Proc. Natl. Acad. Sci. USAVol. 84, pp. 2170-2174, April 1987Biochemistry

Pathway for inositol 1,3,4-trisphosphate and1,4-bisphosphate metabolismROGER C. INHORN, VINAY S. BANSAL, AND PHILIP W. MAJERUS*Division of Hematology-Oncology, Departments of Internal Medicine and Biological Chemistry, Washington University School of Medicine,St. Louis, MO 63110

Contributed by Philip W. Majerus, December 8, 1986

ABSTRACT We prepared [3H]inositol-, 3-[32P]phosphate-and 4-[32P]phosphate-labeled inositol phosphate substrates toinvestigate the metabolism of inositol 1,3,4-trisphosphate andinositol 1,4-bisphosphate. In crude extracts of calf brain,inositol 1,3,4-trisphosphate is first converted to inositol 3,4-bisphosphate, then the inositol 3,4-bisphosphate intermediateis further converted to inositol 3-phosphate. Similarly, inositol1,4-bisphosphate is converted to inositol 4-phosphate, and noinositol 1-phosphate is formed. We partially purified an en-zyme that we tentatively name inositol polyphosphate 1-phosphatase. This cytosolic enzyme converts inositol 1,3,4-trisphosphate to inositol 3,4-bisphosphate and also convertsinositol 1,4-bisphosphate to inositol 4-phosphate. The enzymedoes not utilize inositol 1,3,4,5-tetrakisphosphate, inositol1,4,5-trisphosphate, or inositol 1-phosphate as substrates.Thus we propose a new scheme for inositol phosphate metab-olism. According to this pathway inositol 1,4,5-trisphosphateand inositol 1,4-bisphosphate are degraded to inositol 4-phosphate. Inositol 1-phosphate, which is the major inositolmonophosphate formed in stimulated brain, is derived eitherfrom phospholipase C cleavage of phosphatidylinositol or fromthe degradation of inositol cyclic phosphates.

Inositol phospholipids are membrane lipids that are rapidlymetabolized in response to extracellular agonists to liberateseveral second messenger molecules and precursors of mes-senger molecules (for review, see refs. 1-3). A phospholipaseC enzyme hydrolyzes all three inositol phospholipids (4) toyield mixtures of cyclic and noncyclic phosphate esters (5-7),namely, inositol 1-phosphate (Ins 1P), inositol 1,4-bisphos-phate [Ins(1,4)P2], inositol 1,4,5-trisphosphate [Ins(1,4,5)P3],and the (1:2) cyclic counterparts of each. The metabolism ofthe water-soluble inositol phosphates is complex. An inositolphosphate 5-phosphomonoesterase converts Ins(1,4,5)P3and its cyclic counterpart to Ins(1,4)P2 (8-10) and cyclicinositol 1:2,4-bisphosphate (11), respectively. Additionally,Ins(1,4,5)P3 is metabolized to inositol 1,3,4,5-tetrakisphos-phate [Ins(1,3,4,5)P4] by a 3-kinase and subsequently con-verted to a different inositol trisphosphate isomer, inositol1,3,4-trisphosphate [Ins(1,3,4)P3], by the same 5-phospho-monoesterase (12-17).

In the current study, we have investigated the furthermetabolism of Ins(1,3,4)P3 in crude extracts from calf brainutilizing substrates with radiolabels in the inositol ring, the3-phosphate, or the 4-phosphate. We find that inositol 3,4-bisphosphate [Ins(3,4)P2] is the first product formed fromIns(1,3,4)P3. In addition, we find that the enzyme that utilizesthis substrate also converts Ins(1,4)P2 to inositol 4-phosphate(Ins 4P). Based on these results, a new pathway for inositolphosphate metabolism is proposed.

MATERIALS AND METHODSMaterials. [3H]Ins(1,4)P2 and [3H]Ins(1,4,5)P3 were from

New England Nuclear. [-32P]ATP was from Amersham.Matrix gel PBA-60 (phenyl boronate agarose) was fromAmicon. DEAE-Sepharose CL-6B was from Sigma. TheBio-Gel TSK DEAE-5 PW HPLC column, AG 1-X8 Dowex(formate form, 200-400 mesh) and Chelex 100 (sodium form,200-400 mesh) were from Bio-Rad. The Whatman Partisil 10SAX column was from Cobert Associates (St. Louis, MO).All other materials were from Sigma or Fisher.

Preparation of Radiolabeled Ins 1P, Ins(1,4)P2, andIns(1,4,5)P3. [3H]Ins 1P was prepared by incubation of lipidvesicles containing phosphatidyl[3H]inositol with ram semi-nal vesicle phospholipase C (18, 19). Ins(1,4-[4-32P])P2 andIns(1,4,5-[4,5-32P])P3 were prepared from erythrocytes asdescribed by Downes et al. (9).

