Metabolism of Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate by the Oocytes of Xenopus laevis
1998; Elsevier BV; Volume: 273; Issue: 7 Linguagem: Inglês
10.1074/jbc.273.7.4052
ISSN1083-351X
AutoresChristopher E. Sims, Nancy L. Allbritton,
Tópico(s)Genetics, Aging, and Longevity in Model Organisms
ResumoThe 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), IP3was 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. 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), IP3was 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. Ca2+ signals regulate a diverse array of cellular functions including secretion, cytoskeletal rearrangement, and gene transcription (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6186) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2271) Google Scholar). Modulations in [Ca2+] 1The 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; IP≤2, 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. are used to transduce signals in nearly all cells including those of plants and animals (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6186) Google Scholar, 2Clapham D.E. Cell. 1995; 80: 259-268Abstract Full Text PDF PubMed Scopus (2271) Google Scholar, 3Allen G.J. Muir S.R. Sanders D. Science. 1995; 268: 735-737Crossref PubMed Scopus (305) Google Scholar). Generation and degradation of the second messenger IP3, which opens the IP3receptor/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 (4Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholar, 5Shears S.B. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 63-92PubMed Google Scholar, 6Irvine R.F. Biochem. Soc. Trans. 1995; 23: 27-35Crossref PubMed Scopus (17) Google Scholar). 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 IP3yielding inositol 1,4-bisphosphate (4Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholar, 5Shears S.B. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 63-92PubMed Google Scholar, 6Irvine R.F. Biochem. Soc. Trans. 1995; 23: 27-35Crossref PubMed Scopus (17) Google Scholar, 7Mitchell C.A. Connolly T.M. Majerus P.W. J. Biol. Chem. 1989; 264: 8873-8877Abstract Full Text PDF PubMed Google Scholar, 8Hansen C.A. Johanson R.A. Williamson M.T. Williamson J.R. J. Biol. Chem. 1987; 262: 17319-17326Abstract Full Text PDF PubMed Google Scholar). IP3 is also a substrate of the 3-kinase which phosphorylates IP3 to form IP4 (9Irvine R.F. Letcher A.J. Heslop J.P. Berridge M.J. Nature. 1986; 320: 631-634Crossref PubMed Scopus (400) Google Scholar, 10Biden T.J. Wollheim C.B. J. Biol. Chem. 1986; 261: 11931-11934Abstract Full Text PDF PubMed Google Scholar, 11Ryu S.H. Lee S.Y. Lee K.Y. Rhee S.G. FASEB J. 1987; 1: 388-393Crossref PubMed Scopus (54) Google Scholar, 12Johanson R.A. Hansen C.A. Williamson J.R. J. Biol. Chem. 1988; 263: 7465-7471Abstract Full Text PDF PubMed Google Scholar, 13Daniel J.L. Dangelmaier C.A. Smith J.B. Biochem. J. 1988; 253: 789-794Crossref PubMed Scopus (11) Google Scholar). The binding of Ca2+/calmodulin to the 3-kinase enhances its activity (14Biden T.J. Peter-Riesch B. Schlegel W. Wollheim C.B. J. Biol. Chem. 1987; 262: 3567-3571Abstract Full Text PDF PubMed Google Scholar, 15Yamaguchi K. Hirata M. Kuriyama H. Biochem. J. 1987; 244: 787-791Crossref PubMed Scopus (32) Google Scholar, 16Morris A.J. Downes C.P. Hardin T.K. Mitchell R.H. Biochem. J. 1987; 248: 489-493Crossref PubMed Scopus (31) Google Scholar). 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 (6Irvine R.F. Biochem. Soc. Trans. 1995; 23: 27-35Crossref PubMed Scopus (17) Google Scholar). Like IP3, IP4 may be involved in the regulation of the [Ca2+] (17Loomis-Husselbee J.W. Cullen P.J. Dreikausen U.E. Irvine R.F. Dawson A.P. Biochem. J. 1996; 314: 811-816Crossref PubMed Scopus (39) Google Scholar, 18Parker I. Ivorra I. J. Physiol. (Lond .). 1991; 433: 207-227Crossref Scopus (30) Google Scholar, 19Ferguson J.E. Han J.K. Kao J.P.Y. Nuccitelli R. Exp. Cell Res. 1991; 192: 352-365Crossref PubMed Scopus (19) Google Scholar). 