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. 1998 Jan;116(1):291–297.

Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds

Brian Q Phillippy 1,*
PMCID: PMC35169

Abstract

In extracts of immature soybean (Glycine max [L.] Merr.) seeds inositol tetrakisphosphate was formed from [3H]inositol 1,3,4-trisphosphate but not from [3H]inositol 1,4,5-trisphosphate. Inositol 1,3,4-trisphosphate kinase was purified to a specific activity of 3.55 min−1 mg−1 by polyethylenimine clarification and anion-exchange chromatography. The partially purified enzyme converted [3H]inositol 1,3,4-trisphosphate to inositol 1,3,4,5-tetrakisphosphate as the major product and inositol 1,3,4,6- and/or 1,2,3,4-tetrakisphosphate as the minor product. Subsequent experiments revealed a separate inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, which could link these enzymes to inositol hexakisphosphate synthesis via the previously reported inositol 1,3,4,5,6-pentakisphosphate 2-kinase. The apparent Km values for inositol 1,3,4-trisphosphate kinase were 200 ± 0 nm for inositol 1,3,4-trisphosphate and 171 ± 4 μm for ATP, and the reaction was not reversible. The kinetics were such that no activity could be detected using unlabeled inositol 1,3,4-trisphosphate and [γ-32P]ATP, which suggested that other kinases may have been observed when less purified fractions were incubated with radiolabeled ATP. Inositol 1,3,4-trisphosphate kinase was nonspecifically inhibited more than 80% by various inositol polyphosphates at a concentration of 100 μm.

Inositol phosphates are important in plants because of the role of d-Ins(1,4,5)P3 in signal transduction (Drobak, 1992; Cote and Crain, 1993) and because seeds contain an extraordinary amount of InsP6, which is commonly known as phytic acid (Reddy et al., 1989). Since these two compounds may be synthesized by the same pathway, albeit a circuitous one, in animal cells, it would be of interest to determine their relationship in plants. The biosynthetic pathway in seeds has been an unresolved dilemma for many years. Although kinases that can synthesize InsP5 and InsP6 from Ins(1)P and Ins(2)P, respectively, have been reported (Chakrabarti and Biswas, 1981), another possibility is that the inositol is partially or completely phosphorylated in a bound form, most likely as a lipid conjugate (Asada et al., 1969; Drobak, 1992).

The most direct approaches to identifying the pathway of InsP6 synthesis are to identify the presence of the intermediates or the corresponding enzymes in the seeds. Intermediates could not be observed during InsP6 synthesis in rice and wheat (Asada et al., 1968; Graf, 1983). Kinases that phosphorylate various InsP5 isomers have been identified in mung bean (Biswas et al., 1978; Stephens et al., 1991) and soybean seeds (Phillippy et al., 1994), but the earlier reactions have not yet been identified.

Although the phosphoinositide pathways may vary among different types of cells, clues to the pathway of InsP6 synthesis in seeds may be obtained from those intermediates and enzymes identified elsewhere. Suspension cells of rice were reported to produce Ins(1)P, Ins(2)P, Ins(1,3)P2, Ins(2,4)P2, Ins(1,3,5)P3, Ins(2,4,5)P3, Ins(1,3,4,5)P4, Ins(1,2,4,5)P4, Ins(1,2,4,5,6)P5, Ins(1,2,3,4,5)P5, and Ins(1,3,4,5,6)P5 (Igaue et al., 1982). In duckweed InsP6 may be formed by sequential phosphorylation of Ins(3)P, Ins(3,4)P2, Ins(3,4,6)P3, Ins(3,4,5,6)P4, and Ins(1,3,4,5,6)P5 (Brearly and Hanke, 1996). Ins(1,4,5)P3 6-kinase activity has been described in pea roots (Chattaway et al., 1992), and the intermediates in Dictyostelium sp. are Ins(3)P, Ins(3,6)P2, Ins(3,4,6)P3, Ins(1,3,4,6)P4, and Ins(1,3,4,5,6)P5 in the cytosol (Stephens and Irvine, 1990) and Ins(1,4,5)P3, Ins(1,3,4,5)P4, and Ins(1,3,4,5,6)P5 in the nucleus (Van der Kaay et al., 1995). In animal cells the multibranched signaling pathway contains Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(3,4,6)P3, Ins(1,3,4,5)P4, Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(3,4,5,6)P4, and Ins(1,3,4,5,6)P5, but the major pathway for the de novo synthesis of InsP6 may start with the direct phosphorylation of myo-inositol (Sasakawa et al., 1995). The intent of the following work was to search for enzyme activities that might contribute to the synthesis of InsP6 in soybean seeds and thereby reveal the likely InsP3 and InsP4 intermediates.

