From the Department of Physiology and Biophysics, University of California, Irvine, California 92697-4560
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The pathway and kinetics of inositol
1,4,5-trisphosphate (IP3) metabolism were measured in
Xenopus laevis oocytes and cytoplasmic extracts of oocytes.
Degradation of microinjected IP3 in intact oocytes was
similar to that in the extracts containing comparable concentrations of
IP3 ([IP3]). The rate and route of metabolism of IP3 depended on the [IP3] and the
intracellular free Ca2+ concentration
([Ca2+]). At low [IP3] (100 nM)
and high [Ca2+] (1 µM), IP3
was metabolized predominantly by inositol 1,4,5-trisphosphate 3-kinase
(3-kinase) with a half-life of 60 s. As the [IP3]
was increased, inositol polyphosphate 5-phosphatase (5-phosphatase) degraded progressively more IP3. At a [IP3]
of 8 µM or greater, the dephosphorylation of
IP3 was the dominant mode of IP3 removal irrespective of the [Ca2+]. At low [IP3]
and low [Ca2+] (both
400 nM), the
activities of the 5-phosphatase and 3-kinase were comparable. The
calculated range of action of IP3 in the oocyte was ~300
µm suggesting that IP3 acts as a global messenger in
oocytes. In contrast to IP3, inositol
1,3,4,5-tetrakisphosphate (IP4) was metabolized very
slowly. The half-life of IP4 (100 nM) was 30 min and independent of the [Ca2+]. IP4 may
act to sustain Ca2+ signals initiated by IP3.
The half-life of both IP3 and IP4 in Xenopus oocytes was an order of magnitude or greater than
that in small mammalian cells.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ca2+ signals regulate a diverse array of cellular
functions including secretion, cytoskeletal rearrangement, and gene
transcription (1, 2). Modulations in
[Ca2+]1 are
used to transduce signals in nearly all cells including those of plants
and animals (1-3). Generation and degradation of the second messenger
IP3, which opens the IP3
receptor/Ca2+ channel on the endoplasmic reticulum,
regulates the formation and termination of Ca2+ signals.
Knowing the rates and pathways of IP3 degradation is fundamental to understanding Ca2+ wave formation. The
metabolism of IP3 and its products by phosphatases and
kinases is unfolding as an increasingly complex process (4-6). Two
primary degradative pathways exist for IP3, but they differ in their relative importance among cell types and between species. Several isoforms of the 5-phosphatase dephosphorylate IP3
yielding inositol 1,4-bisphosphate (4-8). IP3 is also a
substrate of the 3-kinase which phosphorylates IP3 to form
IP4 (9-13). The binding of Ca2+/calmodulin to
the 3-kinase enhances its activity (14-16). The 5-phosphatase also
metabolizes IP4 to inositol 1,3,4-trisphosphate (I-1,3,4-P3). Current evidence suggests that the major
function of the 5-phosphatase in the phosphoinositide cycle is to
decrease the [IP3] and the concentration of
IP4 ([IP4]). In contrast, the role of the
3-kinase is to generate another second messenger, IP4, as
well as to decrease the [IP3]. An increasing amount of evidence suggests that IP4 is an important regulatory
molecule in cells (6). Like IP3, IP4 may be
involved in the regulation of the [Ca2+] (17-19).
IP4 binds to the IP3 receptor/Ca2+
channel and releases Ca2+ from the endoplasmic reticulum
although with a 10-fold lower potency than IP3. More
intriguing, IP4 binds with high affinity to several
intracellular proteins, synaptotagmin I and II, Gap1, Btk, and
centaurin- (20-24). The Ras GTPase-activity of Gap1 is stimulated
by IP4, and IP4 may interact with synaptotagmin
to inhibit synaptic transmission (20, 25). The pathway selected to
metabolize IP3 may not only influence its rate of removal
but also alter subsequent signal transduction within the cell.
