Planta 9 Springer-Verlag 1989
Planta (1989) 177:261~64
Phosphoribosyl pyrophosphate and the measurement of inorganic pyrophosphate in plant tissues Jane E. Dancer* and Tom ap Rees Botany School, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK
Abstract. This work provides further evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi), and that breakdown of phosphoribosyl pyrophosphate (PPRibP) does not contribute significantly to the PPi detected in plant extracts. Inorganic pyrophosphate in extracts of the roots of Pisum sativum L., clubs of the spadices of Arum maculatum L., and the developing endosperm of Zea mays L. was assayed with pyrophosphate fructose 6-phosphate l-phosphotransferase (EC 220.127.116.11), and with sulphate adenyltransferase (EC 18.104.22.168). The two different assays gave the same value for PPi content, and for recovery of added PPi. It was shown that P P R i b P is converted to PPi during the extraction of PPi. However, the amounts of P P R i b P in clubs of A. maculatum and the developing endosperm of Z. mays were negligible in comparison with the contents of PPi. Key words: Arum Inorganic pyrophosphate (content) - Pisum (pyrophosphate assay) - Phosphoribosyl pyrophosphate - Pyrophosphate measurement - Sulphate adenyltransferase - Zea (pyrophosphate assay)
The evidence that plants contain appreciable amounts of inorganic pyrophosphate (PPi) is a sur* Present address." Botanisches Institut der Universit~it Bayreuth, Lehrstuht Pflanzenphysiologie, Postfach 101251, D-8580 Bayreuth, FRG
EDTA = ethylenediaminetetraacetic acid; PFK(PPi)=pyrophosphate fructose 6-phosphate 1-phosphotransferase; PPi=inorganic pyrophosphate; P P R i b P = p h o s phoribosyl pyrophosphate Abbreviations:
prising development in our understanding of plant metabolism (Edwards et al. 1984; Smyth and Black 1984; ap Rees etal. 1985; Chanson et al. 1985; Huber and Akazawa 1986; Weiner et al. 1987). There are two weaknesses in this evidence. First, all of it rests on the specificity and purity of two enzyme preparations. Pyrophosphate fructose 6phosphate 1-phosphotransferase [EC 22.214.171.124; PFK(PPi)] has been used to assay PPi in plant extracts on the assumption that no substance other than PPi can act as a phosphoryl donor for the formation of fructose-l,6-bisphosphate by this enzyme. Careful studies of the isolated enzyme support this view (Kombrink et al. 1984; Yan and Tao 1984) but do not eliminate the possibility that some untested component of plant extracts can also act as a phosphoryl donor. Commercial preparations of inorganic pyrophosphatase have been used to confirm that PPi is the component of plant extracts that reacts with PFK(PPi). The purity and specificity of these preparations have not been examined rigorously. The second criticism made of the evidence that plants contain PPi is that the procedures used to extract PPi from plants are likely to cause breakdown of phosphoribosyl pyrophosphate (PPRibP) to PPi. Thus PPi measured in extracts could be an artefact resulting from the destruction of P P R i b P (Van Schaftingen 1987). We have investigated these criticisms by using an alternative assay for PPi and by estimating the extent to which breakdown of P P R i b P contributes to PPi in plant extracts. The alternative assay shares no step with the PFK(PPi) assay and depends upon the specificity of sulphate adenyltransferase (EC 126.96.36.199) for PPi (Drake et al. 1979). The assay involves: Adenyl sulphate + PPi a , ATP + sulphate
J.E. Dancer and T. ap Rees: Measurement of inorganic pyrophosphate
ATP + glucose b , glucose 6-phosphate + A D P Glucose 6-phosphate + N A D P c , N A D P H + 6-phosphogluconate,
where a, b and c, are, respectively, sulphate adenyltransferase, hexokinase (EC 188.8.131.52) and glucose-6phosphate dehydrogenase (EC 184.108.40.206). Materials and methods Materials. Enzymes, co-enzymes and ATP were from Boehringer, Lewes, Sussex, UK, except that PFK(PPi), sulphate adenyltransferase, orotate phosphoribosyltransferase (EC 220.127.116.11) and substrates were from Sigma (London) Chemical Co., Poole, Dorset, UK. Maize (Zea mays L. cv. Golden Bantam) was grown in John Innes No. 3 Compost, one plant per 15-cmdiameter pot, in a greenhouse at 15-25 ~ C in light with a photon fluence rate of 350-500 photons-m -2.s 1 of photosynthetically active radiation and a 16-h photoperiod. Cobs were harvested 22 d after pollination and the endosperm was removed by dissection, immediately frozen in liquid nitrogen and stored in the latter for up to eight weeks before analysis. Peas (Pisum sativum L. cv. Kelvedon Wonder) were grown as described by Smith and ap Rees (1979), except that the seedlings were in water not CaC12. Complete inflorescences of Arum maculatum L. were collected from local natural sites. Within an hour of this collection the clubs, the swollen end of the appendix, were excised and used at once. The stages of development have been defined (ap Rees et al. 1976).
