for Plant Science Research, Colney Lane, Norwich NR4 7UH, Great Britain (B.K.D., ... School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, ...
Received for publication July 18, 1990 Accepted October 3, 1990
Plant Physiol. (1991) 95, 412-419 0032-0889/91/95/041 2/08/$01 .00/0
Metabolism of Inositol(1,4,5)trisphosphate by a Soluble Enzyme Fraction from Pea (Pisum sativum) Roots B. K. Dr0bak*, P. A. C. Watkins, J. A. Chattaway, K. Roberts, and A. P. Dawson Department of Cell Biology, John Innes Institute, A.F.R.C. Institute of Plant Science Research, John Innes Centre for Plant Science Research, Colney Lane, Norwich NR4 7UH, Great Britain (B.K.D., P.A.C.W., J.A.C., K.R.); and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, Great Britain (J.A.C., A.P.D) via ROCs' or, alternatively, Ca2+ is released into the cytosol from one or more intracellular stores. The latter hypothesis has gained in popularity since the finding that D-myo-inositol(1,4,5)trisphosphate is able to release Ca2+ from intracellular stores (presumably the vacuole or a compartment associated with the vacuole) in plant cells (8, 25). A key function for Ins(1,4,5)P3 in mediating responses to external signals by modulating intracellular Ca2+ levels/fluxes is now established in many eukaryotic cells (e.g. mammalian cells, yeast, and slime moulds). In these cells Ins(1,4,5)P3 is produced by receptor activated phosphoinositidase C-mediated cleavage of phosphatidylinositol(4,5)bisphosphate (Ptdlns(4,5)P2). The hydrolysis of Ptdlns(4,5)P2 leads to a concomitant production of 1,2-diacylglycerol (DG) which activates protein kinase C (1). Several pieces of evidence for the structural presence of a similar signal transducing system (the phosphoinositide-or PI-system) in plant cells have emerged. These include the presence of: (a) phosphatidylinositol(4)phosphate and phosphatidylinositol (4,5)bisphosphate (2, 9, 13, 15); (b) kinases and phosphatases involved in polyphosphoinositide metabolism (9, 24); and (c) enzymes which in many respects resemble mammalian phosphoinositidase(s) C (19, 28) and protein kinase(s) C (10). Possible functional roles for the PI-system in processes such as light-induced, motor-cell activation in Samanea pulvini (21, 22) and responses to auxin and gibberellin (1 1, 23) have recently been reported. However, the crucial event in the conversion of extracellular signals into cytosolic [Ca2+] elevation, i.e. the agonist-provoked production of Ins(l,4,5)P3, has still not been adequately demonstrated in any plant system. Three main obstacles complicate the pursuance of this line of investigation. (a) Nothing is presently known either about the timing or extent of the putative Ins(1,4,5)P3 production. (b) Several InsP3 isomers other than Ins( 1 ,4,5)P3 may exist in 'unstimulated' plant cells which complicates the assay of the 1,4,5-isomer by conventional methods. (c) Perhaps of greatest importance, hardly anything is known about the potential enzyme system(s) involved in metabolism of Ins(1,4,5)P3 in plant systems. Due to the rapid metabolism of Ins( 1 ,4,5)P3 in mammalian cells, Ins( 1 ,4)P2, Ins( 1 ,3,4)P3, and
ABSTRACT
Metabolism of the putative
messenger
molecule D-myo-inosi-
tol(1,4,5)trisphosphate [Ins(1,4,5)P3] in plant cells has been studied using
a
soluble fraction from
enzyme source and
pea
(Pisum sativum) roots
as
[5-32P]lns(1,4,5)P3 and [2-3H]lns(1,4,5)P3 as
tracers. lns(1,4,5)P3 was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P2J whereas inositol(1,4)bisphosphate [Ins(1,4)P2] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites
produced including inositol and inositol(1,4,5,X)P4. Dephosphorylation of lns(1,4,5)P3 to lns(4,5)P2 was dependent on lns(1,4,5)P3concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of lns(1,4,5)P3to lns(4,5)P2 and lns(1,4,5,X)P4was inhibited by 55 micromolar Ca2 This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting lns(1,4,5)P3and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.
were
.