Much of our mechanistic knowledge of Ca2+ wave propagation is derived from studies of Ca2+ waves in oocytes and eggs of Xenopus laevis. Formation of the Ca2+ wave, which follows the fertilization of an egg and which follows the activation of plasma-membrane receptors in oocytes, requires IP3 (26, 27). Work to date on IP3 metabolism in Xenopus oocytes has yielded conflicting results. Microinjection of concentrated [3H]IP3 into single Xenopus oocytes followed by separation of the metabolites by ion-exchange chromatography has suggested that the 5-phosphatase pathway prevailed (28). However, the addition of [3H]IP3 to a homogenate of oocytes or microinjection into ovarian follicles followed also by high pressure liquid chromatography (HPLC) separation have provided strong evidence for the 3-kinase as the primary route of IP3 degradation (29, 30). These discrepancies remain unresolved, despite the central role that the Xenopus oocyte has played in understanding Ca2+ wave propagation. All three of the studies discussed above found a surprisingly prolonged degradation time for IP3, 20 min or longer. This contrasts with smaller cells that degrade IP3 in a few seconds (31, 32). Establishing both the rate and pathway of IP3 metabolism in oocytes is required to understand the role of the phosphoinositide pathway in the generation and termination of Ca2+ signals and in interactions with other signal transduction cascades. The purpose of this investigation was to determine how IP3 and IP4 were degraded in X. laevis oocytes. Moreover, the goal was to quantitate the kinetic properties of these metabolic pathways in the cytoplasmic milieu.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials-- Tritiated inositol and inositol phosphates were purchased from NEN Life Science Products. Nonradioactive IP3 was purchased from Calbiochem and Alexis (Woburn, MA), and nonradioactive IP4 and calpain inhibitor I (N-Ac-Leu-Leu-norleucinal) were purchased from Calbiochem. Phorbol 12-myristate 13-acetate was supplied by Alexis (Woburn, MA). EGTA "puriss" grade, was obtained from Fluka (Ronkonkoma, NY). Rhod-2, BAPTA, and calcium green-5N were supplied by Molecular Probes (Eugene, OR). All other reagents were purchased from Fisher.
EGTA-buffered Ca2+ Solutions-- An equimolar solution of EGTA and Ca2+ was prepared by the method of Tsien and Pozzan (33). Cytoplasmic extracts with varying [Ca2+] were made by addition of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+. The [Ca2+] in the EGTA-buffered extracts was estimated from the [Ca2+] in buffer A (135 mM KCl, 5 mM NaCl, 1 mM MgCl2, 10 mM HEPES, pH 7.4) which approximated the intracellular ionic environment and contained the same mixture of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+ as the EGTA-buffered extract. The [Ca2+] in the buffer solution was measured using the fluorescent Ca2+ indicators, rhod-2 and calcium green-5N as described by Allbritton and colleagues (34) and Haugland (35). The [Ca2+] in buffer A containing 10 mM EGTA and 10 mM Ca2+ was 10 µM.
Preparation of Oocytes and Cytoplasmic Extracts-- Female X. laevis frogs were purchased from Nasco (Modesto, CA). Oocytes were surgically obtained and prepared as described previously (34, 36). Cytoplasmic extracts were also made as described previously with the following exceptions (34, 37). To minimize proteolysis the oocytes and extract were maintained at 4 °C throughout the preparation. Calpain inhibitor I (10 µg/ml) was added to the extract to block the Ca2+-activated protease calpain. After isolation, the cytoplasmic extract was used immediately in experiments. Prolonged delays prior to use diminished the ability of the extract to metabolize inositol phosphates. This cytoplasmic preparation is ~90% pure cytoplasm (90 mg/ml protein) (34).
Measurement of IP3 Degradation in Intact Oocytes-- Oocytes were microinjected with 5-30 nl of [3H]IP3 (2 µM) contained in buffer A with or without BAPTA (100 mM). The volume of injectate was determined from the total number of counts contained in the oocyte. The oocytes were incubated at room temperature for the indicated times. Just prior (~10 s) to the end of the incubation period, the buffer solution surrounding the oocyte was removed to determine how much of the tritium label had leaked from the oocyte. Cells that leaked greater than 10% of the radioactive label were excluded from experiments. The intracellular reactions of the oocyte were terminated by rapid freezing with powdered dry ice. The frozen oocyte was then homogenized in chloroform:methanol (50 µl of 1:2) and buffer B (50 µl of 25 mM tetrabutylammonium hydrogen sulfate, 20 mM KH2PO4, pH 3.5) with 80 µg of hydrolyzed phytic acid added as a carrier to prevent nonspecific loss of inositol phosphates. Hydrolyzed phytic acid was prepared as described by Irvine and colleagues (38). After addition of chloroform (50 µl), the mixture was centrifuged at 15,000 × g, and the aqueous phase was separated by reverse phase HPLC.