Assay of PPi. Samples of single or half (cut lengthwise) clubs of Arum, and of 20 2-cm apices of roots of 5-d-old pea seedlings were freeze-clamped and ground to a powder in liquid nitrogen with a pestle and mortar. Samples (0.2~?.8 g fresh weight) of the frozen maize endosperm were ground in liquid nitrogen with a pestle and mortar. Then each powdered sample was transferred with liquid nitrogen to a 50-ml centrifuge tube and almost all the nitrogen was allowed to evaporate before 1 ml 3.1 M HC104 was added to give a suspension that was kept in ice for 60 min and then centrifuged at 25000.g for 2 min at 2 ~ C. The pellet was rinsed with two 0.25-ml portions of ice-cold water: the washings and the supernatant were combined and neutralized with 5 M K2CO3. The precipitate of KC104 was removed by centrifugation and washed with two 0.25-ml portions of water: the supernatant and washings were combined and assayed for PPi. Assay with PFK(PPi) was as described by Edwards et al. (1984). We checked routinely that N A D production in this assay was abolished by treating the extract with 0.1 unit inorganic pyrophosphatase (EC 18.104.22.168) but not affected by adding 0.1 unit adenosine 5'-triphosphatase (EC 22.214.171.124). Assay of PPi with sulphate adenyltransferase was as described by Drake et al. (1979), in a reaction mixture, 1 ml, that contained: 40 mM 2-(N-morpholino)ethanesulphonic acid (Mes), pH6.5; 200mM glucose; 1 0 m M MgClz; 0.66mM N A D P ; 0.2 mM adenyl sulphate; 1 unit glucose-6-phosphate dehydrogenase from yeast; 1 unit hexokinase. The reaction was started by the addition of 1 unit sulphate adenyltransferase.
Assay of PPRibP. Tissue was freeze-clamped and treated as for the assay of PPi up to the point when the liquid nitrogen had almost evaporated. Then 2 ml of boiling 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid (Hepes), pH 7.5;
10 mM ethylenediaminetetraacetic acid (EDTA) was added to the sample, which was placed in a boiling-water bath for exactly 2 min and then centrifuged at 0~ C at 25000.g for 2 rain. The supernatant was assayed at once according to Kornberg et al. (1954), in a reaction mixture that contained in 1 mh 50 mM 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris), pH 8.0; 0.2 mM orotic acid; 50 mM MgC12. The reaction was started by adding 1 unit orotate phosphoribosyltransferase (EC 126.96.36.199) and the disappearance of orotate was followed spectrophotometrically at 295 nm.
Recovery experiments. In each experiment we prepared duplicate samples of the same batch of endosperm or pea roots. For Arum we cut a single club lengthwise into halves to give duplicate samples. Each sample was carefully weighed, then one of the duplicates was freeze-clamped, killed and extracted as described above. The other of the duplicates was treated similarly, except that a measured amount of PPi or PPRibP was added once the sample had been frozen. The amounts added varied according to the tissue and the size of the sample, and were adjusted to ensure that they were equal to the amounts of PPi found in the untreated sample of tissue. The amounts were: PPi, 20 nmol and 10-15 nmol, respectively, for pea roots and endosperm; PPRibP, 58 nmol and 40 80 nmol, respectively, for endosperm and Arum club.