The Ca2" ion acts as an important messenger linking extracellular signals to intracellular responses in mammalian and many other eukaryotic cells. The Ca2"-activity in the cytoplasm of unstimulated mammalian cells is typically 50 to 100 nm and recent studies using Ca2"-indicator dyes and microelectrodes have shown that this is likely also to be the case in plant cells (4, 12). The concept of Ca2' being a messenger is based on the finding that in many cell types cytosolic Ca2+ activities are rapidly elevated when agonists interact with plasma membrane associated receptors (6). This rise in cytosolic Ca2' activity leads to activation of a multitude of Ca2+/ Ca2+-calmodulin-dependent intracellular response elements. A similar chain of events has for some time been suggested to occur in plant cells. It is, however, only recently that a direct link between arrival of extracellular agonists and a rise in cytosolic Ca2' activities has been demonstrated (18). The nature of the agonist-sensitive releasable Ca2+ pool(s) still remains an open question. Two basic possibilities exist: either Ca2' enters the cytosol from the extracellular medium
Abbreviations: ROC, receptor-operated channels; Ins, inositol; Pi, orthophosphate; Ins( l)P, o-myo-inositol-l-monophosphate, other myo-inositolpolyphosphates are given in the D-notation unless otherwise stated; p-NPP, p-nitrophenylphosphate; G-6-P, glucose 6-phosphate; 2,3-DPG, 2,3-diphosphoglycerate. 412
METABOLISM OF INOSITOL(1,4,5)TRISPHOSPHATE IN PLANT EXTRACTS
Ins(1,3,4,5)P4 are frequently found to be the prevalent inositol-phosphate isomers shortly after agonist stimulation (27). As such, these isomers leave a 'foot-print' in the cellular inositol phosphate pool bearing witness of the Ins(1,4,5)P3 transient. Some work has already been carried out to investigate the metabolic pathways of inositol phosphate degradation in plant cells and tissues (16, 17, 20). To shed some further light on the possible fate of Ins(1,4,5)P3 during or after its presumed cytoplasmic migration in plant cells, we have investigated the metabolism of 32P/3H-Ins(1,4,5)P3 using a soluble fraction from pea roots as enzyme source. Our data demonstrate that the metabolism of Ins(1,4,5)P3 is complex and may differ significantly from that of other eukaryotes.
MATERIALS AND METHODS
Materials D-myo-[5-32P]inositol(1,4,5)P3,(specific activity 4.5 TBq/ mmol) was from New England Nuclear and D-myo-[2-3H] inositol(1,4,5)P3 (specific activity 1.67 TBq/mmol) was from Amersham International. D-myo-Ins( 1 ,4,5)P3 and [2-3H] Ins(1,3,4,5)P4 was a generous gift from Dr. R. F. Irvine, Babraham, U.K. Radiolabeled standards were produced as follows: Ins(4,5)P2, incubation of 32P/3H-Ins(1,4,5)P3 with alkaline phosphatase; Ins( 1 ,4)P2, incubation of 32P/3HIns( 1 ,4,5)P3 with inositol( 1 ,4,5)P3-5-phosphohydrolase from human erythrocyte ghosts (7). For use as internal standards InsP2s were purified by HPLC and desalted on Amprep (Amersham) columns (triethylamine-bicarbonate elution/ freeze-drying). 2,3-Diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate were obtained from Sigma and were analytical grade. [2-3H]Ins, [2-3H]Ins(1)P, and [2-3H] Ins(4)P were obtained from New England Nuclear. Preparation of Soluble Enzyme Extracts Pea seeds (Pisum sativum cv Kelvedon wonder) were imbibed in running water overnight and germinated in darkness (25°C) on moist vermiculite. When the primary root had reached a length of approximately 2 cm (72-84 h germination) 150 to 200 segments (1.5 cm) were cut from the apical end of the roots and transferred to 5 mL of ice-cold extraction buffer (50 mM K-Hepes, 1 mM MgCl2, 2 mm EGTA, 30 mM mercaptoethanol [pH 7.2]). Root segments were homogenized in a mortar and pestle in the presence of acid-washed sand. The homogenate was strained through cheesecloth and centrifuged at 2,000g for 10 min to remove sand and debris. The supernatant was recovered and centrifuged at 100,000g for 1 h. The 100,000g supernatant was recovered and kept on ice until start of incubation. All steps in the extraction procedure were carried out at 4°C. Incubation Procedure Nine hundred microliter aliquots of the soluble enzyme extracts were transferred to microfuge tubes which were placed in a 25°C waterbath for temperature equilibration. The incubation was started by addition of the following components: ATP (final concentration 5.0 mM), Ins(1,4,5)P3 (final concentration 5 AM) spiked with 32P-and/or 3H-Ins(1,4,5)P3