Preparation of Unlabeled Ins(1,4,5)P3. Unlabeled Ins(1,4,5)-P3 was prepared from Folch fraction I (Sigma) by the methodof Irvine et al. (20) as detailed (21).Dowex Chromatography of Inositol Phosphates. Reaction

mixtures were diluted to 1 ml with H20 containing 50 nmolPi and poured onto a 1.5 x 11 cm Dowex-formate columnequilibrated in 50 mM NH4COOH (pH 3.45). Columns wereeluted with linear gradients (200 ml) of 0.05-1.5 MNH4COOH. Fractions (2 ml) were collected, and radioactiv-ity was estimated by liquid scintillation counting withScintiverse I.Rat Brain Homogenate. Crude rat brain cytosol was pre-

pared by the method of Irvine et al. (14) as described (17).Briefly, rat brain cortex in 160 mM sucrose/10 mM Hepes,pH 7.5, was homogenized with a Teflon-glass homogenizer,and the homogenate was centrifuged at 50,000 x g for 30 min.The supernatant was used as a crude source ofboth Ins(1,4,5)-P3 3-kinase and inositol phosphate 5-phosphomonoesterase.In some cases the 3-kinase was partially purified as described(17).

Preparation ofRadiolabeled Ins(1,3,4,5)P4 and Ins(1,3,4)P3.Reaction conditions were found in which incubation ofIns(1,4,5)P3 and ATP with crude rat brain cytosol yieldedprimarily Ins(1,3,4,5)P4 at early time points. Ins(1,3,4,5)P4was further converted to Ins(1,3,4)P3 with longer incuba-tions.

3H-labeled substrates. Tritium-labeled Ins(1,3,4,5)P4 andIns(1,3,4)P3 were prepared by incubating [3H]Ins(1,4,5)P3 (3nmol, 0.2 ,ACi/nmol; 1 Ci = 37 GBq) in 50 Al containing 50mM glycine-NaOH (pH 10.0), 20mM MgCl2, 10mM ATP, 40mM Na2HPO4, and crude rat brain cytosol (50 ,ug of protein)

Abbreviations: Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins 1P,inositol 1-phosphate; Ins(1,4)P2, inositol 1,4-bisphosphate;Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; Ins(1,3,4)P3,inositol 1,3,4-trisphosphate; Ins 3P, inositol 3-phosphate; Ins 4P,inositol 4-phosphate.*To whom reprint requests should be addressed at: Division ofHematology-Oncology, Washington University School of Medi-cine, 660 South Euclid, St. Louis, MO 63110.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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for 45 min at 370C. The products of this reaction wereseparated by Dowex-formate chromatography (17) usingIns(1,4,5-[4,5-32P])P3 as an internal standard. Approximately2/3 of the radioactivity was recovered in peaks migrating inthe positions of Ins(1,3,4)P3 and Ins(1,3,4,5)P4 with equalamounts of both products. These products result from the3-kinase and 5-phosphatase activities in this preparation. The[3H]Ins(1,3,4,5)P4 product was further characterized by con-verting a sample of it to [3H]Ins(1,3,4)P3 with the 5-phospha-tase enzyme from platelets (10). The [3H]Ins(1,3,4)P3 wascharacterized by showing that it was resistant to 5-phosphomonoesterase and that it yielded altritol after period-ate oxidation, borohydride reduction, and dephosphorylation(13, 17, 20).4-Labeled Ins(1,3,4)P3. Ins(1,3,4-[4-32P])P3 was prepared

in the same way except that Ins(1,4,5-[4,5-32P])P3 preparedfrom erythrocyte ghosts was used as the initial substrate. Theerythrocyte substrate is labeled with 32p in both the 4- and5-phosphates, with -2/3 of the radioactivity in the 5-position(9). The 5-phosphate is removed by the 5-phosphomonoes-terase activity in the rat brain cytosol, thereby yieldingIns(1,3,4)P3 specifically labeled in the 4-position. Startingwith 90 nmol of Ins(1,4,5-[4,5-32P])P3 (250,000 cpm,Cerenkov) we obtained 35,000 cpm in Ins(1,3,4-[4-32P])P3product. This reflects =40% yield with a specific activity ofabout 900 cpm/nmol.3-Labeled Ins(1,3,4)P3. [3H]Ins(1,3,4-[3-32P])P3 was pre-

