition of Fru-P2ase (Fig. We considered that the absence of a report describing PFP 1) and was effective at relatively low concentrations (Ki = 0.13 in green leaves ofsuch plants could be related to its dependency puM) (Fig. 2). The capability of undergoing inhibition by Fru- on Fru-2,6-P2, a regulatory metabolite unknown until quite re- 2,6-P2 is thus another property that cytoplasmic Fru-P2ase from cently. This hypothesis was confirmed: in cell-free preparations leaves shares with Fru-P2ase from mammalian sources (6, 7). from spinach leaves, we were readily able to detect high levels Under the conditions used here, cytoplasmic Fru-P2ase from of PFP activity, but only in the presence of Fru-2,6-P2. We. spinach leaves was inhibited about 50% by 0.01 mM AMP (cf. found no evidence that PFP occurs in chloroplasts. In partially ref. 15). As found previously for mammalian Fru-P2ases (6, 7), from PFP showed a strong re- the inhibition by AMP complemented that produced by Fru- purified preparations leaves, 2,6-P2, and, at. low Mg2' concentrations (<1 mM), the two in- hibitors acted synergistically. Significantly, results similar to 0.04

.D r 0.03 o.. S e o as v PLOg 0.02

0.01

0 0.05 0.10 0 20 40 60 80 100 Fru-2,6-P2, AM Fru-1,6-P2, JAM FIG. 3. Activation of spinach cytoplasmic PFP by Fru-2,6P2. The assay mixture contained 1.0 mM Fru-6-P and 0.5 mM pyrophosphate. FIG. 1. Effect ofFru-2,6-P2 on cytoplasmic Fru-P2ase from spinach The reaction was started by adding 25 Zg of PFP to the assay mixture leaves. Exceptfor varying Fru-1,6-P2 concentration, experimental con- containing Fru-2,6-P2 as indicated. The Ka calculated from the Inset ditions were as given in Materials and Methods. is 0.012 AM. Downloaded by guest on September 25, 2021 Botany: Cse'ke et d Proc. Natl. Acad. Sci. USA 79 (1982) 4325 Table 1. Summary of effects of Fru-2,6-P2 on target cytoplasmic enzymes of spinach leaves 0.04 Enzyme Effect of Fru-2,6-P2 Fru-P2ase Inhibits competitively >-, a . -4 PFP Relieves pyrophosphate inhibition . P.'.4 0.03 . .4 E 1-1 Increases affinity for Fru-6-P and Mg+ t 4 Cd M Changes Mg2e and Fru-6-P kinetics from 04 1 44 0.02 cooperative to Michaelian C14 0.01 7-5 0 (30 nmol/g of leaves), thus making the Fru-2,6-P2 content of spinach and pea leaves roughly equivalent to that ofmammalian 0 tissue (32). On the basis offactors developed by Stitt et aL (33), 6.5 7.0 7.5 8.0 8.5 these values correspond to a maximal concentration in the me- sophyll cytoplasm of300 AM. Significantly, free Fru-2,6-P2 was pH not detected in significant amounts in isolated mesophyll chlo- roplasts. On the basis of these results, it would seem FIG. 4. EffectofpH on Fru-2,6-P2-linked activation of cytoplasmic unlikely PFP from spinach leaves. Except for varying pH as indicated, assay that Fru-2,6-P2 plays an important regulatory role in chloroplasts. conditions were as given in Materials and Methods. CONCLUDING REMARKS quirement for Fru-2,6-P2 (Ka for Fru-2,6-P2 = 0.012 AM) (Fig. The present results provide evidence that Fru-2,6-P2, arecently 3). The concentration of Fru-2,6-P2 required for activation of discovered regulatory metabolite that is widely distributed in cytoplasmic PFP from leaves is similar to that observed with animal cells (32), functions differentially in the regulation of PFK from mammalian sources (3, 5, 26). The Fru-2,6-P2-linked sugar synthesis and degradation in the cytoplasm of leaves of activation of PFP was observed over broad ranges of pH and C3 plants. Fru-2,6-P2 was found to activate PFP, a pyrophos- substrate/ concentrations, but the extent of activation phate-linked phosphofructokinase of glycolysis, and to com- was most pronounced at lower pH values (Fig. 4) and high py- petitively inhibit Fru-P2ase, an enzyme of sugar (sucrose) syn- rophosphate concentrations (>0.5 mM) (Fig. 5). Limiting Fru- thesis in the cytoplasm. The basis for the difference in Fru-2,6- 6-P and Mg2e were also conducive to enhancing activation by P2 concentration required to achieve modulation of these two Fru-2,6-P2 (Fig. 5). The data indicate that Fru-2,6-P2 promotes enzymes (Ka = 0.012 ,uM for PFP, Ki = 0.13 AM for Fru-P2ase) the activation of PFP through relieving substrate inhibition by is not clear, but it is noteworthy that a similar situation holds pyrophosphate and increasing affinity for Mg2e and Fru-6-P. for the regulation of corresponding enzymes from mammalian In the latter case, the addition of Fru-2,6-P2 decreased the K,,, cells [Ka = 0.01-0.024 AM for PFK (26), Ki = about 0.5 .M for Fru-6-P substrate from 3.3 to 0.05 mM. The effects of Fru- for Fru-P2ase (7)]. Significantly, -in contrast to mammalian tis- 2,6-P2 on the kinetics ofcytosolic PFP and Fru-P2ase from spin- sue, we observed no effect ofFru-2,6-P2 on PFK activity in leaf ach leaves are summarized in Table 1. preparations. Occurrence ofFru-2,6-P2 in Leaves. The above results dem- Under the conditions used here, PFP extracted from spinach onstrate that two enzymes of leaf cytoplasm, Fru-P2ase and leaves showed a higher activity (10 units/100 g offresh leaves) PFP, show a regulatory response to Fru-2,6-P2 at low concen- than that reported for cytoplasmic PFK from the same source trations. Ifthese findings are to be physiologically meaningful, (3-6 units/100 g of fresh leaves) (30). Accordingly, it would the cytoplasm of leaf cells should contain Fru-2,6-P2. This was seem that PFP plays at least as important a role in cytoplasmic found to be the case: when spinach and pealeaves were analyzed glycolysis ofthe leafas does PFK, provided that sufficient con- by the mammalian PFK procedure, Fru-2,6-P2 was found to be centrations ofpyrophosphate are present. Future experiments present, specifically in the cytoplasmic fraction of mesophyll are necessary to determine ifthis is indeed the case, and ifso, cells, the most important photosynthetic cells in C3 plants. The the source of the pyrophosphate needed by the enzyme must Fru-2,6-P2 content varied with the lot of leaves, with values be identified. A related unanswered question of fundamental ranging up to 3 nmo/mg of protein recovered in leaf extracts importance concerns the nature ofthe mechanism that controls