413
(final specific activity approximately 20 Bq/nmol), and CaCl2 (1.7 mm, final free Ca2+ concentration was 55 ,uM, determined by a Ca-sensitive electrode), KCI (final concentration 75 mM), MgCl2 (5 mm), and NaCl (final concentration 210 mM). At appropriate times samples were taken from the incubation mixture and quenched with an equal volume of ice-cold HC104 (10 % w/v). Extraction, neutralization, and partial desalting of Ins( 1 ,4,5)P3 and other inositol-phosphates was by the method of Sharps and McCarl (26). Extracts were stored at -20°C until analysis. Protein was determined according to the method of Bradford (3). HPLC of Labeled Compounds Labeled compounds were separated by HPLC on a Partisil10 SAX column (Waters, 25 cm long, 0.46 cm i.d.) using the following gradient system of water (solvent A) and ammonium formate (3.06 M, adjusted to pH 3.70 with H3PO4, solvent B): (a) 0 to 14 min, 0% B; (b) 14 to 60 min, a linear gradient 0 to 23% B; (c) 60 to 89 min, a linear gradient from 23 to 100% B; (d) 89 to 100 min, 100% B. The column was equilibrated with water before the start of each run. A flow rate of 1.25 mL/min was maintained and 1.25 mL fractions were collected. Five microliter aliquots of 5 mM AMP, 5 mM ADP, and 5 mm ATP were included as internal standards in each run and their elution was monitored by absorbance at 259 nm to ensure consistency between separations. Isocratic HPLC was carried out as described by Wreggett and Irvine (30). Radioactivity was determined in individual samples by liquid scintillation spectrometry (1214 Rackbeta, LKB, Bromma, Sweden) using Picofluor 15 and Hionic-fluor (Canberra-Packard) scintillation fluid. Isomeric Configuration All myo-inositol phosphate isomers are, unless specified otherwise, numbered using the D-configuration.
RESULTS Figures 1 and 2 show HPLC profiles of 32P/3H-labeled compounds obtained after 1 min incubations of [32p] Ins(1,4,5)P3 and [3H]Ins(1,4,5)P3 with cell free extracts. In Figure 1, four labeled compounds are present [Pi, Ins(4,5)P2, Ins(1,4,5)P3, and Ins(1,4,5,X)P4]. In contrast, Figure 2 shows that 11 labeled compounds are present when [3H]Ins( 1 ,4,5)P3 is used as precursor. Assignment of Labeled Compounds Although stepwise phosphorylation of inositol is well known to occur in plant cells (17) the approach used in these studies ensures that none of the phosphomonoesters derived by kinase action will contain any label in these experiments when [32P]Ins( 1,4,5)P3 is used as precursor. Inositol and Orthophosphate Inositol was identified by cochromatography with authentic standards. Pi was identified by cochromatography with authentic standards and was the only compound which in duallabeling experiments contained 32P but no 3H.
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proach used in these studies facilitates tentative identification. Four 3H-labeled compounds elute in the InsP2 HPLC region. One contains 32P/3H in the ratio 1:1 relative to Ins(1,4,5)P3 whereas the other three compounds do not contain 32p. Three isomers can result from dephosphorylation of Ins(1,4,5)P3, i.e. Ins( 1 ,4)P2, Ins( 1 ,5)P2, and Ins(4,5)P2. Only the latter two will contain any 32P-label. Using the Wreggett and Irvine
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Figure 1. HPLC profile of water soluble 32P-labeled compounds obtained after 1 min incubation of [5-32P]lns(1,4,5)P3 with soluble enzymes from P. sativum root tips. Sampling was started immediately after injection of the sample (t = 0) and 1.25 mL samples were collected. Assignment of labeled compounds is described in the text.