Results and discussion
Measurement of PPi. Solutions containing 8, 16, 24, and 32 nmol PPi were assayed by the sulphateadenyltransferase method. One nmol of N A D P was reduced per nmol PPi, and there was a linear relationship between PPi assayed and N A D P reduced throughout the range of concentrations of PPi. The suitability of the assay for use with plant extracts was checked by measuring both content and recovery of PPi in six separate clubs of a-stage spadices of A. maculatum. Values (means __ SE) of 23.5 _+2 nmol. (g fresh weight)- 1 and 92 _+3% were obtained. These are very similar to the previously published estimates obtained with the PFK(PPi) assay, 21.1 _+ 1.8 nmol. (g fresh weight)- 1 and 90% (ap Rees et al. 1985). We made further comparisons between the sulphate-adenyltransferase and PFK(PPi) assays by measuring PPi content and recovery in three plant tissues (Table 1). For each tissue the sulphate-adenyltransferase assay gave the same values for both content and recovery as did the PFK(PPi) assay. These recoveries are satisfactory. Treatment of the extracts referred to in Table 1 with alkaline pyrophosphatase abolished their ability to reduce N A D P in the sulphate-adenyltransferase assay. The complete similarity between the results obtained with the two entirely different assays greatly reduces the likelihood that the positive results obtained with the PFK(PPi) assay were caused by the enzyme using some component of the extracts other than PPi as a phosphoryl donor. We argue that the evidence that plant
J.E. Dancer and T. ap Rees: Measurement of inorganic pyrophosphate Table 1. Content and recovery of PPi in plant tissues as assayed with PFK(PPi) and with sulphate adenyltransferase. Each comparison represents a single experiment with duplicate samples, to one of which PPi was added to measure recovery. Extracts of both samples were subjected to each assay Tissue
Developing endosperm of maize
Pea root apices
t-Stage club of Arum maculatum
PPi content (nmoI-(g F W ) - 1 )
Recovery of added PPi (%)
33 59 32 31
27 54 31 29
115 71 114 99
101 71 130 98
35 34 34
36 38 31
100 100 90
106 103 110
32 57 49
36 56 55
~) l S ii
PPRibP A S S A Y E D [nmol]
Fig. 1. Relationship between amount of PPRibP assayed and change in absorbance at 295 nm
extracts contain significant amounts of PPi is acceptable.
Measurement of PPRibP. We subjected solutions that contained 17 nmol PPRibP to the procedure used to kill and extract samples of tissue for analysis of PPi. In two experiments we found that 8.1 and 7.8 nmol PPi were formed from the PPRibP. When 17 nmol PPRibP were added to halves of v-stage clubs of Arum maculatum, the amounts of PPi detected in the extracts were 17 nmol greater than were found for the untreated halves of the clubs. We conclude that the techniques that we have used to measure PPi in plant tissues lead to substantial, if not complete, breakdown of PPRibP to PPi. Much of this breakdown is chemical but, because it was greater in the presence of tissue, a good deal appears to be enzymic. The extent to which measurements of PPi are affected by the breakdown of PPRibP depends upon how much PPRibP is present in plant tissues. We investigated this by assaying PPRibP by measuring the disappearance of orotate in the presence of orotate phosphoribosyltransferase, which catalyses : PPRibP+orotic phate
Disappearance of orotic acid was measured by the change in absorbance at 295 rim. Figure 1 shows that there was a linear relationship between this change and the amount of PPRibP assayed.
Extraction of PPRibP from plants requires a compromise between the need to inactivate enzymes in the homogenate and the need to avoid harsh treatments that destroy PPRibP. We followed Henderson and K h o o (1965) and used boiling to inactivate enzymes. We found that 50 mM Hepes (pH 7.4), 10 m M EDTA was an effective extraction medium. We optimized the time for which the sample was boiled in this medium by measuring the recovery of 58 nmol PPRibP from samples (0.7 g fresh weight) of the developing endosperm of maize. Optimum recovery, 80-90%, was found after boiling for 120 s. This time is crucial. Boiling for less than 120 s gave very low recoveries of the added PPRibP, presumably because degradative enzymes in the extract were not inactivated quickly enough. Boiling for longer than 120 s also reduced the recovery, probably because the longer exposure to high temperature caused chemical breakdown of the PPRibP. We used the optimized assay to measure the content and recovery of PPRibP for samples of 7-stage clubs of A. maculatum and developing endosperm of maize. In none of the six clubs or three different samples of endosperm did we detect any PPRibP (Table 2). The recoveries for the two tissues, 63 and 94% for clubs and endosperm, respectively, were adequate. The amounts of PPRibP added in the recovery experiments were comparable to the amounts of PPi detected in the extracts. The lowest limit of measurement of PPRibP in our assays corresponds to a value of 3 nmol.(g
J.E. Dancer and T. ap Rees: Measurement of inorganic pyrophosphate
Table 2. Estimation and recovery of P P R i b P in developing endosperm of maize and clubs of y-stage spadices o f A r u m maculaturn. For each experiment duplicate samples of tissue were prepared: P P R i b P in 0.1 M EDTA was added to one of each duplicate (58 and 4C~80 nmol for endosperm and club, respectively) and 0.1 M EDTA was added to the other duplicate. Data are presented for each pair of samples. N D = n o t detectable Tissue
P P R i b P ( n m o l . ( g FW) -1)
in extracts made
Recovery ofadded PPRibP
Without added P P R i b P
With added P P R i b P
Endosperm of maize
ND ND ND
83 105 102
87 96 99
ND ND ND ND ND ND
22 19 22 15 55 53
61 59 59 47 81 73
fresh weight)-1 for both Arum clubs and maize endosperm. As relatively little P P R i b P was lost in our recovery experiments we suggest that the content of P P R i b P in Arum clubs and maize endosperm is small when compared with the estimates of PPi. We argue that, although the procedures used to assay PPi will cause P P R i b P to break down to PPi, the content of P P R i b P is likely to be so low that the effect on PPi measurements is not significant. Certainly this appears to be so for the two tissues we have examined. Indirect support for our view that the P P R i b P content of plant tissues is low is provided by low values for estimates of the Km for P P R i b P reported for orotate phosphoribosyl transferase, 1.6 gm (Ashihara 1978), and uracil phosphoribosyl transferase, 11 gm (Bressan et al. 1978). The published values for P P R i b P content of plant tissues appear to be restricted to 2.5 and 46 nmol.(g fresh weight) -1, respectively, for cells of Catharanthus roseus (Hirose and Ashihara 1983) and pea cotyledons (Ross and Murray 1971). Neither estimate is accompanied by recovery data and in both analyses the metabolism of the tissue samples was not quenched instantly prior to extraction. The estimate of Catharanthus cells is consistent with our argument. The estimate for pea cotyledons indicates that this tissue has a much higher content of P P R i b P than the tissues that we analysed. P r o o f that this is so must await a clear demonstration that the assay and procedure used in the studies of pea cotyledons did measure the amounts of P P R i b P present in the intact cotyledon.
