Pyrophosphate Dependent Phosphofructokinase of. Citrullus lanatus: Molecular Forms and. Expression of Subunits1. Anna-Maria Botha and Frederik C. Botha*.
Received for publication November 8, 1990 Accepted March 7, 1991
Plant Physiol. (1991) 96, 1185-1192 0032-0889/91/96/1185/08/$01 .00/0
Pyrophosphate Dependent Phosphofructokinase of Citrullus lanatus: Molecular Forms and Expression of Subunits1 Anna-Maria Botha and Frederik C. Botha* Department of Botany, University of the Orange Free State, Bloemfontein, South Africa In several species, increased PFP activity is associated with the tissues in which a net sucrose breakdown occurs (32 and references therein). In certain tissues, like castor bean endosperm (2 1) and the cotyledons of Citrullus lanatus (7), however, maximum PFP activity during germination coincides with the peak in gluconeogenesis. Whether these changes in PFP activity during specific physiological conditions (6, 1 1, 17, 21, 32) are due to activation of existing enzyme or to induced gene expression is unclear. The increase in PFP activity during germination in the cotyledons of C. lanatus is due to an increase in the PFP concentration (7), presumably by increased gene expression. However, interconversion between different molecular forms, as was reported for the PFP from pea (31) and potato (20), might also contribute to changes in PFP activity. In the latter studies, it was shown that PFP can be reversibly converted from a high to a low molecular form depending on the presence of PPi and Fru-2,6-P2 (20, 31). The enzyme from potato dissociates into a dimer (120 kD) in the presence of PPi, and aggregates into a tetramer (260 kD) in the presence of Fru-2,6-P2 (20). The PFP protein from a variety of plant tissues appears to contain two types of subunits with mol wts of approximately 60,000 to 66,000 (9, 20, 33). Although there is a 60% homology at the amino acid level between the coding regions of the PFPa and PFP3 genes in potato and castor bean (1 3), the two subunits differ in their immunological properties (20, 33). In certain tissues, however, PFP apparently contains only one type of subunit (4, 28). In wheat, PFP is present as two isoforms, one a homodimer (f2 composition) and the other a heterotetramer (a232 composition) (33). In mung bean, both PFP subunits have a mol wt of 60,000 (15). The presence of isoenzymes of PFP in wheat (33) and tomato (29), a novel type of PFP in the tap root of carrot (28), as well as a PFP in Phaseolus mungo (3) and Phaseolus vulgaris (1 1) with kinetic properties distinctly different from other PFPs, suggest that PFP has variable properties when isolated from different tissues. Its properties can also change in the same tissue at different stages of development. Here we report that different isoforms of PFP are present in the cotyledons of C. lanatus and that the ratio between the isoenzymes differs depending on the stage of development. The form in which PFP is present appears to be dependent on the concentration of the subunits, which are not coordinately expressed.
ABSTRACT During germination and seedling establishment, the total pyrophosphate-dependent phosphofructokinase (PFP) activity in the cotyledons increases. Two types of subunits with molecular weights of 68 (a-subunit) and 65 (,-subunit) kilodaltons are present. The increase in activity coincides with an approximately 10fold increase in #-subunit and twofold increase in a-subunit content. Different isoforms of PFP are present at all stages of incubation, but the ratio between the isoforms significantly changes. A linear relationship exists between the ratio of the two PFP subunits and the ratio of the two isoforms of the enzyme. The more anionic (peak 2) isoform of the enzyme apparently is favored by a high ratio of total #-subunit to a-subunit content. The is- to a-subunit ratio of the peak 2 isoform is also approximately fivefold higher than that of the peak 1 (less anionic) isoform. It is evident that the two subunits are not coordinately expressed and the level of expression of each subunit appears to be the primary factor determining the molecular form in which the enzyme is present. In some tissues, only the 65 kilodalton polypeptide is expressed in large amounts. The peak I isoform has a higher affinity for pyrophosphate than the peak 2 isoform, while the affinity for fructose-6-phosphate is similar. Both molecular forms are activated by fructose-2,6-bisphosphate.
