Plant Physiol. (1990) 94, 499-506 Received for publication January 5, 1990 0032-0889/90/94/0499/08/$01 .00/0 Accepted May 4, 1990 -6-P 1- from Tomato Fruit1

Evidence for Change during Ripening

Joshua H. Wong, Ferenc Kiss2, Ming-X WU3, and Bob B. Buchanan* Department of Plant Biology, Genetics and Plant Biology Building, University of California, Berkeley, California 94720

ABSTRACT from most plant sources is highly stimulated by Fru-2,6-P2 Three forms of pyrophosphate fructose-6-phosphate 1-phos- (1, 2, 5, 6, 8-10, 15-18, 20, 21), the from leaves of photransferase (PFP) were purified from both green and red CAM plants responds only sluggishly to this activator (5, 8, tomato (Lycopersicon esculentum) fruit: (a) a classical form (des- 10). In another case (wheat seedlings), PFP activity has been ignated 02) containing a- (66 kilodalton) and ,B- (60 kilodalton) reported to be associated with two structurally different en- subunits; (b) a form (01) containing a ,-doublet subunit; and (c) zyme forms (21). Finally, in a recent study (18), our laboratory a form (Q0o) that appeared to contain a ,B-singlet subunit. Several has identified in carrot roots a form of PFP that differs both lines of evidence suggested that the different forms occur under kinetically and structurally from other forms of the enzyme physiological conditions. Q2 was purified to apparent electropho- studied to date. retic homogeneity; Q1 and Q0 were highly purified, but not to As a result of this unusual kinetic and structural diversity, homogeneity. The distribution of the PFP forms from red (versus green) tomato was: Q2, 29% (90%); 01, 47% (6%); and Q0, 24% we have considered the possibility that the properties of PFP (4%). The major difference distinguishing the red from the green depend on the functional nature of the parent tissue (18). tomato was the fructose-2,6-bisphosphate (Fru-2,6-P2)- That is, the form of PFP present in one tissue with a given induced change in Km for fructose-6-phosphate (Fru-6-P), the metabolic function may differ significantly from the enzyme 'green forms' showing markedly enhanced affinity on activation in a tissue with another function. We have put this idea to a (Km decrease of 7-9-fold) and the 'red forms' showing either little further test using pericarp of tomato fruit-a tissue known to change (Q0o, Q1) or a relatively small (2.5-fold) affinity increase change from a starch-storing to a sugar-storing function dur- (02). The results extend our earlier findings with carrot root to ing ripening (7, 14). The results indicate that, while occurring another tissue and indicate that forms of PFP showing low or no in analogous enzyme forms, the PFPs from green and red Fru on activation 01 affinity increase for 6-P by Fru-2,6-P2 (here on and Qo) are associated with sugar storage, whereas the classical fruit show a pronounced difference activation by Fru-2,6- form (02), which shows a pronounced affinity increase, is more P2. The activated green forms are characterized by a marked important for starch storage. increase in affinity for Fru 6-P, whereas the red forms show either little or no change in this connection. A preliminary account of this work has been published (19).

MATERIALS AND METHODS One of the puzzles in our understanding of PFP,4 PFP Plant Materials Fructose-6-P + PPi z fructose-1,6-P2 + Pi (1) Mg2+ Mature green and red stage fruits were harvested from an enzyme probably universally distributed in higher plants, tomato (Lycopersicon esculentum cv VFNT Cherry) plants is the wide variation in its properties. For example, while PFP grown in a greenhouse in U. C. mix and watered as needed with half-strength Hoagland solution. Fruit was harvested at 'This work was supported by grants from the Cellular Biochem- either the mature green or fully ripe stage (13). istry, U.S.-Hungary, and U.S.-China Programs of the National Sci- ence Foundation. 2 Permanent address: Department of Biology, Gyorgy Bessenyei Reagents College, Nyiregyhaza, Hungary. Biochemicals and lyophilized coupling enzymes were ob- Permanent address: Shanghai Institute of Plant Physiology, 300 tained Fonglin Road, Shanghai, People's Republic of China 200032. from Sigma Chemical Co. (St. Louis, MO). Solutions 4 Abbreviations: PFP, pyrophosphate fructose-6-P 1 -phosphotrans- of Fru-6-P and Fru- 1,6-P2 were prepared after acid treatment ferase; Fru-6-P, fructose-6-phosphate; Fru- 1,6-P2, fructose- 1 ,6-bis- to remove any Fru-2,6-P2 contamination (15 min in 0.25 N phosphate; Fru-2,6-P2, fructose-2,6-bisphosphate; FPLC, fast protein HCI then neutralized by 0.25 N NaOH). Other reagents were liquid chromatography. purchased from commercial sources and were of the highest 499 500 WONG ET AL. Plant Physiol. Vol. 94, 1990