pared using purified 3-kinase and 5-phosphomonoesteraseenzymes. [y-32P]ATP (0.33 uCi/nmol) was applied to a 2-mlphenyl boronate agarose column to remove radiocontami-nants from the ATP. The column was washed with 12 ml of0.2 M triethylammonium bicarbonate, pH 8.8, containing 15mM MgCl2. ATP was eluted with H20 and lyophilized.[3H]Ins(1,4,5)P3 (3 nmol, 0.33 ,Ci/nmol) and [y- 2P]ATP(0.25 ,umol) were incubated at 37°C for 10 min in 50 ,ul of 50mM Tris maleate (pH 7.5), 10 mM MgCl2, 100 mM sodiumphosphate (pH 7.5), and partially purified inositol trisphos-phate 3-kinase (17). The reaction was stopped by adding 1 mlof 50 mM NH4COOH (pH 3.45), and the products wereseparated by Dowex-formate chromatography (17). Frac-tions containing Ins(1,3,4,5)P4 were pooled, diluted to 200 mlwith H20, then rechromatographed on Dowex-formate tofurther resolve the [3H]Ins(1,3,4,5-[3-32P])P4 from contami-nating labeled ATP. The [3H]Ins(1,3,4,5-[3-32P])P4 (210,000cpmof3H]) was combined with 70 nmol unlabeled Ins(l ,3,4,5)-P4 prepared in a parallel reaction, then desalted (22). The[3H]Ins(1,3,4,5-[3-_2p])p4 was incubated 1 hr at 37°C withinositol phosphate 5-phosphomonoesterase (7 nmol/min), 50mM Mes (pH 6.5), and 3 mM MgCl2 in 400 ,d. The productwas chromatographed on Dowex-formate as describedabove. [3H]Ins(1,3,4-[3-32P])P3, which was separated fromunreacted Ins(1,3,4,5)P4, was desalted as before. The finalyield ofproduct was 52,000 cpm of tritium and 55,000 cpm of32p with a specific activity of 3000 cpm of 3H per nmol.

Inositol Monophosphate Phosphatase Assay. Inositolmonophosphate phosphatase activity was assayed by mea-suring the formation of [3H]inositol from [3H]Ins 1P asdescribed (10).

Inositol Polyphosphate 1-Phosphatase Assay. Inositolpolyphosphate 1-phosphatase activity was assayed by one oftwo methods.Assay 1: 4-Phosphate release. Inositol polyphosphate

1-phosphatase converts Ins(1,4)P2 to Ins 4P, then inositolmonophosphate phosphatase hydrolyzes the 4-phosphate asdescribed below. Since crude homogenates and fractionsfrom early steps of the inositol polyphosphate 1-phosphatasepurification contain contaminating inositol monophosphatephosphatase, 1-phosphatase activity in these fractions can beestimated by measuring 4-phosphate release. Samples wereincubated in 3 mM MgCl2, 50 mM Hepes (pH 7.5), and

Ins(1,4-[4-32P])P2 (10-200 ,uM) in 50 ud at 370C. Reactionswere initiated by the addition of enzyme and stopped byaddition of 75 /.l of 1.67 M perchloric acid. Inorganicphosphate was complexed with 1% ammonium molybdate,extracted with 2 volumes ofisobutanol/toluene, 1:1 (vol/vol)(9, 23), and measured as Cerenkov radioactivity. This assaywas linear until 35% of the substrate was hydrolyzed. Mostassays were for 15 min.Assay 2: Coupled 4-phosphate release. Samples were

assayed as in assay 1 with the addition of partially purifiedinositol monophosphate phosphatase (1 nmol of Ins 1Phydrolyzed per min) (see below). The reaction was initiatedby the addition of inositol polyphosphate 1-phosphatase andcarried out for 15 min. [32P]Pi release was measured asdescribed for assay 1. Inositol monophosphate phosphatasealone hydrolyzed no [32P]Pj from Ins(1,4-[4-32P])P2.

Purification of Phosphatases. Inositol polyphosphate 1-phosphatase and inositol monophosphate phosphatase werepartially purified from calf brain (unpublished results). Brief-ly, 10 calf brains from a local abattoir were rapidly frozen ondry ice. Brains were shredded in a commercial vegetableshredder and homogenized in buffer (1:2, wt/vol) with aPolytron homogenizer in 20 mM Hepes, pH 7.5/10 mMEGTA/2 mM EDTA/0.25 M sucrose/100 ,uM phenylmeth-ylsulfonyl fluoride. The homogenate was centrifuged 1 hr at30,000 X g, then the supernatant was recentrifuged 1 hr at48,400 x g. The supernatant was chromatographed sequen-tially on a DEAE-Sepharose CL-6B column and a Bio-GelTSK DEAE-5 PW HPLC column. Inositol polyphosphate1-phosphatase and inositol monophosphate phosphatase ac-tivities were partially resolved on the DEAE-resin usingHPLC. The separated pooled fractions of each enzyme wereindividually rechromatographed on the DEAE-resin byHPLC, and the resulting further-resolved peaks of activitywere used for the experiments described below. The inositolmonophosphate phosphatase pool was heated to 70°C for 30min to inactivate small amounts of contaminating inositolpolyphosphate 1-phosphatase activity (see Results).