MATERIALS AND METHODS

[γ-32P]ATP (3000 Ci/mmol), [α-32P]ATP (3000 Ci/mmol), [3H]Ins(1,3,4)P3 (21 Ci/mmol), [3H]Ins(1,4,5)P3 (21 Ci/mmol), and [3H]Ins(1,3,4,5)P4 (21 Ci/mmol) were from NEN. Ins(1,3,4)P3, Ins(1,4,5)P3, and Ins(1,3,4,5)P4 were purchased from LC Laboratories (Woburn, MA), which was recently acquired by Alexis Corp. (San Diego, CA). Ins(1,3,4,6)P4 was from Calbiochem-Novabiochem Corp. (La Jolla, CA). Ins(1,2,3,6)P4 and Ins(1,2,5,6)P4 were prepared by hydrolysis of InsP6 with wheat phytase (Phillippy, 1989). Adenosine 5′-tetraphosphate from equine muscle and sodium phytate were obtained from Sigma. Phytic acid (40 weight% solution in water) was obtained from Aldrich, and ScintiSafe Econo 2, ScintiVerse II, and ScintiSafe Plus 50% were purchased from Fisher Scientific. Soybean (Glycine max [L.] Merr.) seeds from Pioneer Hi-Bred International (Johnston, IA) were planted outdoors, and green immature seeds were harvested at maximum size and stored at −80°C until used.

Enzyme Preparation

Five grams of immature seeds was homogenized for 10 s with 25 mL of 20 mm Hepes buffer (pH 7.8) containing 2 mm EDTA, 10 mm β-mercaptoethanol, 0.1 mm PMSF, and 5% glycerol. The homogenate was filtered through several layers of cheesecloth and centrifuged for 30 min at 10,000g. Coagulated fat was removed with a spatula and 10% polyethylenimine adjusted to pH 7.8 was added to the supernatant at a final concentration of 0.1%. After stirring on ice for 10 min the suspension was centrifuged for 20 min at 10,000g. The supernatant was loaded onto a 2.5- × 5.0-cm DEAE Toyopearl 650M column (TosoHaas, Montgomery-ville, PA) and eluted with a 100-mL gradient of 0 to 0.3 m KCl in homogenization buffer at 1.5 mL/min. Ten 10-mL fractions were collected and assayed for activity with Ins(1,3,4)P3 as described below, and protein was determined according to Bradford (1976) using ovalbumin as the standard.

Inositol Trisphosphate Kinase Assay

One microliter of enzyme was incubated in a total volume of 50 μL of 20 mm Hepes buffer (pH 7.0) containing 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, 5 mm MgCl2, 500 μm ATP, and 4 nm [3H]Ins(1,3,4)P3 for 30 min at 30°C. The reaction was stopped by dilution with 1 mL of H2O containing 40 μg of InsP6 hydrolysate (from the 40 weight % solution of phytic acid, which apparently had degraded during storage to give the pattern of a random hydrolysate) to improve recoveries. Ins(1,3,4)P3 kinase activity was determined after purification of InsP4 on a 400-μL AG 1-X8 column (Wilson and Majerus, 1996). The diluted reaction mixture was loaded onto the column and washed with four 2-mL aliquots of 0.8 m ammonium formate adjusted to pH 3.5 with formic acid. InsP4 was eluted with 2 mL of 1.6 m ammonium formate, and radioactivity was counted with 15 mL of ScintiSafe Plus 50% or ScintiVerse II in plastic vials. Activity was expressed in terms of the first-order rate constant using the equation: k = −ln([S]/[S]o)/t, where k = activity in min−1, [S]o = initial [3H]Ins(1,3,4)P3 cpm, [S] = [S]o − [3H]InsP4 cpm, and t = time in min (Wilson and Majerus, 1996).