In addition to the ATP-dependent phosphofructokinase, plants also contain a pyrophosphate-dependent phosphofructokinase, PFP,2 that catalyzes the conversion of Fru-6-P to Fru-2,6-P2 in the cytosol. Since the discovery in 1979 of PFP in
pineapple leaves (14), significant levels of this
enzyme have
been found in a variety of plant tissues (1, 3, 4, 11, 17, 2 1, 23, 24, 28, 29, 33). Although the kinetic properties of PFP from several plant species have been studied (4, 8, 11, 19, 24-26, 28), the physiological role of PFP in plants is still in doubt. Major functions that have been attributed to the enzyme include rapid equilibration of the hexose phosphate/triose phosphate pools (16), regulation of the cytosolic PPi levels (1, 2, 6, 18, 26), and an adenylate bypass for glycolysis during Pi starvation ( 1 7). ' This project is financially supported by the Foundation for Research Development and the University of the Orange Free State. 2Abbreviations: PFP, PPi:D-fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90); Fru-6-P, fructose-6-phosphate; Fru-2,6-P2, fructose-2,6-bisphosphate; IgG, immunoglobulin.
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MATERIALS AND METHODS
Material
Seeds of Citrullus lanatus (Thunb.) Matsumura and Nakai (colocynth or bitter melon) were obtained and stored as previously described (10). The different tissues of C. lanatus plants were obtained from plants grown in a glass house. The Bio-Gel DEAE-5-PW anion exchange column was obtained from Bio-Rad. Immunochemicals, substrates, and cofactors were from the Sigma Chemical Company, coupling enzymes from Boehringer Mannheim, and nitrocellulose (0.45 Mm pore size) from Amersham. Antisera against purified potato PFP were obtained and purified as previously described (9). Germination Seeds were soaked in distilled water for 4 h at room temperature before the outer seed structures (seed coat and underlying seed membrane) were removed as previously described (10). The 4 h soaking period was not considered as part of the total incubation time, i.e. removal of the outer seed structures was taken as 0 h of incubation. Germination tests were conducted in 500 mL Schott Duran glass bottles capped with cotton wool. Seeds were incubated at 270C on a single layer of Schleicher and Schuell No. 595 filter paper moistened with 4 mL of water. A seed was considered germinated when radicle elongation was visible. Extraction and Assay of PFP All extraction procedures were carried out on ice or at 40C. Crude enzyme extracts were prepared by homogenizing the tissue (1 g of tissue in 2 mL buffer) in 100 mm Tris-acetate buffer (pH 8.0) containing 2 mM MgCl2, 2 mm EDTA, 14 mM 2-mercaptoethanol, 2 mm PMSF, and 10% (v/v) glycerol. The homogenate was centrifuged at 12,000g for 15 min. The supernatant was decanted and again centrifuged as described. Aliquots of the resulting supernatant were directly used for enzyme assays, protein determination, and immunoblotting. The PFP activity was measured in the presence of saturating Fru-2,6-P2 concentrations (10 gM) as previously described (21). The presence of possible PFP activators or inhibitors was determined by preparing series of extracts, each containing at least two different tissues. In all cases, the measured activity was between 95 ± 6% of that measured in tissues extracted separately. In addition, no significant differences were found between the PFP activity of crude extracts and desalted extracts. The addition of protease inhibitors, other than 2 mM PMSF, resulted in no significant difference in PFP activity. Purification of PFP Cotyledons were extracted for total PFP activity as described above. To the crude extract, solid PEG-8000 (5 g 100 mL-') was slowly added with continuous stirring. The preparation was allowed to stand for 30 min and then was centrifuged at 27,000g for 15 min. A further 10 g 100 ml-' PEG8000 was added to the resulting supernatant with continuous stirring and, after 1 h, centrifuged as described above. The
Plant Physiol. Vol. 96, 1991
resulting pellet was dissolved in 10 mM Hepes buffer (pH 7.5) containing 1 mM MgCl2, 14 mM 2-mercaptoethanol, and 10% (v/v) glycerol and was dialyzed overnight against the same buffer. After centrifugation at 27,000g for 15 min, the resulting supernatant was applied to the Bio-Gel DEAE-5-PW column (150 x 7.5 mm) equilibrated with the same buffer. The column was washed with four bed volumes of buffer and then developed with a 25 mL linear KCl gradient (0-0.4 M) at a flow rate of 1 ml min-'. Fractions of 300 ,uL were collected and assayed for PFP activity. Kinetic Analysis
The kinetic properties of the partially purified isoforms of PFP were determined at the pH optima (7.5) as previously described (8). The kinetic data were analyzed using nonlinear regression (8). The final specific activity of the peak 1 isoform was 2.4 ,umol mg-' protein min-' and that of the peak 2 isoform was 1.6 Mmol mg-' protein min-'.