quality available. DEAE (DE-52) cellulose and pnosphocel- 100 0 lulose (P-I1) were purchased from Whatman, Inc. (Clifton, 0 10.6 NJ). Q Sepharose Fast Flow, Superose 12, and Mono Q were x4 obtained from Pharmacia LKB Biotechnology. c E 0.4 Enzyme Assays 0 a,

All assays were carried out at 25°C in a Beckman DU cuu Lo 0.2 spectrophotometer equipped with a Gilford automatic sample - 0 changer and recorder. PFP activity was assayed in the forward I and reverse directions as described previously (6, 18).

0 pH Study Fraction Number The optimal pH for each of the enzyme forms was deter- Figure 1. Elution profile of red tomato fruit PFP from a phosphocel- mined using Mes-Tris buffer (100 mm total concentration) at lulose column. 0.2 unit intervals in the pH range from 5.6 to 9.0.

Kinetic Measurements system. Spectra/pore dialysis membrane (VWR Scientific, San Francisco, CA) with a 12 to 14 kD cutoff was used for Kinetic measurements were conducted at the optimal pH dialysis against buffer solutions or, for concentration against of each form of PFP, i.e. pH 8.0 for both forward and reverse sucrose, glycerol or PEG. Highly purified samples were con- directions. There were 10 to 12 substrate () concen- centrated using a Centricon 30 (Amicron, Danvers, MA) with trations in each set of determinations. The concentration a 30 kD cutoff membrane. Buffers were adjusted to the range in the assay mixture (mM) was: Fru-6-P, 0 to 10; PPi, 0 indicated pH at room temperature. to 2.0; MgCl2, 0 to 5.0; Fru-1,6-P2, 0 to 3; Pi, 0 to 10. The concentrations for Fru-2,6-P2 in ,iM were: 0, 0.02, 0.04, 0.1, Preparation of Crude Extract 0.2, 0.4, 1.0, and 2.0. For kinetic measurements, the reaction was initiated by adding enzyme. Each data point represented Five kg ofpericarp tissue was obtained by manually remov- the mean of two determinations. Values for Vmax, Km, and Ka ing pulp (locules and seeds) with a spatula. After washing were calculated using the equation for Michaelis Menten once with distilled water, pericarps, in 1 kg lots, were added Kinetics that was included in a nonlinear regression data to 1 L of a homogenization solution that contained 75 mM analysis program (ENZFITTER from Elsevier-Biosoft). The Tris buffer (pH unadjusted), 10% glycerol, 14 mm 2-mercap- coefficient for each set of data was 0.95 or better. toethanol, 1 mM EDTA. The mixture was blended for 2 x 30 s in a Waring Blendor (1 gallon capacity), the pH after Purification of PFP from Ripe Tomato Fruit blending was approximately 7.5. The homogenate was filtered through four layers of nylon cloth (30 threads/cm) and the All preparative procedures were carried out at 4°C unless retentate was discarded. The filtered extract was adjusted to otherwise stated. FPLC on Mono Q and Superose 12 columns pH 8.0 with 1 M KOH and centrifuged (30 min, 13,7000g). was carried out at room temperature using a Pharmacia The dark red precipitate was discarded and the light red