Measurement of Inositol Phosphate Degradation in Cytoplasmic Extracts-- [Ca2+] in the cytosol was buffered at the indicated concentration by addition of 10 mM EGTA and 10 mM EGTA with 10 mM Ca2+. In some instances, phorbol 12-myristate 13-acetate (50 ng/ml) was included in the cytoplasmic mixture. [3H]IP3 or [3H]IP4 was then added to the oocyte cytosol. After the addition of each reagent to the cytosolic extract, it was gently mixed over a 5-s period by pipetting the mixture up and down. The cytoplasmic extract was incubated for the indicated time at room temperature (~20 °C). The reaction was stopped with a 16-fold excess volume of trifluoroacetic acid (10%) containing hydrolyzed phytic acid (0.8 mg/ml) and centrifuged at 15,000 × g. The supernatant was dried, resuspended in buffer B, and separated by reverse phase HPLC.
Reverse Phase HPLC-- Inositol phosphates were separated on an analytical C18 column (Alltech, San Jose, CA) maintained at 45 °C with an elution protocol modified from that of Shayman and Bement (39) and Sulpice et al. (40). The flow rate was 1 ml/min. During the first 22 min, fractions were collected every 0.25 min and thereafter every 1.25 min. To monitor the separation efficiency of the column, a set of standards previously added to and then extracted from cytosol was chromatographed each day. [3H]Inositol coeluted with inositol 1-phosphate as did the other isomers of inositol phosphate. The isomers of inositol bisphosphate were not distinguishable.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To qualitatively determine the rate of IP3 metabolism in vivo, stage V/VI oocytes were microinjected with [3H]IP3 (2 µM) (n = 11). After incubation for 30 s, 1 min, or 5 min, the oocytes were rapidly frozen to terminate intracellular reactions. The inositol phosphates were extracted from the cytoplasm and separated by HPLC. Representative standard and experimental traces are shown in Fig. 1, A-C. Oocytes microinjected with small volumes of [3H]IP3 metabolized half of the [3H]IP3 in approximately 1 min (Fig. 1B). Longer times were required to degrade half of the [3H]IP3 when larger volumes were microinjected (Fig. 1C). In all of the experiments, the metabolic fraction that contained the most tritium was that of IP4 or IP3. In vivo and at an [IP3] of 2 µM and lower, IP3 was metabolized predominantly by the 3-kinase.
|
In other species the activity of the 3-kinase is increased by the binding of calmodulin and Ca2+ (11, 14-16). To determine whether [Ca2+] regulated the metabolism of IP3 in Xenopus oocytes, [3H]IP3 (2 µM) was coinjected with BAPTA (100 mM) (n = 10) to diminish the IP3-mediated increase in [Ca2+]. After a 1-, 5-, 15-, or 30-min incubation time, the inositol phosphates were extracted and separated. Remarkably, very little of the [3H]IP3 was metabolized by 5 min (Fig. 1D). The half-life of the microinjected IP3 was ~13 min. In contrast to the oocytes without BAPTA, substantial amounts of tritium did not accumulate in the IP4 fraction (Fig. 1, D and E). The activity of the 3-kinase was dramatically decreased in the oocytes containing BAPTA presumably due to a decrease in [Ca2+]. In these in vivo experiments, both diffusion and degradation altered [IP3] during the time course of the measurements. One minute after microinjection in the absence of degradation, the [IP3] at the microinjection site would still be 10 times greater than the final equilibrium concentration (41). Since [IP3] was also altered by diffusion, these experiments could not be used to measure quantitatively the rates and pathways of IP3 metabolism.