SDS-PAGE and Protein Blotting
Polypeptides were resolved on 12% polyacrylamide gels with a 3% stacking gel (ratio of acrylamide to N'N'-methylene-bisacrylamide, 100:1) according to the method of Laemmli (22). The mol wt standards that were used included phosphorylase B (92,500), BSA (66,700), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,000), and lysozyme (14,400). The separated polypeptides were transferred to nitrocellulose for 1 h at 100 V in a BioRad minigel blot system. The transfer buffer contained 25 mM Tris-HCl (pH 8.3), 192 mM glycine, and 20% (v/v) methanol. Nitrocellulose filters were quenched in 1% milkpowder in Tris saline buffer (10 mm Tris-HCl, pH 8.0, containing 150 mM NaCl and 0.05% NaN3) at room temperature for 1 h. Incubation in primary antibody, washing, and probing with secondary antibody were done as previously described (7). Cross-reacting polypeptides were identified by the enzymatic cleavage of the phosphate group of 5-bromo-4-chloroindolyl-phosphate and using nitroblue tetrazolium as a stain enhancer (5). To quantify the amount of PFP on the filters, the nitrocellulose membranes were scanned after immunostaining using a laser densitometer (LKB 2202 Ultrascan) linked to an Apple IIE computer. The specificity and immunological cross-reactivity of the purified potato IgG with C. lanatus PFP has been described (7, 9). The serum is highly efficient to immunoinactivate the PFP from C. lanatus (9). The serum not only specifically cross-reacts with the a- and /3-subunits of potato PFP, but also hybridizes with the same two polypeptides from a variety of species. The only species in which an apparent third polypeptide is present is in some tissues of C. lanatus (7). Extraction of Proteins Using Trichloroacetic Acid
Different tissues (0.5 g) were collected and extracted using
a modified method of Wu and Wang (30). The plant tissue was homogenized with a glass tissue grinder in 1.6 mL 10%
(m/v) TCA. The resulting homogenate was incubated at 4°C for 30 min. Precipitated protein was collected by centrifuga-
MOLECULAR FORMS OF PYROPHOSPHATE DEPENDENT PHOSPHOFRUCTOKINASE
0
0"
1187
0.967) between the peak areas and the amount of PFP protein of any crude extract loaded on the gels, provided that no more than 80 ,g of total protein per tract was applied (not shown).
tI.N
kD 97.4
42.7 Figure 1. Protein blot analysis of PFP in crude extracts of C. lanatus cotyledons after different incubation periods at 270C in the dark.
tion at 1 2,000g for 10 min, then washed once with 5% (m/v) TCA and twice in prechilled (-20°C) 90% (v/v) acetone. The precipitates were dried and then solubilized in 62.5 mM TrisHCl (pH 6.8) containing 10% (v/v) glycerol, 2% (m/v) SDS, and 2% (v/v) mercaptoethanol with constant stirring. Insoluble materials in the samples were removed by centrifugation at 12,000g for 2 min. Prior to electrophoretic analysis, the samples were heated in boiling water for 5 min. Other
Protein concentrations were determined using the dyebinding assay technique (12). RESULTS
Protein blots of the crude extracts from the cotyledons probed with the anti-PFP IgG revealed two cross-reacting polypeptides of 65 ± 4 kD and 68 ± 3 kD at all stages of incubation (Fig. 1). The 68 kD polypeptide will be referred to as the a-subunit and the 65 kD polypeptide as the (-subunit. To estimate the relative abundance of the subunits of PFP, the protein blots were scanned with a laser densitometer. As previously described (7), there was a linear relationship (r =
Effect of Protease Inhibitors on PFP Activity and Subunit Content The inclusion of protease inhibitors resulted in an increase in the specific activity of PFP and a decrease in the ratio between the fl- and a-subunits (Table I). No significant differences in either the specific activity or subunit ratios were observed between extracts made in the presence of PMSF, amino-n-caproic acid, and benzamidine-HCl. Phenanthroline was the only inhibitor that had no effect. Furthermore, the ratio between the ,3- and a-subunits of extracts made in TCA did not differ significantly from extracts made in the presence of 2 mm PMSF, amino-n-caproic acid, and benzamidine-HCl. The higher (3-/a-subunit ratio in the presence of the protease inhibitors or TCA clearly indicates that the a-subunit especially is susceptible to protease cleavage. Total Activity and Isoforms in the Cotyledons The PFP activity in the cotyledons sharply increased after 12 h of incubation and reached a maximum of 48.6 nmol cot pair-' min-' at 84 h (Fig. 2). Thereafter, the activity decreased, and after 140 h attained an apparent constant level of 24 nmol cot pair-' min-'. The increase in PFP activity was also evident on a specific activity basis (Table II). The total soluble protein slightly increased over the first 60 h of incubation; thereafter, it decreased (not shown). The large increase in the specific activity of PFP when total protein marginally changes and the large decrease in specific activity that coincides with the total decrease in protein, therefore, most probably reflects different levels of PFP expression. These changes in PFP activity follow the same pattern precisely as that of the gluconeogenic flux and isocitrate lyase activity in the cotyledons (F. C. Botha, unpublished result).