Table I. Summary of Purification of PFPs from Red Tomato Fruit Total Total Specific Purification Step Protein Activity Activity Recovery mg ua U/mga % -fold 1) Initial extract 7292 1054 0.15 100 1 2) 1st DEAE-cellulose 1659 589 0.36 56 2 3) 2nd DE-52 646 269 0.42 25.5 3 4) Phosphocellulose 30 123 4.12 11.7 28 5) 0 Sepharose Peak A 8.4 58.6 6.98 Peak B 6.8 52.9 7.78 Total 15.2 111.5 7.34 10.6 51 6) Mono 0 FPLCb QO 4.2 22.7 5.40 0, 7.0 43.4 6.20 Q2 3.1 27.0 8.71 Total 14.3 93.1 6.51 8.8 45 a U = gmol Fru-6-P formed/min. b PFP activity from peaks A and B was further separated at this step. MULTIPLE FORMS OF TOMATO PFP 501

0- 0 volume was 800 mL). Fractions of 6.9 mL were collected. A cJ single PFP activity peak was eluted to 0.44 M KCI. Fractions showing PFP activity were pooled and concentrated by di- aL) alysis against 50% glycerol and 1 ,um Fru-2,6-P2. 011.4 IN, Q Sepharose Chromatography -a0 'a- The concentrated sample was diluted two times (to 96 mL) with buffer A and applied to a Q Sepharose column (2.5 x C) 15 cm) that was equilibrated with buffer A. The column was washed with 1 volume of buffer A, and PFP was then eluted with 300 mL of a linear 0 to 0.4 M KCI gradient that was a- U- o followed by a wash with 100 mL of 1 M KCI. Fractions of 6 a- 0 10 20 30 40 50 60 mL were collected. Two PFP activity peaks (designated A and Fraction Number B) were recovered-one eluting at 0.20 to 0.26 M KCl and the other at 0.26 to 0.31 M KCl. The fractions under each Figure 2. Elution profile of red tomato fruit PFP from a Q Sepharose peak were combined separately and concentrated by dialysis column. against 30% PEG in buffer A. supernatant fraction was subjected to batchwise adsorption Fast-Protein Liquid Chromatography with Mono Q Column onto DEAE-Cellulose without delay so that the extract did The concentrated PFP peaks were separately applied to a not gel. Mono Q HR 5/5 column equilibrated with buffer A. The Batchwise Adsorption on DEAE-Cellulose Moist DEAE-cellulose (coarse), which had been equili- brated with homogenization solution adjusted to pH 8.0 with HCI, was added to the clarified extract (10 g wet weight/100 80 mL extract). The mixture was stirred for 30 min and then left to stand an additional 30 min. The supernatant solution was removed by filtration under vacuum and treated with another 60 lot of matrix as before. This step was repeated. The three lots of matrix were combined and washed once with 1 L ofbuffer o 40 A containing 20 mM Tris-HCl (pH 8.0), 10% glycerol, 14 mM 0 2-mercaptoethanol, and 1 mM EDTA. The bulk of the PFP c activity was then eluted with buffer A containing 0.5 M KCI @ 20 0.2 > in three 1-L washes. The combined DEAE-cellulose elutates were concentrated by dialysis against PEG. 10un 0.1 S o DE-52 Chromatography E0 rn The concentrated PEG fraction was diluted 10 times with buffer A and centrifuged (25 min, 27,000g). The clarified sample (366 mL) was applied at a flow rate of 3 mL/min to z> 80 - a DE-52 column (2.7 x 28 cm) that was equilibrated with a- U- buffer A. The column was washed with 200 mL buffer A and a- ':10- PFP was then eluted with a linear 0 to 0.5 M KCI gradient in buffer A (total volume was 1 L). Fractions of 9 mL were collected. A single peak of PFP activity was eluted at 0.2 M KCI. Fractions containing the bulk of PFP activity were combined and concentrated by dialysis against buffer A that was modified to contain 50% glycerol and 1 gM Fru-2,6-P2. v Phosphocellulose Chromatography Ile The concentrated PFP sample was diluted three times with 5 10 15 20 25 buffer A and applied (3 mL/min) to a phosphocellulose Fraction Number column (2.7 x 33.5 cm) equilibrated with buffer A. The Figure 3. Chromatography of 0 Sepharose PFP fractions (red to- column was washed with 300 mL buffer A, and PFP was then mato fruit) on Mono 0. Upper panel, elution profile of 0 Sepharose eluted with a linear 0 to 0.6 M KCI gradient in buffer A (total peak A; lower panel, peak B. 502 WONG ET AL. Plant Physiol. Vol. 94,1990