To provide a quantitative description of the metabolism of IP3, subsequent experiments were performed in a cytosolic extract. In past experiments, cytosolic preparations were greatly diluted during preparation, and the compartmentalization of proteins and membranes was abolished by homogenization (29, 30). To eliminate these disadvantages, we used a cytosolic extract that is ~90% of the concentration of undiluted cytoplasm (34). The cytosol contains nearly all of the intracellular organelles, and they retain many of their normal functions including the ability to transit through the cell cycle (34, 37, 42). These observations suggested that this extract could be used to define how [IP3] and [Ca2+] regulate the removal of IP3 in an environment similar to the cytoplasmic milieu. [3H]IP3 (100 nM) was added to a cytoplasmic extract that contained 10 mM EGTA with 10 mM Ca2+ ([Ca2+] ~10 µM). At varying times an aliquot of this cytosolic mixture was removed, and the inositol phosphates were extracted and chromatographed. The half-life of the [3H]IP3 was 60 s (Fig. 2B). Under these conditions most of the [3H]IP3 was converted to IP4. The half-life of IP4 was considerably longer than that of IP3. For incubation times of 5 min or less, the metabolism of IP4 was negligible (see also Fig. 4). By 10 min much of the tritium (~30-40%) initially added to the cytosol was no longer soluble after acid extraction; the [3H]inositol was recycled into a cellular component other than inositol or an inositol phosphate. These results are consistent with those obtained in the intact oocytes microinjected with [3H]IP3 suggesting that the cytosolic extract is a very good model for the intact oocyte.
|
For experimental time points of 5 min or less, the cytosolic reactions
of IP3 could be segregated into two distinct pathways without common metabolites (Fig. 2A). In the 3-kinase
pathway, IP4 accumulated with very little conversion of
IP4 to the lower inositol phosphates. Consequently,
[IP4] was used as an estimate of the activity of the
3-kinase for time points of 5 min or less. Inositol bisphosphate
(IP2) was then formed almost exclusively by
dephosphorylation of IP3 rather than by dephosphorylation
of I-1,3,4-P3. Inositol and inositol monophosphate
(IP1) were formed by subsequent dephosphorylation of that
IP2. The products of the 5-phosphatase pathway were
inositol, IP1, and IP2, and the sum of the
concentrations of these three inositols ([IP2]) was used to estimate the activity of the 5-phosphatase toward
IP3. For times less than 5 min, neither inositol,
IP1, nor IP2 accrued to a substantial degree
(Fig. 2B). Neither of these inositol fractions exceeded 10%
of the total tritium in the cytosol. The metabolite of IP4,
I-1,3,4-P3, did not accumulate either. The chromatographic peak representing this isomer never contained more than 6% of the
added tritium. Although both the 3-kinase and 5-phosphatase were active
in the extract, the conversion of IP3 to IP4
dominated at these low [IP3] and for incubation times
less than 5 min. Since the activity of the 5-phosphatase was much lower
than that of the 3-kinase, the kinetic properties of the 3-kinase were
estimated from the rate of formation of IP4. The kinetics
of this reaction appeared first order; the rate of formation of
IP4 was proportional to [IP3] (43). The first
order rate constant (k) obtained by fitting the
[IP4] versus time trace to an exponential was
10
2 s
1.
Since the prior in vivo experiments suggested that
[Ca2+] regulated the metabolism of IP3 in
oocytes, [3H]IP3 (100 nM) was
added to a cytoplasmic extract which contained 10 mM EGTA
decreasing [Ca2+] to less than 100 nM (Fig.
2C). Since the half-life of IP4 remained much
greater than 5 min (see also Fig. 4), the data were displayed identically to that in Fig. 2B. At low [Ca2+]
the half-life of IP3 was increased markedly to 10 min. In
agreement with the in vivo results, the longer half-life was
due to a decrease in the activity of the 3-kinase. Less than 7 and 25%
of the IP3 was converted to IP4 in 1 and 10 min, respectively. As before, I-1,3,4-P3 did not
accumulate. The rate of accrual of IP4 and IP2 was similar and linear over time (0.04 nM/s) and nearly identical to the rate of accumulation of
IP
2 in the presence of high [Ca2+] (Fig. 2,
B and C). Formation of IP
2 was
independent of [Ca2+] suggesting that the 5-phosphatase
of Xenopus oocytes was not regulated by
[Ca2+]. The unchanged production of
[IP
2], despite a markedly decreased [IP4], also suggests that the lower inositol phosphates
resulted predominantly from sequential dephosphorylation of
IP3 rather than IP4.