J.E.D thanks the Science and Engineering Research Council for a research studentship.
References ap Rees, T., Fuller, W.A., Wright, B.W. (1976) Pathways of carbohydrate oxidation during thermogenesis by the spadix of Arum maeulatum. Biochim. Biophys. Acta 437, 22-35 ap Rees, T., Green, J.H., Wilson, P.M. (1985) Pyrophosphate: fructose 6-phosphate 1-phosphotransferase and glycolysis in non-photosynthetic tissues of higher plants. Biochem. J. 227, 299 304 Ashihara, H. (1978) Orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase of black gram (Phaseolus mungo) seedlings. Z. Pflanzenphysiol. 87, 225 241 Bressan, R.A., Murray, M.G., Gale, J.M., Ross, C.W. (1978) Properties of pea seedling uracil phosphoribosyltransferase and its distribution in other plants. Plant Physiol. 61,442446 Chanson, A., Fichmann, J., Spear, D., Taiz, L. (1985) Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles. Plant Physiol. 79, 159-164 Drake, H.L., Gross, N.H., Wood, H.G. (1979) A new, convenient method for the rapid analysis of inorganic pyrophosphate. Anal. Biochem. 94, 117 120 Edwards, J., ap Rees, T., Wilson, P.M., Morrell, S. (1984) Measurement of inorganic pyrophosphate in tissues of Pisum sativum L. Planta 162, 188 191 Henderson, J.F., Khoo, M.-K.Y. (1965) Synthesis of 5-phosphoribosyl-l-pyrophosphate from glucose in Ehrlich Ascites tumor cells in vitro. J. Biol. Chem. 240, 2349-2357 Hirose, F., Ashihara, H. (1983) Content and availability of 5-phosphoribosyl-l-pyrophosphate in cultured ceils of Catharanthus roseus. Z. Pflanzenphysiol. 110, 183-190 Huber, S.C., Akazawa, T. (1986) A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiol. 81, 1006-1013 Kombrink, E., Kruger, N.J., Beevers, H. (1984) Kinetic properties of pyrophosphate: fructose-6-phosphate phosphotransferase from germinating castor bean endosperm. Plant Physiol. 74, 395401 Kornberg, A., Lieberman, I., Simms, E.S. (1954) Enzymatic synthesis and properties of 5-phosphoribosylpyrophosphate. J. Biol. Chem. 215, 389-402 Ross, C., Murray, M.G. (1971) Development of pyrimidinemetabolizing enzymes in cotyledons of germinating peas. Plant Physiol. 48, 62~%630 Smith, A.M., ap Rees, T. (1979) Effects of anaerobiosis on carbohydrate oxidation by roots of Pisum sativum. Phytochemistry 18, 1453-1458 Smyth, D.A., Black, C.C. (1984) Measurement of the pyrophosphate content of plant tissues. Plant Physiol. 75, 86~864 Van Schaftingen, E. (1987) Fructose 2,6-bisphosphate. Adv. Enzymol. 59, 315 395 Weiner, H., Stitt, M., Heldt, H.W. (1987) Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves. Biochim. Biophys. Acta 893, 13-21 Yan, T.-F.J., Tao, M. (1984) Multiple forms of pyrophosphate: D-fructose-6-phosphate l-phosphotransferase from wheat seedlings. J. Biol. Chem. 259, 5087-5092
Received 14 July; accepted 2 September 1988