Table II. Summary of Purification of PFPs from Green Tomato Fruit Step Total Total Specific Protein Activity Activity Recovery Purification mg Ua U/mga % -fold 1) Initial extract 3994 193.8 0.05 100 1 2) DE-52 1293 139.2 0.11 72 2 3) Phosphocellulose 45.84 124.2 2.71 64 56 4) 0 Sepharose Peak A 3.8 15.2 3.98 Peak B 6.3 91.7 14.46 Total 10.1 106.9 10.10 53 208 5) Mono Q FPLCb Q0 1.01 3.45 3.42 01 1.37 6.20 4.53 02 5.40 88.66 16.42 Total 7.78 98.31 12.64 50 261 6) Superose 12 FPLC 00 0.66 2.42 3.66 01 0.23 1.94 8.42 02 3.38 76.84 22.73 Total 4.27 81.22 19.01 42 372 a U = ,smol Fru 6-P formed/min. b PFP activity from A and B was further separated at this step. column was eluted sequentially with 1 mL of buffer A, 5 mL blended in 1 L of homogenization buffer, filtered, and centri- of a linear salt gradient from 0 to 150 mm KCl, 4 mL of 150 fuged as described above ("Preparation of Crude Extract"). mM KCl, 5 mL of a linear salt gradient from 150 to 200 mM The resultant crude extract, in 1.5 L, was applied directly to KCl, 5 mL of 200 mm KCl, 8 mL of a linear gradient from a DE-52 column (1.6 x 15 cm) that was developed as de- 200 to 300 mm KCl, 7 mL of a linear gradient from 300 to scribed above ("DE-52 Chromatography"). The procedures 1000 mm KCl, and finally 5 mL of 1000 mm KCI, all in buffer used for chromatography on phosphocellulose, Q Sepharose, A. Fractions of 1 mL were collected. Peak A from Q Sepharose Mono Q, and Superose 12 were also the same as those was resolved into two PFP activity peaks on Mono Q: the respectively described above for red tomato. first peak (designated Qo) eluting between 11O to 150 mM KCl and the second (designated Q,) between 150 to 160 mm KCl. Forms of PFP in Crude Extracts Peak B from Q Sepharose was resolved into one minor and Crude extracts were obtained from 30 g of pericarp tissues two major activity peaks on Mono Q: the minor peak eluted from green fruit or red fruit, from a 1:1 mixture of the two as Qo; the first major peak eluted as Q, and the second major fruit types (15 g each). The procedure was as described above peak (designated Q2) eluted between 200 to 230 mM KCl. for preparation of crude extract, except the extraction buffer Each activity peak was combined separately, concentrated by had 2 mm each of benzamidine, E-amino-n-caproic acid, and Centricon 30, and stored at -20°C. 0.5 mM PMSF. After centrifugation (20 min, 46,000g) and desalting through Bio-Rad PDlO columns, an equal amount Fast-Protein Liquid Chromatography with Superose 12 of each extract was subjected to FPLC Mono Q chromatog- Each of the concentrated PFP samples from Mono Q was raphy as described and analyzed for PFP activity. applied to a Superose 12 (HR 10/30) column equilibrated and eluted with buffer A containing 150 mM KCl. Fractions Protein Determination of 250 AL were collected. Activity peaks were combined and Protein was determined by the Bradford method (4), using concentrated by Centricon 30. Each PFP sample from Mono Bio-Rad reagent and bovine -y-globulin as standard. Q was found to contain a single mol wt species. The concen- trated PFP samples could be stored for several weeks at -20°C Subunit Molecular Mass Determination without appreciable loss of activity. Long-term storage re- SDS-PAGE was performed with 7.5 to 15% (w/v) slab gels quired the presence of 50% glycerol, 14 mm 2-mercaptoetha- as described by Laemmli (12) with specifics previously de- nol (or 1 mm DTT), and 1 ,uM Fru 2,6-P2. scribed (18). Protein was stained with Coomassie blue R-250 or silver stain. Purification of PFP from Mature Green Tomato Fruits A modified purification procedure (omitting batchwise ad- Native Molecular Mass Determination sorption on DEAE-Cellulose) for green tomato gave better Native molecular mass ofthe individual enzyme forms was yield. In the modified procedure 1 kg of pericarp tissue from determined by gel filtration on a Superose 12 column (HR1O/ mature green tomato fruit (free of locules and seeds) was 30) as previously ( 18). MULTIPLE FORMS OF TOMATO PFP 503