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-α (20Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar, 21Fukuda M. Aruga J. Niinobe M. Aimoto S. Mikoshiba K. J. Biol. Chem. 1994; 269: 29206-29211Abstract Full Text PDF PubMed Google Scholar, 22Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6262Crossref PubMed Scopus (495) Google Scholar, 23Fukuda M. Kojima T. Kabayama H. Mikoshiba K. J. Biol. Chem. 1996; 271: 30303-30306Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 24Hammonds-Odie L.P. Jackson T.R. Profit A.A. Blader I.J. Turck C.W. Prestwich G.D. Theibert A.B. J. Biol. Chem. 1996; 271: 18859-18868Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The Ras GTPase-activity of Gap1 is stimulated by IP4, and IP4 may interact with synaptotagmin to inhibit synaptic transmission (20Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar, 25Llinas R. Sugimori M. Lang E.J. Morita M. Fukuda M. Niinobe M. Mikoshiba K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12990-12993Crossref PubMed Scopus (88) Google Scholar). 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 (26Nuccitelli R. Yim D.L. Smart T. Dev. Biol. 1993; 158: 200-212Crossref PubMed Scopus (179) Google Scholar, 27Lechleiter J. Girard S. Peralta E. Clapham D. Science. 1991; 252: 123-126Crossref PubMed Scopus (575) Google Scholar). 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 (28Shapira H. Lupu-Meiri M. Oron Y. J. Basic Clin. Physiol. Pharmacol. 1992; 3: 119-128Crossref PubMed Scopus (8) Google Scholar). 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 (29McIntosh R.P. McIntosh J.E.A. Biochem. J. 1990; 268: 141-145Crossref PubMed Scopus (8) Google Scholar, 30McIntosh R.P. McIntosh J.E.A. Biochem. Biophys. Res. Commun. 1990; 166: 380-386Crossref PubMed Scopus (5) Google Scholar). 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 (31Wang S.S. Alousi A.A. Thompson S.H. J. Gen. Physiol. 1995; 105: 149-171Crossref PubMed Scopus (99) Google Scholar, 32Burgess G.M. McKinney J.S. Irvine R.F. Putney J.W. Biochem. J. 1985; 232: 237-243Crossref PubMed Scopus (114) Google Scholar). 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. 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. An equimolar solution of EGTA and Ca2+ was prepared by the method of Tsien and Pozzan (33Tsien R. Pozzan T. Methods Enzymol. 1989; 172: 230-262Crossref PubMed Scopus (399) Google Scholar). 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 mmEGTA 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 (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar) and Haugland (35Haugland R. Spence M.T.Z. Handbook of Fluorescent Probes and Research Chemicals. 6th Ed. Molecular Probes, Inc., Eugene, OR1996: 511-518Google Scholar). The [Ca2+] in buffer A containing 10 mm EGTA and 10 mmCa2+ was 10 μm. FemaleX. laevis frogs were purchased from Nasco (Modesto, CA). Oocytes were surgically obtained and prepared as described previously (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar, 36Zagotta W.N. Hoshi T. Aldrich R.W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7243-7247Crossref PubMed Scopus (122) Google Scholar). Cytoplasmic extracts were also made as described previously with the following exceptions (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar, 37Murray A.W. Kirschner M.W. Nature. 1989; 339: 275-280Crossref PubMed Scopus (868) Google Scholar). 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) (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar). 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 (38Wreggett K.A. Howe L.R. Moore J.P. Irvine R.F. Biochem. J. 1987; 245: 933-934Crossref PubMed Scopus (63) Google Scholar). After addition of chloroform (50 μl), the mixture was centrifuged at 15,000 ×g, and the aqueous phase was separated by reverse phase HPLC. [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. 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 (39Shayman J.A. Bement D.M. Biochem. Biophys. Res. Commun. 1988; 151: 114-122Crossref PubMed Scopus (24) Google Scholar) and Sulpice et al. (40Sulpice J.C. Bachelot C. Gascard P. Giraud F. Irvine R.F. Methods in Inositide Research. Raven Press, New York1990: 45-63Google Scholar). 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. To qualitatively determine the rate of IP3 metabolismin 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.1 B). Longer times were required to degrade half of the [3H]IP3 when larger volumes were microinjected (Fig. 1 C). 