A B C 0.04 W Fru-2,6-P2 Fru-2,6-P2 -Fru-2p6P2

0.03 la Control C 0.02

p14 0.01 N Control Control c I I 0 / I I 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 1.0 2.0 3.0 4.0 5.0

Fru-6-P, mM Pyrophosphate, mM MgCl2, mM FIG. 5. Activation of cytoplasmic PFP from spinach leaves by Fru-2,6-P2. Except for varying Fru-6-P (A), pyrophosphate (B), and MgCl2 (C) as indicated, assay conditions were as given in Materials and Methods. Downloaded by guest on September 25, 2021 4326 Botany: Cs6ke et aL Proc. NatL Acad. Sci. USA 79 (1982) the formation of Fru-2,6-P2, which, in turn, modulates the en- 12. Pfleiderer, C. & Klemme, J. H. (1978) Z. Naturforsch. 35c, zymes described here. Mammalian cells have recently been 229-238. to link the activity of a specific , catalyzing syn- 13. Macy, J. M., Ljungdahl, L. G. & Gottschalk, J. (1980)J. Bacte- shown riot 134, 84-91. thesis of Fru-2,6-P2 from Fru-6-P and ATP, to the hormone 14. Carnal, N. W. & Black, C. C. (1979) Biochem. Biophys. Res. glucagon viaacAMP-dependent phosphorylation ofthe enzyme Commun. 86, 20-26. (34-36). In recent experiments we have obtained evidence that 15. Zimmermann, G., Kelly, G. J. & Latzko, E. (1978)J. Biol Chem. spinach leaves contain akinasewith a similarcatalyticcapability. 253, 5952-5956. The regulatory properties ofthe spinach kinase are not yet fully 16. Harbon, S., Foyer, C. & Walker, D. A. (1981) Arch. Biochem. Biophys. 212, 237-246. known. 17. Turner, J. F. & Turner, D. H. (1975) Annu. Rev. Plant Physiot A final point worthy ofmention is the current evidence that 26, 159-186. Fru-2,6-P2 is apparently neither present in chloroplasts nor 18. Pontis, H. G. (1977) in Plant Biochemistry II, ed. Northcote, D. functional in the regulation ofphotosynthesis. Support for this H. (University Park Press, Baltimore), Vol. 13, pp. 79-117. conclusion comes not only from the experiments described 19. Kalberer, P. P., Buchanan, B. B. & Arnon, D. I. (1967) Proc. above but also from related experiments in which we were un- Natt Acad. Sci. USA 57, 1542-1549. of Fru-2,6-P2 in oxygenic 20. Arnon, D. I., McSwain, B. D., Tsujimoto, H. Y. & Wada, K. able to detect significant quantities (1974) Biochim. Biophys. Acta 357, 231-245. photosynthetic prokaryotes [(Nostoc muscorum, acyanobacteri- 21. Cseke, C., Nishizawa, A. & Buchanan, B. B. (1982) Plant Phys- um, and Prochloron, a chlorophyll b-containing unicellular or- iol, in press. ganism (37, 38)]. The evidence currently available thus indicates 22. Wirtz, W., Stitt, M. & Heldt, H. W. (1980) Plant Physiot 66, that, at least in the case ofphotosynthetic organisms, Fru-2,6- 187-193. P2 occurs and functions mainly, ifnot exclusively, in . 