To determine the range of [Ca2+] that potentiated the 3-kinase's activity, we varied the [Ca2+] in the extract by altering the ratio of 10 mM EGTA to 10 mM EGTA with 10 mM Ca2+. After addition of [3H]IP3 (100 nM) to the cytosolic preparation for 1 min, the inositol phosphates were extracted and chromatographed. As the [Ca2+] was increased from <100 nM to >2 µM, the amount of IP3 degraded increased from 7 to 40 nM. Half-maximal activation of the 3-kinase occurred at a [Ca2+] of 390 nM and maximal activation appeared by 1 µM (Fig. 2D). Several previous reports indicated that the 5-phosphatase was activated after phosphorylation by protein kinase C, whereas other experiments suggested that the 5-phosphatase activity was inactivated by protein kinase C (44-48). Addition of phorbol 12-myristate 13-acetate (50 ng/ml), a potent activator of protein kinase C, to cytosolic extracts with varying [Ca2+] did not alter the rate or pathway of IP3 metabolism (data not shown). Under these conditions, activation of protein kinase C did not alter the dephosphorylation of IP3 by the 5-phosphatase or phosphorylation of IP3 by the 3-kinase.
The Michaelis constant (Km) of the 5-phosphatase for
IP3 is greater than 10 times that of the 3-kinase in many
different tissues (4, 5). Therefore, the metabolic pathway of
IP3 may depend on [IP3]. To determine how the
route of degradation in Xenopus oocytes changed with
[IP3], we varied the starting [IP3] in the
cytosolic preparation from 100 nM to 30 µM.
[3H]IP3 was incubated in the extract for 5 min followed by extraction and separation of the inositol phosphates.
In the first series of experiments, [Ca2+] was maintained
at 10 µM by addition of 10 mM EGTA with 10 mM Ca2+. For a starting [IP3] of
less than 8 µM, the major metabolic pathway was
conversion to IP4 by the 3-kinase (Fig.
3A). At an initial
[IP3] of approximately 2 µM and greater,
the rate of formation of IP4 was independent of
[IP3] consistent with [IP3] being much greater than the Km of the 3-kinase. The rate of
generation of IP4 at this high [IP3] was used
to estimate the maximal velocity (Vmax) of the
3-kinase for IP3 (4 nM/s). For a starting
[IP3] greater than 8 µM,
[IP2] continued to increase despite a plateau in the
generation of IP4 (Fig. 3A). The additional
IP
2 must originate from dephosphorylation of
IP3 by the 5-phosphatase. At high [IP3], most
of the IP
2 was formed by dephosphorylation of
IP3 rather than IP4; IP3 was
degraded chiefly by the 5-phosphatase.
|
To determine how a decrease in [Ca2+] altered
IP3 metabolism at different [IP3], the
initial [IP3] was varied from 100 nM to 30 µM in a cytosolic preparation containing 10 mM EGTA (Fig. 3B). The
[3H]IP3 was incubated in the extract for 5 min followed by acid extraction and chromatographic separation. For all
starting [IP3], the rate of formation of IP4
was greatly decreased compared with that in the presence of micromolar
[Ca2+]. In contrast, the rate of accrual of
[IP2] was nearly unchanged from that in the presence of
high [Ca2+]. As before the 5-phosphatase was independent
of [Ca2+]. Moreover, [IP
2] did not depend
on [IP4] indicating that these lower inositol phosphates
resulted mainly from the action of the 5-phosphatase on
IP3. The rate formation of IP4 became independent of [IP3] when the initial [IP3]
was ~10 µM or greater. This rate was used to estimate
the Vmax of the 3-kinase at low [Ca2+] (1 nM/s). For most initial
[IP3], IP3 was degraded mainly by dephosphorylation, and the rate of formation of [IP
2]
was linearly related to the initial [IP3] (Fig.