Inositol-Monophosphates Only one 3H-containing peak was found to elute in the region of inositol-monophosphates using the HPLC system described above. Traces of 32p were found in this peak in some experiments when [32P]InsP3 was employed. The ratio of 32p to 3H was so low that the possibility can be excluded that this peak is pure Ins(5)P formed by dephosphorylation of Ins(1,4,5)P3. If the compound(s) is a dephosphorylation product of Ins( 1 ,4,5)P3 it must either be Ins( 1 )P or Ins(4)P or a mixture of these. Using the isocratic HPLC system developed by Wreggett and Irvine (30) we get a separation of authentic Ins(1)P and Ins(4)P of 14 fractions. As shown in Figure 3, no label is found to coelute with Ins(1)P whereas the majority of label coelutes with Ins(4)P. A smaller peak elutes between Ins( 1 )P and Ins(4)P. Although this peak elutes where Ins(5)P would be expected, the amount of 32p in total InsP and the known ratio of 32p to 3H in Ins( 1 ,4,5)P3 suggests that the amount of Ins(5)P is at least 10-fold lower than that of Ins(4)P and is approaching the detection limit of these experiments. Hence, Ins(5)P cannot account completely for the 3H-label in this peak and we find it likely that there is a contribution from Ins(2)P, formed by phosphorylation of inositol. We conclude that the peak labeled InsP in Figure 2 predominantly contains Ins(4)P and that this isomer is the major InsP formed by stepwise dephosphorylation of Ins( 1 ,4,5)P3.
Inositol-Bisphosphates Inositol-bisphosphates were identified by cochromatography with authentic standards and by analysis of data obtained from dual-labeling experiments. Although 15 isomers of Dmyo-inositolbisphosphate are possible, the experimental ap-
isocratic HPLC separation system (30) it is possible to achieve a clear separation of Ins( 1 ,4)P2, Ins( 1 ,5)P2, and Ins(4,5)P2. As shown in Figure 3,the 32P-labeled InsP2 comigrates exactly with authentic Ins(4,5)P2. Ins( 1,5)P2 is not currently commercially available but it is known that it elutes between Ins( 1 ,4)P2 and Ins(4,5)P2 using the system described above (RF Irvine, personal communication; see also legend to Fig. 3). The presence of small amounts of 32P-Ins(1,4,5,X)P4 in these experiments necessitates that consideration is given to the possibility that the 32P-InsP2 could be derived from Ins( 1 ,4,5,X)4 by dephosphorylation. Six isomers are possible, i.e. Ins(1,4)P2, Ins(1,5)P2, Ins(4,5)P2, Ins(1,X)P2, Ins(4,X)P2, and Ins(5,X)P2 where X represents a P-ester in the D-2, 3, or 6 position. Apart from the isomers discussed above, only Ins(2,5)P2, Ins(3,5)P2, and Ins(5,6)P2 would contain any 32p. As D-Ins(3,5)P2 (L-Ins( 1 ,5)P2) is a stereoisomer ofD-Ins( 1 ,5)P2, and as the P-esters in Ins(2,5)P2 are in the paraposition, it is highly unlikely that these isomers would cochromatograph with Ins(4,5)P2 on isocratic HPLC. We cannot completely rule out that the 32P-InsP2 is a stereoisomer of D-Ins(4,5)P2 (i.e. D-Ins(5,6)P2) but, as will be discussed later, kinetic data do not favor this possibility. We conclude that the 32P-InsP2 beyond reasonable doubt is D-Ins(4,5)P2. Apart from Ins(1,4)P2, one other 3H-labeled compound elutes before Ins(4,5)P2 and two 3H-labeled compounds elute just after Ins(4,5)P2. These three compounds have been named InsP2-A, InsP2-B, and InsP2-C. We have not as yet identified their isomeric configuration but they are clearly not hydrolysis
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time (min) Figure 2. HPLC profile of water soluble 3H-labeled compounds obtained after 1 min incubation of [2-3H]lns(1,4,5)P3 with soluble enzymes from P. sativum root tips. Sampling was started immediately after injection of the sample (t = 0) and 1.25 mL samples were collected. Assignment of labeled compounds is described in the text.