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, IP3was metabolized predominantly by the 3-kinase. In other species the activity of the 3-kinase is increased by the binding of calmodulin and Ca2+ (11Ryu S.H. Lee S.Y. Lee K.Y. Rhee S.G. FASEB J. 1987; 1: 388-393Crossref PubMed Scopus (54) Google Scholar, 14Biden T.J. Peter-Riesch B. Schlegel W. Wollheim C.B. J. Biol. Chem. 1987; 262: 3567-3571Abstract Full Text PDF PubMed Google Scholar, 15Yamaguchi K. Hirata M. Kuriyama H. Biochem. J. 1987; 244: 787-791Crossref PubMed Scopus (32) Google Scholar, 16Morris A.J. Downes C.P. Hardin T.K. Mitchell R.H. Biochem. J. 1987; 248: 489-493Crossref PubMed Scopus (31) Google Scholar). 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.1 D). 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 vivoexperiments, 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 (41Crank J. The Mathematics of Diffusion. Clarendon Press, Oxford1989: 29Google Scholar). Since [IP3] was also altered by diffusion, these experiments could not be used to measure quantitatively the rates and pathways of IP3metabolism. 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 (29McIntosh R.P. McIntosh J.E.A. Biochem. J. 1990; 268: 141-145Crossref PubMed Scopus (8) Google Scholar, 30McIntosh R.P. McIntosh J.E.A. Biochem. Biophys. Res. Commun. 1990; 166: 380-386Crossref PubMed Scopus (5) Google Scholar). To eliminate these disadvantages, we used a cytosolic extract that is ∼90% of the concentration of undiluted cytoplasm (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar). 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 (34Allbritton N.L. Meyer T. Stryer L. Science. 1992; 258: 1812-1815Crossref PubMed Scopus (919) Google Scholar, 37Murray A.W. Kirschner M.W. Nature. 1989; 339: 275-280Crossref PubMed Scopus (868) Google Scholar, 42Newmeyer D.D. Wilson K.L. Methods Cell Biol. 1991; 36: 607-634Crossref PubMed Scopus (130) Google Scholar). 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]IP3was 60 s (Fig. 2 B). 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.Figure 4Metabolism of IP4 by cytoplasmic extracts of oocytes. [3H]IP4 (10 μm) was added to a cytoplasmic extract which was then incubated for varying times. The inositol phosphates were isolated and quantitated. The concentrations of IP≤2 (▴, ▵), IP4 (•, ○), and I-1,3,4-P3 (▪, □) are plotted against the incubation time. [Ca2+] in the extract was buffered to 10 μm (▵, ○, □) or to less than 100 nm (▴, •, ▪). The lines through the IP4 and IP≤2 data points are fits to an exponential function, and the line through the I-1,3,4-P3 data points is hand drawn.View Large Image Figure ViewerDownload (PPT) 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. 2 A). 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 ([IP≤2]) 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. 2 B). 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 IP4dominated 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] (43Segel I.H. Biochemical Calculations. 2nd Ed. John Wiley and Sons, New York1975: 225-229Google Scholar). 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.2 C). 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. 2 B. 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 IP≤2 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 mmEGTA 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 2 μm, the amount of IP3degraded 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. 2 D). 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 (44Biden T.J. Valler L. Wollheim C.B. Biochem. J. 1988; 251: 435-440Crossref PubMed Scopus (29) Google Scholar, 45King W.G. Rittenhouse S.E. J. Biol. Chem. 1989; 264: 6070-6074Abstract Full Text PDF PubMed Google Scholar, 46Connolly T.M. Lawing W.J. Majerus P.W. Cell. 1986; 46: 951-958Abstract Full Text PDF PubMed Scopus (199) Google Scholar, 47Lin A.N. Barnes S. Wallace R.W. Biochem. Biophys. Res. Commun. 1990; 170: 1371-1376Crossref PubMed Scopus (13) Google Scholar, 48Sim S.S. Kim J.W. Rhee S.G. J. Biol. Chem. 1990; 265: 10367-10372Abstract Full Text PDF PubMed Google Scholar). 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 (4Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholar, 5Shears S.B. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 63-92PubMed Google Scholar). 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.3 A). 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, [IP≤2] continued to increase despite a plateau in the generation of IP4 (Fig. 3 A). 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. 3 B). The [3H]IP3 was incubated in the extract for 5 min followed by acid extraction and chromatographic separ
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