Ion Chromatography

Gradient ion chromatography was used to identify the inositol phosphate isomers as described previously (Phillippy and Bland, 1988). In experiments to identify the presence of inositol polyphosphate kinase activities, 5- or 10-μL enzyme samples were assayed in a total volume of 200 μL of 20 mm Hepes (pH 7.0) containing 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, 5 mm MgCl2, and 520 pm [γ-32P]ATP for 10 min at 30°C. The reaction was stopped by the addition of 200 μL of 0.75 n HCl, and the mixture was passed through a 0.45-μm filter. Fifty-microliter aliquots were separated on AG3 and AS3 (guard and analytical, respectively) columns (Dionex, Sunnyvale, CA) with a 25-mL gradient of 0 to 0.155 n HNO3 followed by 5 mL of 0.155 n HNO3, and 0.5-mL fractions were collected. In similar experiments [γ-32P]ATP was replaced with 100 μm unlabeled ATP and 12 nm [3H]Ins(1,3,4)P3, [3H]Ins(1,4,5)P3, or [3H]Ins(1,3,4,5)P4, and 160 μg InsP6 hydrolysate was added prior to ion chromatography to improve recoveries. [γ-32P]ATP was determined by Cerenkov counting, and the [3H] fractions were counted with 5 mL of ScintiSafe Econo 2 liquid-scintillation cocktail.

RESULTS

To identify the inositol polyphosphate kinases in immature soybeans, assays were conducted using isomeric mixtures of inositol phosphates obtained via ion-exchange chromatography of a phytic acid hydrolysate and [γ-32P]ATP. When a mixture containing predominantly InsP3 and InsP4 isomers was incubated with the soybean extract, both InsP4 and InsP5 appeared to be formed (results not shown). Since the InsP4 peak could have contained one or several different isomers, it was necessary to identify either the precursor(s) or the product(s). The simplest way would have been to try different purified possible precursors, except that when a control with no added inositol phosphate substrate was assayed, peaks at 12 and 15 min, tentatively identified as InsP3 and InsP4, were observed (Fig. 1B).

Figure 1.

Figure 1

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Kinase activity of an immature soybean seed extract utilizing [γ-32P]ATP. The extract was prepared and partially purified as detailed in “Materials and Methods,” except that the polyethylenimine clarification step was omitted. Twenty-six micrograms of protein was incubated for 10 min at 30°C in 200 μL of 520 pm [γ-32P]ATP (3000 Ci/mmol), 5 mm MgCl2, 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, and 20 mm Hepes, pH 7.0. Reactions from control preheated at 95°C for 15 min (A) and active (B) extracts were terminated by the addition of 200 μL of 0.75 n HCl, and 50-μL aliquots were analyzed by ion chromatography.

There were two possible explanations for the formation of the apparent InsP4 peak. First, the crude enzyme fraction may have contained the inositol trisphosphate precursor. Second, the product may have been an artifact formed from the [γ-32P]ATP by some other mechanism. The latter possibility was ruled out two ways. First, when the enzyme was heated at 95°C for 15 min and assayed, no products were observed (Fig. 1A). Second, when [γ-32P]ATP was replaced with [α-32P]ATP, no products such as adenosine 5′-tetraphosphate were observed. However, when a phytic acid hydrolysate was fractionated on a DEAE Toyopearl 650M column by the same procedure used to prepare the enzyme, InsP2, InsP3, and InsP4 eluted in fractions with the same retention volumes as those observed to have kinase activity.

Instead of attempting to remove the copurifying inositol phosphates from the kinases, it was decided to first test the commercially available radiolabeled inositol phosphates that are in the phosphoinositide pathways leading to InsP6 in other types of cells. No kinase activity was detected using [3H]Ins(1,4,5)P3, but [3H]Ins(1,3,4)P3 was phosphorylated to a InsP4 peak with a leading shoulder that was suspected of harboring more than one product (Fig. 2). Further attempts to detect Ins(1,4,5)P3 kinase activity using a polyethylenimine-clarified extract were also unsuccessful.

Figure 2.

Figure 2

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Kinase activity of an immature soybean seed extract utilizing [3H]Ins(1,3,4)P3. The extract was prepared and partially purified as detailed in “Materials and Methods,” except that the polyethylenimine clarification step was omitted. Forty-one micrograms of protein was incubated in 200 μL of 12 nm [3H]Ins(1,3,4)P3 (21 Ci/mmol), 100 μm ATP, 5 mm MgCl2, 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, and 20 mm Hepes, pH 7.0. Reactions incubated for 0 (A) and 10 (B) min at 30°C were terminated by the addition of 200 μL of 0.75 n HCl containing 160 μg of InsP6 hydrolysate, and 50-μL aliquots were analyzed by ion chromatography.