60 -
100
50 C:
Table I. The Influence of Different Protease Inhibitors on the PFP Activity and the PFP Cross-Reactivity (Peak Area) after Blotting of Crude Protein from C. lanatus Cotyledons (120 h Incubation) Loaded on an SDS-PAGE Subunit Ratio Protease Inhibitor Activity
E 0
Q
0-
z
30 -
o C:
units mg-1 protein ± SD 5.37 ± 0.54
z 0
40 -
H---
0c
20 -
0
:/a
2.38 ± 0.50 None 7.14 ± 0.78 1.01 ± 0.22 1 mM PMSF 7.54 ± 0.52 0.92 ± 0.15 2 mm PMSF 7.75 ± 0.32 1.14 ± 0.08 2 mm amino-n-caproic acid 7.87 ± 0.33 1.11 ± 0.04 2 mm benzamidine HCI 2 mm 1:1 0-phenanthroline HCl 6.03 ± 0.65 2.09 ± 0.55 a 1.04 ± 0.28 TCA extraction a Due to denaturation, no enzyme activity could be detected.
>10
-
01 i 0
i
I
00
80 120 160 200 INCUBATION TIME (h) 40
Figure 2. PFP activity (0) in C. lanatus cotyledons during germination (U) and early seedling establishment at 270C in the dark.
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Table II. Changes in PFP Activity and Relative Subunit Concentration in C. lanatus Cotyledons during Incubation at 270C in the Dark Incubation
Time
Total Subunit Content a
Total Activity Peak2
Oa
~~~~~~~~~~~~~~~Ratio Peak 1
relativeb
h
0
0.33 ± 0.01
*d*
12
0.43 ± 0.03
0.09 ± 0.01
0.21 ± 0.02
Specific Activity Peak 1 Peak 2
unit cot pair-c ± SD
unit mg' protein ± SD
NDe
ND
ND
ND
24 0.66 ± 0.04 0.22 ± 0.03 0.33 ± 0.08 18.4 3.0 2.24 0.36 60 1.21 ± 0.03 0.86 ± 0.02 0.71 ± 0.15 36.6 12.4 2.38 0.96 84 1.35 ± 0.04 1.00 0.74 ± 0.19 27.6 8.0 3.08 2.54 200 0.49 ± 0.02 0.62 ± 0.01 1.27 ± 0.10 8.0 14.0 0.86 1.60 Ratio of total subunit content present in the tissue ± SD. For the subunit ratio of peak 1 and peak 2, see Table Ill. b Concentration of subunits expressed relative to the ,8-subunit content of cotyledons after 84 h of incubation ± SD. c One unit refers to 1 nmol Fru-1 ,6-P2 d No detectable ,8-subunit. produced min-1. e Not determined.