23. Wolosiuk, R. A. & Buchanan, B. B. (1977) Nature (London) 266, 565-567. Note Added in Proof. It is pertinent to note that a similar insensitivity 24. Lovenberg, W., Buchanan, B. B. & Rabinowitz, J. C. (1963) J. ofchloroplast Fru-P2ase to Fru-2,6-P2 has also been observed by others Biot Chem. 238, 3899-3913. (39). 25. Arnon, D. I. (1949) Plant Physiot 24, 1-15. 26. Uyeda, K., Furuya, E. & Luby, L. J. (1981)J. Biot Chem. 256, This work was supported in part by grants from the National Science 8394-8399. Foundation (to B.B.B.) and the National Institutes ofHealth (to K.U.). 27. Preiss, J., Biggs, M. L. & Greenberg, E. (1967) J. Biot Chem. B.B.B. also gratefully acknowledges a gift from Mr. Frank Weeden. 242, 2292-2294. 28. Buchanan, B. B., Schurmann, P. & Kalberer, P. P. (1971)J. Biot 1. Furuya, E. & Uyeda, K. (1980) Proc. Nati Acad. Sci. USA 77, Chem. 246, 5952-5959. 5861-5864. 29. Zimmermann, G., Kelly, G. J. & Latzko, E. (1976) Eur. J. 2. Van Schaftingen, E., Hue, L. & Hers, H. G. (1980) Biochem.J. Biochem. 70, 361-367. 192, 887-895. 30. Kelly, G. J. & Latzko, E. (1977) Plant Physiot 60, 290-294. 3. Van Schaftingen, E., Hue, L. & Hers, H. G. (1980) Biochem. J. 31. Kelly, G. J. & Latzko, E. (1977) Plant Physiol 60, 295-299. 192, 897-901. 32. Kuwajima, J. & Uyeda, K. (1982) Biochem. Biophys. Res. Com- 4. Uyeda, K., Furuya, E. & Sherry, A. D. (1981)J. Biot Chem. 256, mun. 104, 84-88. 8679-8684. 33. Stitt, M., Wirtz, W. & Heldt, H. W. (1980) Biochim. Biophys. 5. Pilkis, S. J., Raafat El-Maghrabi, M., Pilkis, J., Claus, T. H. & Acta 593, 85-102. Cumming, D. A. (1981) J. Biot Chem. 256, 3171-3174. 34. Van Schaftingen, E. & Hers, H. G. (1981) Biochem. Biophys. Res. 6. Van Schaftingen, E. & Hers, H. G. (1981) Proc. Nati Acad. Sci. Commun. 101, 1078-1084. USA 78, 2861-2863. T. H., Pilkis, J. & Pilkis, S. J. 7. Pilkis, S. J., Raafat El-Maghrabi, M., Pilkis, J. & Claus, T. H. 35. Raafat El-Maghrabi, M., Claus, (1981) J. BioL Chem. 256, 3619-3622. (1982) Proc. Natt Acad. Sci. USA 79, 315-319. 8. Sabularse, D. C. & Anderson, R. L. (1981) Biochem. Biophys. 36. Furuya, E., Yokoyama, M. & Uyeda, K. (1982) Proc. Natt Acad. Res. Commun. 100, 1423-1429. Sci. USA 79, 325-329. 9. Sabularse, D. C. & Anderson, R. L. (1981) Biochem. Biophys. 37. Lewin, R. A. & Withers, N. W. (1975) Nature (London) 256, Res. Commun. 103, 848-855. 735-737. 10. Reeves, R. E. (1976) Trends Biochem. Sci. 1, 53-55. 38. Lewin, R. A. (1976) Nature (London) 261w 697-698. 11. Wood, H. G., O'Brien, W. E. & Michaels, G. (1977) Adv. En- 39. Gottschalk, M. E., Chatterjee, T., Edelstein, I. & Marcus, F. zymot 45, 85-155. (1982)J. Biolt Chem. 257, in press. Downloaded by guest on September 25, 2021