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Plant Physiol. (1998) 116: 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*
United States Department of Agriculture, Agricultural Research
Service, Southern Regional Research Center, 1100 Robert E. Lee
Boulevard, New Orleans, Louisiana 70124
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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 mg1 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 [ -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.
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
[-32P]ATP (3000 Ci/mmol),
[ -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  -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 -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 min1,
[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 -mercaptoethanol, 0.1 mm PMSF, 5 mm MgCl2, and 520 pm [-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 [-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.
[-32P]ATP was determined by Cerenkov
counting, and the [3H] fractions were counted
with 5 mL of ScintiSafe Econo 2 liquid-scintillation cocktail.
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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 [-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).
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| Figure 1.
Kinase activity of an immature soybean seed
extract utilizing [-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 [-32P]ATP (3000 Ci/mmol), 5 mm MgCl2, 5 mm EGTA, 50 mm KCl, 5% glycerol, 5 mm -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.
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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
[-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 [-32P]ATP was
replaced with [-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.
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| Figure 2.
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 -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.
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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.
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).
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| Figure 3.
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 -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.
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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.
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| Figure 4.
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 ``Materials and 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 -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.
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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).
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| Figure 5.
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.
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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
[-32P]ATP as a substrate. However, unlike
[3H]-labeled inositol polyphosphates, which are
relatively easy to analyze on anion-exchange columns,
[-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 [-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 [-32P]ATP as
the labeled substrate. Since the suspected InsP4
peak formed from [-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.
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FOOTNOTES |
*
E-mail bqphil{at}nola.srrc.usda.gov; fax 1-504-286-4419.
Received June 2, 1997;
accepted September 18, 1997.
| |
ABBREVIATIONS |
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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Top
Abstract
Introduction