I, step 2), which represented a true chromatography, PFP was eluted at 0.2 M KCl as a single activity peak. While serving to 0 LO~ concentrate the enzyme, the first two steps brought about (Qo) only a threefold purification with a 25% yield. 40 Chromatography on phosphocellulose proved to be an ef- 0 fective step, resulting in nearly a 10-fold purification and about a 50% activity loss (Table I, step 4). Again, PFP activity E 30 was recovered as a single peak (Fig. 1). It was only at the Q Sepharose step that multiple forms of the enzyme became +Fru 2,6-P2 apparent. Here the preparation was resolved into two activity 20- peaks ofalmost equal proportion (Table I, step 5; Fig. 2). The 0.3 first peak (designated A) eluted between 0.20 and 0.26 M KCI o (ot and the second peak (B) between 0.26 and 0.31 M KCI. The 10 - . -0.2 and extents of U- two peaks showed similar specific activities 0. - Control 0.1 Y purification (Table I, step 5). Further fractionation on a strong anion exchange column, Mono Q, further resolved the en- 0 5 10 15 20 25 zyme into its component forms (Table I, step 6). Peak A was 0 I separated into two activity peaks, the first (designated Qo) peak A;lorPeak B . eluting between 110 to 150 mM KCI, and the second (Q,) 1<120 (2 between 150 to 160 mM KCI (Fig. 3, upper panel). Peak B from the Q Sepharose step was resolved into three PFP activity 90 +Fru 26-P2( peaks on Mono Q, a minor Qo peak, and two major peaks, C a previously unde- E one corresponding to Q, and the other to 0 tected component eluting between 200 to 230 mM KCI (Q2) 60 (Fig. 3, lower panel). This latter form of PFP (Q2) had the protein) representing 0.3 highest specific activity (8.7 units/mg an overall purification of 60-fold. At this stage only the Q2 30 - -- . 0.2-:- component was judged to be highly purified (95%) as deter- Overall, the enzyme was purified 45- These cControl 0.1 mined by SDS-PAGE. U_ 000(Qi)(Qo fold (average specific activity was 6.5 units/mg) with a yield 5 10 15 20 25 of about 9%. Fraction Number Subsequent gel filtration chromatography on Superose 12 removed additional minor contaminants so that Q2 was ho- Figure 4. Chromatography of 0 Sepharose PFP fractions (green mogeneous from both sources, Q, was homogeneous from tomato fruit) on Mono 0. Upper panel, elution profile of 0 Sepharose peak A; lower panel, peak B. 00 RESULTS R G R G R G Our initial data on pericarp PFP were obtained with resus- pended PEG fractions. The PFP activity ofred and green fruit kDa preparations was found to differ with respect to: (a) effect of Fru-2,6-P2 on Km for Fru-6-P; (b) Ka for Fru-2,6-P2; (C) molecular mass of fraction showing major activity; (d) amount of PEG required for precipitation; and (e) stability. a -& - 68 These differences were pronounced to encourage sufficiently -_ai ~A._ 60 us to purify and characterize the PFPs from the two sources. We describe first the purification of the enzymes from the -43 two types of tomato fruit and then present certain of their IMr distinguishing features. Purification of PFP from Ripe Tomato Fruit I- A summary of the purification of PFP from red tomato fruit is shown in Table I. To avoid polymerization of the .- crude extract and the resultant clogging of the column, we adsorbed and eluted the clarified extract from DEAE-cellulose Figure 5. SDS-PAGE analysis of the three forms of tomato fruit PFP, as soon as possible. Timing of this step was critical because it 0Q, Q, and 02- 'R' stands for red and 'G' for green. Molecular mass affected the overall yield. In the subsequent DE-52 step (Table standards (kD) were BSA, 68; catalase, 60; and ovalbumin, 43. 504 WONG ET AL. Plant Physiol. Vol. 94,1990