During chromatography of the total PFP activity from the cotyledons on an anion exchange column, more than one peak of PFP activity was detected (Fig. 3). At 24 h of incubation, 90% of the PFP activity was associated with a peak that eluted at 0.12 M KCl (peak 1) and 10% with a peak eluting at 0.18 M KCl (peak 2). The increase in total PFP activity between 24 and 60 h of incubation (Fig. 2) was due to an increase in both peak 1 and peak 2 activity. After 64 h, the activity associated with peak 1 decreased rapidly, so that after 200 h of incubation, peak 2 was the major form of PFP activity (Fig. 3). Although both peak 1 and peak 2 activity, on a protein and cotyledon basis, increased over the first 60 h of incubation, the increase in peak 2 activity is higher (Table II). The decrease in activity after 84 h was much more pronounced in peak 1 activity. Hence, the ratio between peak 2 and peak 1 increased throughout the incubation period. The total extractable PFP activity was not altered by the inclusion of 10 ,uM Fru-2,6-P2 to the extraction buffer. In addition, the ratio between the two PFP peaks was not altered by adding 10 ,uM Fru-2,6-P2 to all the buffers used during isolation and separation. In both treatments, the recovery of added PFP from the ion exchange column was higher than 93%.
Subunit Content
Both the a- and (3-subunit content increased over the first 84 h of incubation (Table II). The changes in PFP activity coincided with changes in PFP protein content. This is similar to our previous findings, namely that the increase in PFP activity during germination in C. lanatus is due to an increase in PFP concentration (7). The pattern of change in the content of the two subunits, however, differed (Table II). The ratio of (3- to a-subunit content linearly increased throughout the 200 h incubation period. Analysis of the subunit composition of the two PFP peaks that eluted from the anion exchange column indicated that the ratio between the (3- and a-subunit content of peak 1 was substantially lower than of peak 2 (Fig. 4 and Table III). The small amount of a-subunit present in peak 2 may represent contamination of peak 2 activity with peak 1 activity. The third type of polypeptide observed in the cotyledons after 24 h of incubation was also detected in very low quantities in some of the peak 1 and peak 2 preparations. This polypeptide might be a breakdown product of one or both of the subunits. However, the low concentration of this polypeptide in comparison to the total PFP concentration could prevent detec-
16 -
12-
Figure 3. Elution profile of the total PFP activity from C. lanatus cotyledons at different times during incubation at 270C in the dark. Proteins were separated on a Bio-Gel DEAE-5-PW column at pH 7.8.
0 0-
0.4
8-
F-
0.3
A:
0
>
0.2n
4-
CD 1--
0.1
0-
0.0
FRACTION #
MOLECULAR FORMS OF PYROPHOSPHATE DEPENDENT PHOSPHOFRUCTOKINASE 1.75-
0
e-
-Y
1189
r=0.983
1.50-
0
1.25-
(14 1.000
CD
o
0.
0.75
0
0.25-
0 U.UU
.
-
0.25
.
0.50
0.75
1.00
1.50
1.25
Ratio (a/a -subunit)
Figure 5. The relationship between the isoform of PFP in C. lanatus cotyledons and the relative subunit concentration. 0.40
0.43 0.46
0.40
0.43 0.46
Rf Figure 4. Densitograph of the protein blot analysis of the PFP associated with peak 1 (A) and peak 2 (B). Cross-reacting polypeptides were scanned with a laser densitometer. Lanes were loaded with an equal amount of PFP activity. The nitrocellulose filter was probed with a 1:750 dilution of purified IgG against potato PFP.
tion in total crude extracts. There is a linear relationship (r= 0.982) between the ratio of PFP activity associated with peak 2 and peak I and the ratio of to a-subunit (Fig. 5). An increase in ,8-subunit relative to the az-subunit, therefore, appears to favor peak 2 activity. -
Preliminary Kinetics of Isoforms The specific activity of the partially purified peak 1 isoform was 2.6 qmol mg-' prot min-' and for peak 2 was 1.8 umol mg-' prot min-'. This represents a purification of at least 500fold for both isoforms. Both forms of PFP were activated by Fru-2,6-P2 (not shown). The affinity for Fru-6-P of peak 1 and peak 2 was similar (Table V). Peak 1 activity, which is the dominant form in the cotyledons during seedling establishment, has a ninefold higher affinity for PPi than the peak 2 isoform. The possible presence of inorganic pyrophosphatase activity in the PFP preparations was determined by adding 20 nmol PPi to the PFP reaction mixture and allowing the reaction to go to completion. The obtained stoichiometry
Tissue Specific PFP Activity and Subunit Concentration The highest specific activities of PFP in C. lanatus were present in the youngest leaf, root, stems, developing fruit, and reproductive organs, while the lowest specific activity was present in the mature leaves (Table IV). Protein blot analysis of different tissues of C. lanatus clearly indicated that the typical PFP doublet was present in significant quantities in most of the tissues, while the a-subunit was absent in developing fruit, stamens, pistils, and oldest leaves (Fig. 6). No significant differences in the to a-subunit ratio between extracts made with 10% TCA or buffer containing 2 mM PMSF were found.