Using an assay procedure similar to that of Wilson and Majerus (1996), the Ins(1,3,4)P3 kinase was partially purified from a crude seed extract by polyethylenimine clarification and anion-exchange chromatography (Table I). The polyethylenimine treatment removed InsP3, InsP4, InsP5, and InsP6 from the extract, as evidenced by recovery experiments with added inositol phosphates (results not shown). The enzyme was purified 23-fold in fraction 7 from the DEAE Toyopearl 650M column, and that fraction was used to examine some of its properties. The apparent Km values for Ins(1,3,4)P3 and ATP were determined to be 200 ± 0 nm and 171 ± 4 μm, respectively. The reaction did not appear to proceed in reverse when [3H]Ins(1,3,4)P3 and ATP were replaced with equivalent concentrations of [3H]Ins(1,3,4,5)P4 and ADP, respectively. In addition, no InsP4 was observed upon incubation of the enzyme with 20 μm unlabeled Ins(1,3,4)P3 and 1.7 nm [32P]ATP, because the reaction rate calculated from the substrate concentrations and apparent Km values was approximately 103-fold less than when 12 nm [3H]Ins(1,3,4)P3 and 100 μm unlabeled ATP were used.

Table I.

Partial purification of Ins(1,3,4)P3 kinase

Step Total Activity Protein Specific Activity Purification
min−1 mg min−1/mg -fold
Crude supernatant 117 1029 0.114 1
Polyethylenimine 103 166 1.61 14
DEAE Toyopearl 650M 27.8 10.5 2.65 23
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To identify the InsP4 products of Ins(1,3,4)P3 kinase, the resolution of the ion chromatography was increased by omitting the HCl ordinarily used to stop the reaction and by reducing the size of the collected fractions from 0.5 to 0.2 mL. Two InsP4 peaks were completely separated and their retention times were compared with those of standards. The earlier-eluting and smaller peak was identified as Ins(1,3,4,6)P4 and/or Ins(1,2,3,4)P4, and the later-eluting and larger peak was Ins(1,3,4,5)P4 (Fig. 3).

Figure 3.

Figure 3

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Indentification of the products of Ins(1,3,4)P3 kinase. Seven micrograms of partially purified Ins(1,3,4)P3 kinase was incubated in 100 μL of 9.6 nm [3H]Ins(1,3,4)P3 (21Ci/mmol), 100 μm ATP, 5 mm MgCl2, 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, and 20 mm Hepes, pH 7.0. After 30 min at 30°C, 80 μg of InsP6 hydrolysate was added, and a 50-μL aliquot was analyzed by ion chromatography. One hundred twenty 200-μL fractions were collected at 12-s intervals. Retention times were compared with those obtained from [3H]Ins(1,3,4,5)P4, and unlabeled Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, and Ins(1,2,3,6)P4. Ins(1,2,3,4)P4 and Ins(1, 2, 3, 6)P4 are enantiomers and would have identical retention times in this procedure.

When [3H]Ins(1,3,4,5)P4 was incubated with aliquots from a polyethylenimine-clarified extract under conditions similar to those used for [3H]Ins(1,3,4)P3 in Figure 2, a trace amount of InsP5 appeared to be formed. By increasing the incubation time to 30 min and the ATP concentration to 500 μm, an InsP5 peak was clearly observed eluting between 23 and 24 min (Fig. 4). This product was identified as Ins(1,3,4,5,6)P5, since Ins(1,2,3,4,5)P5, the isomer that would result from phosphorylation at the 2 position of Ins(1,3,4,5)P4, has a retention time between 20 and 21 min under the conditions used. No Ins(1,3,4,5)P4 6-kinase activity was detected in the partially purified Ins(1,3,4)P3 kinase preparation, indicating that these reactions were catalyzed by separate enzymes.

Figure 4.

Figure 4

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Kinase activity of an immature soybean seed extract utilizing [3H]Ins(1,3,4,5)P4. The extract was prepared and clarified with polyethylenimine as described in Methods. Thirty-three micrograms of protein was incubated in 100 μL of 10 nm [3H]Ins(1,3,4,5)P4 (21 Ci/mmol), 500 μm ATP, 5 mm MgCl2, 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm β-mercaptoethanol, 0.1 mm PMSF, and 20 mm Hepes, pH 7.0. Reactions incubated for 0 (A) and 30 (B) min at 30°C were terminated by the addition of 100 μL of 0.75 n HCl containing 80 μg of InsP6 hydrolysate, and 50-μL aliquots were analyzed by ion chromatography.