Table Ill. Kinetic Constants of PFPs from Tomato Fruit Parameter Small (Qo) Small (01) Large (02) Forward control +F2,6-P2 control +F2,6-P2 control +F2,6-P2 Red Km, Fru-6-P 0.50 0.60 0.50 0.40 1.00 0.38 Km, PPi 0.07 0.04 0.03 0.02 0.01 0.02 Ka, Fru-2,6-P2a 6.0 13.0 4.0 Green Ki, Fru-6-P 2.30 0.26 1.90 0.22 2.65 0.38 Km, PPi 0.01 0.01 0.02 0.01 0.01 0.02 Ka, Fru-2,6-P2a 6.4 9.5 3.1 Reverse Red Km, Fru-1,6-P2 0.07 0.07 0.04 0.04 0.05 0.05 Km, Pi 0.95 0.95 0.61 0.61 0.80 0.80 Green Km, Fru-1,6-P2 0.05 0.05 0.06 0.06 0.05 0.05 Km, Pi 0.70 0.70 0.75 0.75 1.00 1.00 Unit for Km = mM; Ka = nM. a Fold activations by Fru-2,6-P2 (1 AM) in the forward direction for the different forms of PFP were: red tomato 00 (12.9), 01 (7.4), and Q2 (4.8); green tomato Q0 (8.2), 01 (6.4), and 02 (4.1). No significant (5-15%) activation by Fru-2,6-P2 in reverse direction was observed for any of these forms from either green or red fruit.

green fruit, and the remaining forms (Q, from red fruit, Qo peak B was increased (data not shown). The PFP activity of from green or red fruit) were about 50% pure. In each case, green tomato peak A was resolved into Qo and Q, on the the specific activity and yield were invariably reduced by gel Mono Q column (Fig. 4, upper panel), respectively, account- filtration, possibly due to dilution of the enzyme (data not ing for 4 and 6% of the total activity (Table II, step 5). Peak shown). B represented mainly Q2 (90% of the total activity) (Table II, step 5; Fig. 4, lower panel). Further fractionation of each of Purification of PFP from Mature Green Tomato Fruit the enzyme forms on Superose 12 removed minor protein contaminants resulting in an increase in specific activity and The purification procedure for green tomato PFP is sum- a decrease in recovery (Table II, step 6). marized in Table II. As mentioned above, the omission ofthe batchwise DEAE-cellulose step greatly enhanced recovery. Physical Properties of PFPs from Red and Green Tomato Overall, the enzyme was enriched 372-fold and showed a Fruits specific activity of 19 units/mg protein; the yield of 42% was Subunit four- to fivefold higher than with the preparation from red Composition. tomato. However, like red tomato, green tomato PFP: (a) The purified Q2 form of PFP showed two polypeptides in gave a single activity peak on both DE-52 (step 2) and SDS-PAGE, the larger (66 kD) corresponding to the a-subunit phosphocellulose (step 3); (b) showed the greatest purification and the smaller (60 kD) to the A-subunit (Fig. 5). It is noted on phosphocellulose; and (c) was resolved into two activity that, in most preparations, the Q2 form from green fruit peaks on Q Sepharose-peaks A and B (step 4). Relative to showed a third component, that traveled slightly behind the red tomato, the green tomato PFP peak A was reduced and 3-subunit (Mr = 62 kD) and may correspond to a ,3-doublet.