3B). For an initial [IP3] of 1 µM or greater, [IP3] decreased negligibly
during the course of the reaction (
20%). Since [IP3]
was nearly constant during the reaction time and [IP
2]
was linearly related to the starting [IP3], the reaction
of the 5-phosphatase with IP3 was modeled as a first order
reaction for [IP3] greater than 1 µM. The
k of the 5-phosphatase for IP3 (6 × 10
4 s
1) was then estimated from the rate of
formation of [IP
2]. When [Ca2+] was in
the micromolar range, the metabolic pathway of IP3 depended on [IP3]. At low initial [IP3],
IP3 was metabolized predominantly by the 3-kinase, whereas
at high initial [IP3], the 5-phosphatase dominated. The
crossover point from the 3-kinase to the 5-phosphatase occurred at an
[IP3] of approximately 8 µM.
Since IP4 may be an important signaling molecule, the rate
and pathway of IP4 degradation were determined.
[3H]IP4 (100 nM or 10 µM) was added to a cytosolic preparation containing 10 mM EGTA with 10 mM Ca2+. At the
indicated times the inositol phosphates were extracted from an aliquot
of the reaction mixture and separated. For both initial
[IP4], 50% of the [3H]IP4 was
degraded by dephosphorylation in 30 min (Fig.
4). After a 5-min reaction time, only 5%
of the IP4 was metabolized. For reaction times of 5 min or
less, destruction of IP4 was negligible, and the reaction
scheme depicted in Fig. 2A for IP3 was valid. Varying the [Ca2+] in the extract from <100
nM to 10 µM did not alter the rate of
degradation of IP4. As in prior experiments,
I-1,3,4-P3 did not accumulate to a substantial degree
although [IP2] increased with time. Conversion of
IP4 to IP3 was not observed. Metabolism of
IP4 and formation of IP
2 followed first order
kinetics for initial IP4 concentrations of 100 nM and 10 µM. The k obtained by
fitting the [IP4] or [IP
2] curves to an
exponential was 4 × 10
4 s
1.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intact oocytes and a cytoplasmic extract of oocytes were used to
determine the pathways and estimate the rates of IP3 and IP4 metabolism in Xenopus oocytes. The pathway
of IP3 degradation depended on both [IP3] and
[Ca2+]. The 3-kinase was regulated by
[Ca2+]; the measured Vmax and
k of the 3-kinase decreased as [Ca2+]
decreased. Lowering [Ca2+] from 1 µM to
less than 100 nM diminished the k and the
Vmax by a factor of 25 and 4, respectively. The
k of the 3-kinase for IP3 in the presence of 10 mM EGTA ([Ca2+] < 100 nM) was
estimated from the slope of the IP4 trace in Fig. 2C and the initial slope of the IP4 trace in
Fig. 3B (k = 4 × 104
s
1). The Km calculated from the
Vmax and k increased from 400 nM to 2.5 µM as [Ca2+] declined
from greater than 1 µM to less than 100 nM.
[Ca2+] regulated the 3-kinase by altering both the
Vmax and Km of the enzyme for
IP3. By releasing Ca2+, IP3
enhances its own degradation. The Km values of the
3-kinase and its regulation by Ca2+ in Xenopus
oocytes are close to that reported in other species (4, 5, 10-12).
These similar enzymatic features suggest that the Xenopus
oocyte 3-kinase is regulated by calmodulin as is the case in other
species.
The k value of the 5-phosphatase for IP3 was
approximately twice that of the 3-kinase when [Ca2+] was
low (~100 nM). However, when [Ca2+] was
greater than 1 µM, the k value of the 3-kinase
exceeded that of the 5-phosphatase by almost 20-fold. Although the
Vmax of the 5-phosphatase for IP3
could not be determined, the Vmax must be
greater than 16 nM s1, the fastest rate
measured for the 5-phosphatase (Fig. 3). The Km of
the 5-phosphatase for IP3 must then be greater than 27 µM. The first order behavior of the 5-phosphatase at
IP3 concentrations up to 30 µM also suggests
that the Km is greater than 30 µM. The
lower limit of the Km value and the absence of
regulation by [Ca2+] are consistent with the properties
of the 5-phosphatase in other species (4-8, 48-50). For all
[Ca2+], the Km and
Vmax values of the 5-phosphatase for
IP3 were much greater than those of the 3-kinase for
IP3. The relative values of the Km and
Vmax of the 5-phosphatase and 3-kinase divided
IP3 metabolism into three regions. The regions were defined by their [Ca2+] and [IP3] and were
characterized by their different metabolic routes for IP3.