METABOLISM OF INOSITOL(1,4,5)TRISPHOSPHATE IN PLANT EXTRACTS
415
products derived directly from dephosphorylation of Ins( 1 ,4,5)P3. d.p.m.
Inositol-Trisphosphates Three compounds elute in the InsP3 region of the HPLC profile. One is Ins(1,4,5)P3. The two other InsP3s, named InsP3-A and InsP3-B, elute after Ins(1,4,5)P3 and do not contain 32P. The possible precursor(s) for these compounds are discussed later but neither of these compounds is Ins(1,3,4)P3 as this isomer elutes before Ins(1,4,5)P3 in our HPLC system.
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time (min) Figure 3. Isocratic HPLC of 3H-containing compounds labeled InsP (A) and Ins(4,5)P2 (B). The isocratic system was as described by Wreggett and Irvine (30) and employed 40 mm NaH2PO4 for separation of lnsPs and 240 mm NaH2PO4 for separation of InsP2 isomers. Panel A (upper trace) shows separation of authentic 3H-lns(1)P and 3H-lns(4)P. The flow rate was 1.5 mL/min. Sampling was started at t = 5 min, and 10 s samples were collected. The lower trace shows separation of 3H-lnsPs obtained from incubation of 3H-lns(1,4,5)P3 with cell free extracts. The arrow indicates the elution position of an internal AMP spike. Panel B (upper trace) shows the separation of authentic 3H-lns(1,4)P2 and 3H-lns(4,5)P2. Flow rate was 1.5 mL/min and sampling of 10 s fractions was started at t = 6 min. An 3H-lnsP2 mixture obtained by alkaline phosphatase treatment of 3H-lns(1 ,4,5)P3 was included with the authentic standards. Thus, the small peak eluting between lns(1,4)P2 and lns(4,5)P2 is likely to be lns(1,5)P2. Panel B (lower trace) shows isocratic HPLC of 3H-lns(4,5)P2 obtained from incubations of 3H-lns(1 ,4,5)P3 with cell free extracts. The elution position of an internal ADP spike is indicated with the arrow.
Inositol-Tetrakisphosphates The InsP4 observed in Figures 1 and 2 has been designated an isomeric configuration of Ins( 1,4,5,X)P4. The finding that this compound has a 32P:3H ratio similar to Ins(1,4,5)P3 indicates that it must be formed by phosphorylation of a lower InsP in which the 5'-P originating from Ins(1,4,5)P3 is still present. Four such precursors can be envisaged: Ins(5)P, Ins(1,5)P2, Ins(4,5)P2, and Ins(1,4,5)P3. Although the possibility that Ins(5)P or Ins(1,5)P2 may be precursors cannot be completely excluded, the findings that these isomers are absent at all times during the experimental period makes this very unlikely. The kinetics of Ins(4,5)P2 formation do not favor this compound as precursor either. These considerations have led us to assign a configuration of Ins( 1 ,4,5,X)P4 to this InsP4. When intact plant tissues or cells are incubated for extended periods of time with [2-3H]inositol, label is found to be incorporated into water-soluble compounds other than inositol phosphates-notably, cell wall components (5, 17; BK Dr0bak, unpublished data). However as, in the experiments described here Ins(1,4,5)P3 is used as precursor and short incubation times are employed, it is implausible that highly complex inositol containing compounds are being produced. As illustrated in Figure 4, Ins(1,4,5)P3 is rapidly hydrolyzed at the rate of approximately 1.3 nmol/mg protein during the first minute of incubation. After 5 min this rate is greatly