Table IV. Summary of Characteristics of Red and Green Tomato PFPs Effect of Fru-2,6-P2 on Relative Abundancea PFP Molecular Subunit for Fru-2,6-P2 for Fru-6-P Mass Composition Ka nM Km Green Red G Green Red Form kD Q0 68 ,3-singlet) 6.2 9-x 4 None 4 24 01 68 f-(doublet) 11.3 9-x 4 None 6 47 Q2 443 a-# 3.6 7-x 4 3-x 1 90 29 a These values were estimated from the relative total activity recovered for each form of PFP from the two types of tomato fruits after purification as described in Tables I and I. The corresponding values for Q0, 01, and Q2 observed with desalted crude preparations subjected to Mono Q chromatography were 6, 20, and 74% for green fruit and 7, 34, and 59% for red fruit. MULTIPLE FORMS OF TOMATO PFP 505

The purified Q, form showed two bands, 62 and 60 kD, also DISCUSSION corresponding to a doublet 3-subunit (cf 18); there was no evidence ofan a-subunit. The purified Qo preparation showed The present findings both give answers and raise questions several polypeptide bands in SDS-PAGE; based on a compar- about PFP. First, a comment is in order regarding the phys- ison with the other PFP fractions, Qo contained a d-subunit iological significance of the forms of PFP observed in this but, as with Ql, there was no evidence of an a-subunit. study. The following lines of evidence suggest that the three The a-3 form of PFP (Q2) is similar to the classical form forms characterized (Qo, Ql, Q2) occur under physiological identified in wheat seedlings (21), potato tubers (3, 11, 22), conditions: (a) all three forms were observed in both green bean and cucumber seedlings (3). The 1-doublet form (Q,) is and red fruit irrespective ofthe presence ofprotease inhibitors; similar in to the PFP from carrot (b) the three forms were recovered from both fruit types in subunit composition purified in roots ( 18). While further purification is required for a defini- highly purified preparations and desalted crude extracts tive characterization, it seems possible that the 1-singlet (Qo) fractionated by Mono Q chromatography (the relative per- to earlier seen with centage of the different forms in both types of preparations is form corresponds the minor form wheat in seedlings (21). It should be noted that inclusion of protease given Table IV); and (c) mixing equal amounts ofthe green inhibitors (2 mm each of acid and red tissues prior to blending yielded the forms observed benzamidine, e-amino-n-caproic in and 0.5 mm PMSF) had no significant effect on the distribu- in each tissue separately and approximately the expected tion of enzyme forms seen in red or green fruit. ratio (data not shown). The current study also gives information on the a-subunit. Native Molecular Mass As evident from the overall summary in Table IV, the results provide evidence that the a-subunit increases the affinity Gel filtration profiles determined early in this study re- (lowers the Ka) of PFP for Fru-2,6-P2. Thus the Q2 form, vealed that PFPs from red and green tomato existed in mul- containing an a-subunit showed a Ka one-third to one-half tiple enzyme forms (cf refs. 20-22). In both cases, the form that of the two forms lacking an a-subunit (Ql, Qo). The eluting from Mono Q at high salt (Q2) showed a molecular carrot root enzyme, found here to be ofthe Q, type, also lacks mass of443 kD (Ve identical to that ofapoferritin), suggesting an a-subunit and shows an even higher Ka for Fru-2,6-P2 (22 the enzyme to be a heterologous oligomer (data not shown, nm, ref. 18). In addition, in both green and red fruit the cf 1 1, 21, 22). The corresponding value for both forms eluting presence of the a-subunit (in Q2) significantly increased the at