Since InsP6 accumulates to very high levels, in excess of 1% of the weight of most seeds, whereas the precursors of InsP6 are for the most part difficult to detect, experiments were performed to measure the activity of Ins(1,3,4)P3 kinase in the presence of 100 nm to 100 μm of various inositol phosphates. Formation of InsP4 was inhibited more than 80% by 100 μm Ins(1,4,5)P3, Ins(1,3,4,6)P4, or Ins(1,3,4,5)P4 (Fig. 5A). In other experiments comparable nonspecific inhibition was observed from Ins(1,2,5,6)P4 and Ins(1,2,3,6)P4. InsP6 inhibited the reaction similarly except for a small break at a concentration of 10 μm (Fig. 5B).

Figure 5.

Figure 5

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Inhibition of Ins(1,3,4)P3 kinase by various inositol polyphosphates. One-and-one-half micrograms of partially purified Ins(1,3,4)P3 kinase was assayed in the presence of 0, 0.1, 1, 10, or 100 μm concentrations of Ins(1,4,5)P3, Ins(1,3,4,6)P4, or Ins(1,3,4,5)P4 (A), or InsP6 (B). Data were calculated as the percentage of the activity observed in the absence of the indicated inhibitor.

DISCUSSION

Enzymes that metabolize inositol phosphates are often present in biological tissues at concentrations too low to easily measure without some purification and/or concentration. Even then, the kinetics of the reactions are often such that products do not accumulate in quantities amenable to colorimetric assays. For this reason they are usually labeled with [3H] or [32P] and monitored with radioactivity detectors or scintillation counters. The use of [3H] requires that [3H]inositol be incubated in an appropriate system to synthesize the desired products, which in turn must be purified for further study. Although [32P]Pi can be used in a similar fashion, a more direct method is to use [γ-32P]ATP as a substrate. However, unlike [3H]-labeled inositol polyphosphates, which are relatively easy to analyze on anion-exchange columns, [γ-32P]ATP must be used with the awareness that a variety of nucleotides may co-elute with and thus impede chromatographic resolution of the desired products.

Inositol polyphosphate kinase activities were sought in soybean seeds using [γ-32P]ATP as a substrate. Although the products that eluted in the putative InsP3 and InsP4 fractions were not identified, they were likely to be inositol phosphates, since nucleotides such as adenosine tetraphosphate may be the only other significant polyanionic compounds in seeds that could be retained as strongly on anion-exchange columns under acidic conditions. The formation of adenosine tetraphosphate in the extract was ruled out using [α-32P]ATP as the labeled substrate. Since the suspected InsP4 peak formed from [γ-32P]ATP eluted earlier than [3H]Ins(1,3,4,5)P4, Ins(1,3,4)P3 5-kinase may not have been the predominant InsP3 kinase observed in the crude extract at low concentrations of ATP.

When the commercially available radiolabeled inositol trisphosphates were tested for activity with the soybean extract, InsP4 was formed from Ins(1,3,4)P3 but not from Ins(1,4,5)P3. The lack of detectable Ins(1,4,5)P3 kinase activity is not entirely surprising, since that isomer has not been implicated in signal transduction during seed formation, although functional Ins(1,4,5)P3 receptors have been isolated from mung bean hypocotyl microsomes (Biswas et al., 1995). The major soybean activity was Ins(1,3,4)P3 5-kinase, whereas a smaller amount of Ins(1,3,4)P3 6- and/or 2-kinase was also observed. The multiple products may have resulted from more than one kinase or from a single kinase with mixed specificity. Purified rat liver and calf brain Ins(1,3,4)P3 5/6-kinases yield Ins(1,3,4,6)P4 as the major product (Abdullah et al., 1992; Wilson and Majerus, 1996), which may serve as a precursor of InsP5 and InsP6. However, in animal cells, where Ins(1,4,5)P3 is converted to Ins(1,3,4)P3 via Ins(1,3,4,5)P4, the de novo synthesis of InsP6 is thought to begin with the direct phosphorylation of myo-inositol (Sasakawa et al., 1995).