decreased, possibly indicating instability of hydrolysis enzyme(s) under the assay conditions employed. The hydrolysis is accompanied by the formation of Ins(4,5)P2 and small amounts of Ins( 1 ,4)P2. The Ins(4,5)P2 produced contains 25, 55, and 64% of the label lost from Ins(1,4,5)P3 after 1, 5, and 15 min, respectively, whereas Ins(1,4)P2 at all times during the experiments contains less than 2% of the radioactivity. Figures 5 and 6 show the levels of other labeled compounds produced during the experimental period. Each of these compounds are present after 1 min incubation. Neither Ins, InsP, Ins( 1,4)P2, InsP2-B, nor Ins(1,4,5,X)P4 changed significantly in the period following 1 min incubation. This may indicate that a steady-state of formation and removal is reached. A significant change in levels of InsP2-A, InsP2-C, InsP3-A, and InsP3-B was observed. The level of InsP2-C was found to increase in the period 0 to 5 min, whereas the maximum quantities of the other compounds were present after 1 min followed by a gradual disappearance over the next 14 min. The rate of hydrolysis of Ins(1,4,5)P3 was found to have an approximately linear dependency upon Ins(1,4,5)P3 concen-
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time (min) Figure 4. Hydrolysis of 3H-lns(1,4,5)P3 and formation of Ins(4,5)P2 and lns(1,4)P2 during a 15 min incubation of [2-3H]lns(1,4,5)P3 with soluble enzymes from P. sativum roots. Data for lns(1,4,5)P3 represent the amounts of this compound still present in the reaction mixture after the indicated times. (l, lns(1,4,5)P3; (0), lns(4,5)P2; (0), Ins(1,4)P2. (Bars represent mean ± SE, n = 5).
i-1
I_ 15
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time (min) ._
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a 0)
tration in the region of0 to 25 ,uM. The Km has been estimated ± 0.3 x IO- M (data not shown). The effect on Ins(1,4,5)P3 hydrolysis of 10 mm of the monoester phosphohydrolase substrates 2,3-diphosphoglycerate, glucose 6-phosphate, and p-NPP is shown in Figure 7. The inclusion of either 2,3-DPG, p-NPP, or G-6-P in approximately 2000-fold excess over Ins(1,4,5)P3 resulted in an inhibition of the conversion of Ins(1,4,5)P3 by approximately 50%. No significant difference in effectiveness of inhibition is evident between the three phosphohydrolase substrates. The effect of Ca2" on the rate of Ins( 1 ,4,5)P3 hydrolysis and formation of products is illustrated in Figure 8. Hydrolysis of Ins(1,4,5)P3 and production of Ins(4,5)P2 and Ins(1,4,5,X)P3 is stimulated by lowering the free Ca2+ concentrations to 400 nM. to be 4.6
DISCUSSION The rapid conversion of Ins(1,4,5)P3 found in these experiments is dominated by Ins(1,4,5)P3-1-phosphohydrolase activity-the resulting product being Ins(4,5)P2. In contrast to Ins(1,4,5)P3, Ins(4,5)P2 appears to be metabolized comparatively slowly. This pattern differs significantly from that ob-
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time (min) Figure 5. Formation of 3H-labeled inositol and inositol-monophosphates (InsP) (A) and InsP2-A, lnsP2-B, and lnsP2-C (B) during 15 min incubation of 3H-lns(1,4,5)P3 with soluble enzymes from P. sativum root tips. (0), Ins; (0), InsP, (0), lnsP2-A; (0), InsP2-B; (0), InsP2-C. (Bars represent mean ± SE, n = 3).