low salt (Ql and Qo) was 68 kD (Ve identical to that of specific activity of the enzyme (Tables I and II). Thus, based BSA). on current information, enhancement of Fru-2,6-P2 affinity and increase in specific activity are functions ofthe a-subunit. Kinetic Properties The present results support our earlier suggestion ( 18) that The kinetic properties of the different forms of red and tissues functional in sugar storage contain a form of PFP with green tomato PFP are summarized in Table III. In the forward an altered response to Fru-2,6-P2. Thus, while the PFP of (glycolytic) direction, the control Km values for Fru-6-P and green tomato fruit (Table IV) and other starch storing tissues PPi were similar for the enzyme forms from each source (Qo, (1, 2, 6, 15, 17, 20, 21) shows a marked increase in affinity Q1, Q2). However, the enzyme forms showed pronounced for Fru-6-P substrate on activation by Fru-2,6-P2, the enzyme tissue differences in the Fru-2,6-P2 induced change in Km for in sugar storing tissues shows either little change (red tomato Fru-6-P (values shown in bold type), i.e. a large (7- to 9-fold) fruit, Table IV) or a decrease (carrot root, ref. 18) in affinity decrease in Km for each of the three forms from green tomato for Fru-6-P substrate. As pointed out earlier, these properties and either little change (Qo and Q,) or a relatively small (2.5- may be essential for maintaining the proper balance of PPi/ fold) decrease (Q2) in Km for the forms from red fruit (Table Pi as well as Fru-6-P/Fru-2,6-P2 in cells storing sugars. The III). The Qo and Q, forms from red fruit thus resemble the relatively high abundance of the forms showing this behavior carrot root PFP that showed an actual increase in Km for Fru- in red tomato fruit (Q,, Qo) is in accord with this conclusion. 6-P following activation by Fru-2,6-P2 (18). The present work also raises new questions. It is not clear The forms oftomato fruit PFP exhibited different Ka values how forms of PFP showing similar subunit content, can differ for Fru-2,6-P2: Q2, Q,, and Qo showing respective values of so significantly with regard to Fru-2,6-P2 induced change in about 3, 12, and 6 nM. In this regard, the red and green fruit affinity for Fru-6-P, e.g. (Ql and Qo from red versus green forms showed similar values (Table III). The individual PFPs fruit). Such a difference could result either from a posttrans- from green and red fruit also did not differ greatly from one lational modification of the enzyme or from the synthesis of another with respect to pH optimum (7.5-8.0), Mg2+ require- a different enzyme during ripening. At present there are no ment (Km of 0.06-0.65 mM) and inhibition by Pi (Io.5 of 4.0- results to favor either of these possibilities or to suggest the 6.0 mM) (data not shown). nature of the agent triggering the change. The PFPs from the two fruit types also were similar when analyzed in the reverse (gluconeogenic) direction. As sum- LITERATURE CITED marized in Table III, Fru-2,6-P2 had little effect on the kinetic 1. Black CC, Carnal NW, Paz N (1985) Roles of pyrophosphate parameters tested (values were identical to the minus Fru-2,6- and fructose-2,6-bisphosphate on regulating plant sugar metab- olism. In RL Heath, J Preiss, eds, Regulation of Carbon P, controls), the Km and Ki values being similar in all cases. Partitioning in Photosynthetic Tissue, American Society of Similar findings have been made with PFP from other sources Plant Physiologists, Rockville, MD, pp 45-62 (9). 2. Botha FC, Small JGC, de Vries C (1986) Isolation and charac- 506 WONG ET AL. Plant Physiol. Vol. 94, 1990

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