Table IV. The PFP Activity and PFP Subunit Composition in Different Tissues from C. lanatus Subunit ratios were determined from 10% TCA extracts (see text). Tissue Ratio
units8 mg-1 protein + SD
Developing seed Cotyledonb Cotyledonc First leaf Second leaf Third leaf Mature fruit' Developing fruit0 Rootsc Stemc Petalse Stamene Carpelse Whole flower
1-
Table Ill. The Subunit Composition of the Two Peaks of PFP Activity from the Ion Exchange Column and Total PFP Activity From Cotyledons after 84 h of Incubation The ratio between the PFP activity of peak 1 and peak 2 was 1.53. Fraction
Ratio
Total Peak 1 Peak 2
0.74 ± 0.36 0.34± 0.14 1.59 ± 0.58
1/la
SD
Activity
2.6 ± 0.1 5.6 ± 0.2 6.0 ± 0.4 1.1 ± 0.1 9.2 ± 0.5 31.8 ± 4.3 9.6 ± 0.7 56.8 ± 6.1 48.8 ± 2.9 29.0 ± 3.6 7.7 ± 2.2 42.6 ± 5.4 23.8 ± 3.8 ND'
Subunit SD
13/a
0.67 + 0.17 0.64 + 0.28 *d *
15.86 *0.08 0.74 0.02 7.14 0.56 *
0.24 0.10 0.58 ± 0.18 1.03 ± 0.12 * *
0.45
0.08 b One unit refers to 1 nmol Fru-1,6-P2 produced min-'. From seeds after 84 h of incubation at 270C in the dark. c From a young seedling with three true new leaves and the green cotyledons. The third leaf refers to the youngest leaf. d No detectable asubunit. e From a mature plant growing in a controlled environment. 'Not determined. *
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BOTHA AND BOTHA
of 2:1 between NADH oxidized and added PPi indicates that under the experimental conditions used, no significant pyrophosphatase activity was present.
DISCUSSION It is evident that different isoforms of PFP are present in the cotyledons of C. lanatus. As with the PFP isoforms from tomato fruit (29), these isoforms differ in charge and, therefore, can be separated by ion exchange chromatography. The presence of PFP isoforms was also previously reported in wheat tissue (33). The PFP protein of C. lanatus contains the typical a- and fl-subunits associated with plant PFP (7, 20, 33). The PFP activity associated with peak 1 has a much higher a-subunit content than that associated with peak 2. In contrast, peak 2 is enriched for the fl-subunit. One of the PFP isoenzymes from wheat (33) and tomato fruit (29) and the PFP from carrot tap root (28) and mung bean (4) apparently only contain the fl-type subunit. It is not possible to state precisely the subunit concentration in C. lanatus cotyledons. The experimental protocol used only indicates the relative content of immunological determinants. As the number of epitopes on each of the subunits is not known, we can state only that the relative content during development, as well as the ratio between the two subunits, in the two isoforms differs substantially. The specific activity of the two partially purified PFP preparations and their kinetic properties are similar to those reported previously for other PFP preparations (8, 11, 19, 23, 33). The two PFP isoenzymes from C. lanatus differ in their affinity for PPi. Similarly to wheat, both PFP isoforms of C. lanatus are stimulated by Fru-2,6-P2. All these results indicate that a binding site for Fru-2,6-P2 is probably present on the fl-subunit in these three species. In wheat tissue, it was found that the isoform containing only the f-subunit had a much lower affinity for Fru-2,6-P2. This led the authors to propose that the a-subunit was most probably a regulatory subunit involved in Fru-2,6-P2 binding (33). This proposal was recently confirmed (15).