METABOLISM OF INOSITOL(1,4,5)TRISPHOSPHATE IN PLANT EXTRACTS
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time (min) Figure 6. Formation of 3H-labeled inositol-trisphosphates (A, InsP3A and InsP3-B) and inositol-tetrakisphosphates [Ins(1,4,5,X)P4] (B) during 15 min incubation of lns(1,4,5)P3 with soluble enzymes from P. sativum roots. (0), InsP3-A; (U), InsP3-B; (A), Ins(1,4,5,X)P4. (Bars represent mean ± SE, n = 3).
served in most other eukaryotes. In mammalian cells the removal of Ins( 1 ,4,5)P3 from the cytosol is generally found to be undertaken by a 5-phosphohydrolase and a 3-hydroxykinase, Ins(1,4)P2 and Ins(1,3,4,5)P4 being the resulting products. However, in studies using homogenates from the slime mould Dictyostelium discoideum, Van Lookeren Campagne et al. (29) found that Ins(1,4,5)P3 was rapidly dephosphorylated to 20% Ins(1,4)P2 and 80% Ins(4,5)P2, followed by further dephosphorylation of Ins(4,5)P2 and Ins(1,4)P2 to Ins(4)P and inositol. These findings, when seen in conjunction with the data presented above (see Fig. 4), suggest the presence of a pathway of Ins(1,4,5)P3 hydrolysis in certain lower eukaryotes which differs from that of e.g. mammalian cells. As shown in Figure 7, 10 mm of selected phosphohydrolase substrates partially inhibited the 1-phosphohydrolase activity by approximately 55% during 5 min incubations. Van Lookeren Campagne et al. (29) found that the Ins(1,4,5)P3-1phosphohydrolase of Dictyostelium was inhibited biphasically by 2,3-DPG with a reduction in hydrolysis to less than 10% of control by 10 mM 2,3-DPG. Our data show that the Ins( 1,4,5)P3- I-phosphohydrolase activity in pea roots is considerably less sensitive to 2,3-DPG than the corresponding
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activity in Dictyostelium. Van Lookeren et al. further showed that the Ins(1,4,5)P3-l-phoshohydrolase was inhibited by Li' (half-maximal inhibition at 0.25 mm Li'). Recently, Memon et al. (20) investigated the possibility of Li'-sensitivity of Ins(1,4,5)P3 hydrolysis in soluble and microsomal fractions from suspension cultured carrot cells. It was found that 50 mM Li' had no effect on the rate of hydrolysis of Ins( 1 ,4,5)P3. Although these authors did not investigate the isomeric configuration of the Ins( 1 ,4,5)P3 hydrolysis product(s), it is likely that, at least in part, the dephosphorylation of Ins(1 ,4,5)P3 was due to removal of the 1-phosphate. This suggests that although Ins(1,4,5)P3 metabolizing enzymes exist in slime molds and plant cells which apparently share similar modes of action, their biochemical properties may differ in several aspects. While our experiments were in progress data on inositol phosphate dephosphorylation by gel-filtered extracts from suspension cultured tobacco cells were reported by Joseph et al. (16). These authors found that authentic Ins( 1 )P, Ins(4)P, Ins(1,4)P2, and Ins(1,4,5)P3 were all dephosphorylated to lower inositol phosphates or inositol in a Ca2+-dependent manner. Although a detailed study of Ins( 1 ,4,5)P3 metabolism was not undertaken by Joseph et al. (16), their results indicate that after 2 min Ins( 1,4,5)P3 (7 Mm) was completely hydrolyzed in the absence of Ca21 into roughly equal amounts of Ins(1,4)P2 and Ins(4,5)P2, and in the presence of 1.8 .M Ca2+ the distribution between Ins( 1 ,4)P2 and Ins(4,5)P2 was around 1:2. In the present study we demonstrate that in soluble extracts from pea roots the hydrolysis of Ins( 1 ,4,5)P3 and the formation of Ins(4,5)P2 and Ins(1,4,5,X)P4 is enhanced at low levels of Ca2+ (400 nM), whereas the formation of Ins( 1 ,4)P2 is neglible both under high (55 gM) and low (400 nM) levels of Ca2+. However, not only is the source of experimental material different in our study to that of Joseph et al. (16) but
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.L
a 0. CD
I-
0
C,
0
C.
N a
I
Figure 7. Effect of three monoesterphosphohydrolase substrates upon lns(1 ,4,5)P3 hydrolysis; 10 mm of either 2,3-DPG, p-NPP, or G6-P were included in the reaction mixture and lns(1,4,5)P3 and lns(4,5)P2 levels were determined after 5 min incubation. Data are means of two independent experiments. Vertical bars indicate range.