At high [Ca2+] (
400 nM) but low
[IP3] (
8 µM), IP3 was
predominantly metabolized by the 3-kinase to IP4. The
5-phosphatase and the 3-kinase degraded roughly similar amounts of
IP3 when both [Ca2+] and [IP3]
were low (<400 nM and <1 µM, respectively).
However, the 5-phosphatase was the dominant metabolic enzyme when
[IP3] was greater than 8 µM irrespective of
[Ca2+]. The regions that occur physiologically are not
known since the intracellular range of [IP3] has yet to
be defined.
IP4 was metabolized very slowly with a half-life of approximately 30 min. The k value of the 5-phosphatase for IP4 was similar to that for IP3. The reported Km of the 5-phosphatase for IP4 in other species is approximately 1 µM (7, 51). When the initial [IP4] was 10 µM, the metabolism of IP4 followed first order kinetics. Therefore, it is unlikely that the Km of the 5-phosphatase for IP4 in Xenopus oocytes was as low as 1 µM. When [Ca2+] was elevated, phosphorylation of IP3 to IP4 was much faster than degradation of IP4. Consequently, repeated or persistent stimulation of IP3 production in the presence of high nanomolar to micromolar [Ca2+], could result in IP4 concentrations that are far greater than that of IP3. Although opening of the IP3 receptor/Ca2+ channel requires 10-fold more IP4 than IP3, the accumulation of IP4 may result in physiologically important IP4-mediated Ca2+ release. IP4 may play a necessary role in sustained signal transduction.
The k and Vmax values of the
oocyte's 3-kinase and 5-phosphatase were markedly lower than the
values reported in other species (4-12, 48-50). Since IP3
microinjected into intact oocytes was also metabolized slowly, the long
IP3 degradation times were not an artifact of the cytosolic
preparation. Additionally, IP4 microinjected into intact
oocytes has a half-life similar to that reported here in the cytosolic
extract (29, 30). The lower k and
Vmax values of the Xenopus oocyte
compared with those of mammalian cells may be a reflection of the
different size, temperature, and physiologic role of these cells.
Slowing the degradation of IP3 may be important for signal
transduction from the cell's surface to its interior. The 500-µm
radius of the oocyte makes the transmission of information from the
plasma membrane to the central core formidable. Slowing the metabolism
of IP3 increases its range of action, the distance over
which a spatially localized impulse of IP3 can diffuse
(34). The range of action (R) of IP3 can be
estimated from its diffusion coefficient (D) and half-life
(t1/2) in cytoplasm: r = (2*D*t1/2)1/2.
D for IP3 is 280 µm2
s1, and the t1/2 for IP3
in the oocyte ranges from 1 to greater than 5 min. R for
IP3 is ~300 µm suggesting that IP3 acts as
a long-ranged messenger in the oocyte. In nearly all cell types,
IP3 is a global messenger (Table
I). Whereas the rate of degradation of
IP3 and the size of a cell vary over several orders of
magnitude, the range of action of IP3 is nearly the same as
the cellular dimensions. IP3 transmits signals received at
the plasma membrane throughout the cell, coordinating cell-wide
activities.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Tobias Meyer and Lubert Stryer for stimulating discussions and H. N. Dao for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by the Arnold and Mabel Beckman Foundation, the Searle Scholars Program, and National Institute of Mental Health Grant MH45324.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 714-824-6493;
Fax: 714-824-8540.
1
The abbreviations used are:
[Ca2+], free Ca2+ concentration;
I-1-P1, inositol 1-phosphate; IP1, all isomers
of inositol monophosphate; I-1,4-P2, inositol
1,4-bisphosphate; IP2, all isomers of inositol bisphosphate; IP3, inositol 1,4,5-trisphosphate;
I-1,3,4-P3, inositol 1,3,4-trisphosphate; IP4,
inositol 1,3,4,5-tetrakisphosphate; [IP3], concentration
of IP3; [IP4], concentration of
IP4; IP2, inositol, IP1, and
IP2; [IP
2], the sum of the concentration of
inositol, IP1 and IP2; 5-phosphatase, inositol
polyphosphate 5-phosphatase; 3-kinase, inositol 1,4,5-trisphosphate
3-kinase; HPLC, high pressure liquid chromatography; BAPTA,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|