The identification of kinases that phosphorylate Ins(1,3,4)P3 and Ins(1,3,4,5)P4 raises the possibility of their involvement in InsP6 synthesis in seeds. Recently, Ins(1,3,4)P3 5/6-kinase from Arabidopsis thaliana was expressed in Escherichia coli and found to produce Ins(1,3,4,5)P4 and Ins(1,3,4,6)P4 in a ratio of 3:1 (Wilson and Majerus, 1997). In soybeans Ins(1,3,4,5)P4, which was the major product of Ins(1,3,4)P3 kinase, was converted into Ins(1,3,4,5,6)P5, but the minor product of the reaction was not characterized further. Ins(1,3,4,5)P4 6-kinase has previously been detected in Chlamydomonas eugametos and turkey erythrocytes (Irvine et al., 1992). Although the occurrence of Ins(1,3,4,5,6)P5 2-kinase in soybean and mung bean seeds supports the possibility that Ins(1,3,4)P3 and Ins(1,3,4,5)P4 are precursors of InsP6 in seeds, other InsP5 kinases are present as well (Stephens et al., 1991; Phillippy et al., 1994). Given the myriad of inositol phosphate kinases known to exist in animal cells, it would not be surprising if the pathway for InsP6 synthesis in plants was multibranched, although a primary route might be expected to dominate. The lack of accumulation of precursors of InsP6 in seeds could be due to the high efficiencies of kinases exemplified by Ins(1,3,4)P3 kinase, which had a Km of only 200 nm for Ins(1,3,4)P3. The tight regulation of Ins(1,3,4)P3 kinase via nonspecific inhibition by micromolar concentrations of a variety of inositol phosphates including InsP6 may also serve to limit the formation of its products and predict compartmentation of the InsP6 assembly apparatus.

Ins(1,3,4)P3 has recently been identified as a component of soaked pea flour (Skoglund et al., 1997). In animal cells Ins(1,3,4)P3 can be formed by enzymatic degradation of Ins(1,3,4,5)P4 formed from Ins(1,4,5,)P3 (Shears, 1989). However, recent data indicate that in rat thyroid cells Ins(1,3,4)P3 may also be formed by an alternate mechanism unrelated to Ins(1,4,5)P3 (Singh et al., 1996). Since the soybean seed extract did not phosphorylate the latter, its source of Ins(1,3,4)P3 is likely to be either an InsP2 or phosphatidyl inositol 3,4-bisphosphate, which has been identified in different types of plant cells (Irvine et al., 1992; Brearly and Hanke, 1993; Parmar and Brearly, 1993). The observation that Ins(1,3,4)P3 is found after soaking pea flour for 20 h at 45°C and pH 7.0 (Skoglund et al., 1997) suggests the possible presence of phospholipase C activity, although phosphoinositides phosphorylated at the 3 position are poor substrates for phospholipase C from rat liver and bovine brain (Serunian et al., 1989). Another possibility is that sufficient ATP was regenerated by Ins(1,3,4,5,6)P5 2-kinase (Phillippy et al., 1994) to sustain a low level of InsP2 kinase activity. If that were the case, Ins(1,3,4)P3 could ultimately be derived from the ubiquitous Ins(3)P (Loewus et al., 1982; Stephens et al., 1990). Identification of the spectrum of inositol phosphates present in maturing seeds and the testing of those isomers for their corresponding kinases would shed additional light on whatever diversity may exist in the pathway leading to InsP6 in seeds.

There is much interest in reducing phytate-related water pollution through the addition of phytases to feeds (Wodzinski and Ullah, 1996; Han et al., 1997). An alternate approach would be to reduce the phytate levels in the seeds by genetically blocking one or more of the kinases in the synthetic pathway, either through antisense interference with specific enzymes or by the generation of random mutants. However, the InsP3 and InsP4 isomers implicated in the present work may have biological activities in seeds in addition to phytic acid synthesis. Ins(1,3,4)P3 is capable of mobilizing calcium from hypocotyl microsomes/vacuoles when complexed with mung bean phytase (Dasgupta et al., 1996). Ins(1,3,4,5)P4 can function in animal cells to permit the entry of calcium from the extracellular space (Woodcock, 1997). It is conceivable that these mechanisms of signal transduction may be involved in seed maturation and/or seedling growth and may be regulated to some extent by the synthesis of InsP6 during germination (Mandal and Biswas, 1970; Crans et al., 1995). Hence, blocking the synthetic pathway at a step close to InsP6 may be preferred so as to minimize any unfavorable impact on the physiological functions of the precursors.

Abbreviations:

InsP

InsP2, InsP3, InsP4, InsP5, and InsP6, myo-inositol mono-, bis-, tris-, tetrakis-, pentakis-, and hexakisphosphate, respectively, with appropriate numbering

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