The mechanism involved in controlling the isoform in which PFP will be present is not known. Based on previous reports, an association/dissociation of the protein, controlled by certain metabolites, may be involved. The PFP from peas (31) and potato (20) can aggregate in the presence of Fru-2,6P2 and dissociate in the presence of PPi. However, the physiological significance of this phenomenon in vivo is not clear because unphysiologically high metabolite levels and/or physical conditions were needed to facilitate the aggregation and dissociation. We also observed limited aggregation and dissociation of C. lanatus PFP in the presence of 50 mM PPi and 1 mM Fru-2,6-P2. At physiological concentrations of these metabolites, however, no change in mol wt of PFP was observed (F. C. Botha, unpublished result). In addition, we could find no indication that physiological concentrations of Fru-2,6-P2 had any effect on the ratio between the two forms of PFP observed in this study. No indication of aggregation or dissociation of PFP was found either in PFP preparations from soybean (23) or beans (11). If association/dissociation is the only factor controlling the form in which PFP will be present, the total PFP-subunit concentration should be constant. In C. lanatus, this clearly is not the case. The protein blots show that subunit availability might be the important factor determining the isoform in which PFP is present in C. lanatus. Although the cotyledons contain both a- and f-subunits associated with higher plant PFP, the ratio between the subunits is not constant and varies depending on the developmental stage. In some C. lanatus tissues, apparently only the f-subunit is present, which is similar to carrot tap root (28). As direct extraction of these tissue into TCA also failed to unveil any a-subunit, it must be concluded that our results are not due to rapid proteolytic cleavage of the a-subunit. The large variation in the to subunit ratio in the different tissues clearly indicates that there is not a coordinated expression of the two PFP subunits. Coarse control through differential expression of the subunits, therefore, might be the major factor regulating the prevailing level and form of PFP in C. lanatus and other species. A possibility that cannot be ruled out at present is that the apparent fl-subunit in certain tissues might be heterogeneous and contains both a- and fl-subunits, as was recently shown in mung bean (15). However, as some tissues do contain an a-subunit with a higher mol wt than the fl-subunit, this will mean that at least two copies of the PFPa gene must be f-
a-
Table V. Kinetic Properties of the Two Isoforms of PFP from C.
lanatus Cotyledons at pH 7.5 and in the Presence of 10 AM Fru-2,6-P2 The specific activity of the peak 1 PFP was 2.6 Amol mg-' prot min- and that of the peak 2 activity was 1.8 Mmol mg-' prot min-'. Enzyme was purified from cotyledons after 84 h of incubation. Km Substrate
Peak 2
Peak 1 iuM ± SD
Fru-6-P PPi
285 ± 12 18 ± 4
292 ± 18 161 ± 17
MOLECULAR FORMS OF PYROPHOSPHATE DEPENDENT PHOSPHOFRUCTOKINASE present, and that they are differentially regulated or that the
primary transcript is differently processed in different tissues. This aspect will have to be investigated further. In addition to the major changes in total PFP activity following radicle protrusion (Fig. 1), the ratio between the PFP isoforms also changes. These changes coincide with dramatic alterations in the metabolism from a primarily glycolytic function prior to radicle protrusion, to a gluconeogenic function during early seedling growth (10, 27), and finally to a photosynthetic function. The highest specific PFP activity in the cotyledons occurs at the peak in gluconeogenesis, which is similar to castor bean endosperm PFP (21). Although the function of PFP in the cotyledons is not known, it is most likely responsible for removing the PPi that will be produced during sucrose synthesis. It is interesting, therefore, that the dominant form of PFP in the cotyledon at this stage is peak 1, the form with a high affinity for PPi. The PFP activity of C. lanatus also varies in the different tissues of the plant. In general, it appears that the highest specific activities are associated with sink tissues such as the reproductive organs, stem, and roots. This distribution pattern is in agreement with the concept of PFP being associated with sink tissue and being an indication of sink strength (6, 32). In all these tissues of C. lanatus, carbohydrate (sucrose and/or stachyose) breakdown will be a major metabolic activity. The function of PFP in these tissues could be the provision of PPi to sustain sucrose breakdown through sucrose synthase (1, 2, 18, 26). In most of the strongly defined sink tissues, only the d-subunit is expressed at significant levels. This subunit composition will most probably result in a predominantly peak 2type PFP with a low affinity for PPi. This aspect is currently under investigation. CONCLUSION
Similarly to the PFP from wheat (33), tomato (29), and carrot (28), the PFP of C. lanatus is present in isoforms that differ in molecular form and kinetic properties. The concentration of the PFP subunits varies independently in the different tissues of the plant and in the same tissue at different stages of development. The availability of each subunit appears to be the most important factor controlling the form in which PFP will be present in C. lanatus. LITERATURE CITED
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