DROBAK ET AL.
418
200
175
150
125
100
75
50
time (min) Figure 8. Effect of Ca2+ on Ins(1,4,5)P3 hydrolysis and formation of lns(4,5)P2 and lns(1,4,5,X)P4. Amounts of lns(1,4,5)P3 are those still present in the reaction mixture after the indicated times. Data are from 'high-calcium' experiments and are expressed as percentage of 'low-calcium' data. Data are averages from two independent experiments. Vertical bars indicate range. (E), Ins(1,4,5)P3; (0), Ins(4,5)P2; (A), Ins(1,4,5, X)P4.
there is also substantial differences in experimental conditions (e.g. Mg2+ concentration). A possibility that we cannot rule out is that several phosphohydrolases (noteably a weak 5-phosphohydrolase) may be present in the soluble enzyme fraction but that these are inactivated very rapidly due to denaturation/proteolysis. If this is the case it is conceivable that the observed effect of Ca2+ is dual in nature and can be ascribed, partly, to an effect of Ca2+ upon enzyme stability. The formation of a number of 3H-labeled compounds in addition to Ins(4,5)P2 indicates the presence of several enzyme systems comprising both phosphohydrolases and hydroxykinases with the ability to utilize inositol/inositol phosphates as substrates. Of most interest, perhaps, when the role of Ins(1,4,5)P3 as messenger molecule is considered, is the finding that this isomer can be phosphorylated. Studies in mammalian systems have recently revealed that Ins(1,4,5)P3, and its phosphorylation product Ins( 1 ,3,4,5)P4, may act as a duet in controlling intracellular Ca2+-fluxes, with the function for Ins(1,3,4,5)P4 associated with the refilling of Ins(1,4,5)P3sensitive Ca2+ pools (14). The formation of Ins(1,4,5,X)P4 in our experiments was significant, although this inositol phosphate was only present in small and variable quantities throughout the experimental period. The possibility that
Plant Physiol. Vol. 95, 1991
Ins(4,5)P2, and not Ins(1,4,5)P3, is the precursor for the 32p_ labeled InsP4 cannot be completely excluded but there is nothing in our data which would support such a notion. We conclude that the 32P-InsP4 encountered in these experiments is almost certainly formed by direct phosphorylation of Ins( 1,4,5)P3 and can be represented by the formula Ins(1,4,5,X)P4. An in-depth study of the Ins(1,4,5)P3-kinase(s) of pea roots is currently in progress in our laboratory and preliminary data suggest that more than one kinase may be involved in Ins(1,4,5)P3 phosphorylation (JA Chattaway, BK Dr0bak, unpublished data). The likely initial precursors for InsP2-A to C and InsP3-A and InsP3-B can, on the background of the data in Figures 5 and 6, be limited to either inositol or InsP. Thus, this study demonstrates that products formed by Ins(1,4,5)P3 hydrolysis in plant extracts can act as substrates for kinases and rapidly be converted into higher inositol phosphates with isomeric configurations dissimilar to any of the inositol phosphates normally encountered in, e.g., mammalian systems. A summary of the reactions likely to be responsible for the degradation/formation of the inositol phosphates encountered in this study is illustrated in Figure 9. Two central problems are inherently associated with any study of metabolism where cell free extracts/homogenates are used as enzyme source. These are (a) the question of whether the enzyme(s) under study are membrane associated or soluble and (b) the possibility of cellular compartmentation. The first of these problems is relatively easily solved by separation of extracts into soluble and membrane fractions. In preliminary experiments we compared the rates and pathways of Ins(1,4,5)P3 hydrolysis using both a soluble and a crude membrane fraction from pea roots as enzyme source. The gross pattern of hydrolysis was found to be qualitatively identical in these fractions [i.e. Ins(4,5)P2 was the major breakdown product, and Ins(1,4)P2 formation was small]. Although a potential physiological role for a membrane as-
lns( 1 ,4,5,X)P4 lns( 1 ,4,5)P3
InSP3-A/B lns(4,5)P2
Ins( I ,4)P2 ATP ?
lns(4)P