RESULTSMetabolism of [3H]Ins(1,3,4-[3-32P])P3 in Crude Calf Brain

Supernatant. We prepared [3H]Ins(1,3,4-[3-32P])P3 to inves-tigate the order in which phosphates are removed fromIns(1,3,4)P3. [3H]Ins(1,3,4-[4-32P])P3 degradation was mea-sured at 2, 5, 10, 20, and 60 min after addition of 0.29 mg ofprotein per ml from a calf brain supernatant fraction. Theproducts were analyzed by Dowex-formate chromatography.The earliest product detected was an inositol bisphosphate

eluting before Ins(1,4)P2. In six experiments, the ratio of32P/3H in the unknown inositol bisphosphate was 0.99 ± 0.08(SD), while that in the unreacted substrate Ins(1,3,4)P3 was1.06 ± 0.05. The inositol bisphosphate isomer, therefore,retained the 32P-labeled 3-phosphate, indicating that eitherthe 1- or the 4-phosphate was removed first, yieldingIns(3,4)P2 or Ins(1,3)P2.

Purification of Inositol Polyphosphate 1-Phosphatase. Weset out to purify the enzyme catalyzing the first step in thedegradation of Ins(1,3,4)P3 to determine whether the 1- or the4-phosphate is removed first. As a substrate, we usedIns(1,4-[4-32P])P2 prepared from erythrocyte ghosts due tothe relative ease of its preparation. Our original assumptionwas that the 4-phosphate was removed first from Ins(1,3,4)P3and that the putative 4-phosphatase enzyme would alsoutilize Ins(1,4)P2. Calf brain was used as the starting materialfor enzyme purification. Both the crude brain supernatantand the fractions from the initial chromatography step(DEAE-Sepharose CL-6B) showed activity in the 4-phos-phate release assay (see assay 1). Subsequent HPLC using aDEAE-resin column, however, led to an apparent >90%o loss

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of activity using this assay. A mixture of [3H]Ins(1,4)P2 andIns(1,4-[4-32P])P2 was incubated with a sample of the enzymepooled from this column, and the products were separatedusing Dowex-formate chromatography. The product of thisreaction was an inositol monophosphate with the same32P/3H ratio as the unreacted Ins(1,4)P2 substrate. Thisindicates that the inositol bisphosphatase is in fact a 1-phosphatase that yields Ins 4P as a product. This enzymedoes not utilize Ins1P as a substrate (see below) and will thusbe termed inositol polyphosphate 1-phosphatase.An enzyme that hydrolyzes the phosphate from Ins1P has

been purified from rat (24) and bovine brain (25). Thisenzyme, termed myo-inositol-1-phosphatase (EC 3.1.3.25),has been shown to utilize Ins 4P and inositol 5-phosphate assubstrates (26) in addition to both d- and i-Ins1P. Therefore,this enzyme will be referred to in this paper as inositolmonophosphate phosphatase. Inositol monophosphate phos-phatase was partially purified from the same calf brainpreparation. While the inositol monophosphate phosphatasealone showed no activity with Ins(1,4-[4-32P])P2, mixture ofthe enzyme with HPLC fractions enriched in inositolpolyphosphate 1-phosphatase restored 4-phosphate releaseactivity to the fractions. This result indicates that the inositolmonophosphate phosphatase hydrolyzes the phosphate fromthe Ins 4P intermediate.Heat Lability of Inositol Polyphosphate 1-Phosphatase.

Inositol monophosphate phosphatase is stable at 70'C (24,25). Table 1 shows an experiment in which a fraction from theDEAE-Sepharose CL-6B column containing both inositolmonophosphate phosphatase and inositol polyphosphate 1-phosphatase activities was incubated at 70°C for variouslengths of time. While the rate of hydrolysis of Ins1P wasstable over the time course examined, greater than 90% of theinositol polyphosphate 1-phosphatase activity was inactivat-ed within 5 min.

In further purification of inositol polyphosphate 1-phos-phatase, we used assay 2 to resolve the enzyme from inositolmonophosphate phosphatase. The ratio of the inositolpolyphosphate 1-phosphatase activity to inositol monophos-phate phosphatase activity in the most highly purified en-zyme pool is 110-fold greater than that in the startingsupernatant. The residual activity using Ins1P as substrate isheat stable, suggesting that it results from inositol mono-phosphate phosphatase contamination and not slight activityof inositol polyphosphate 1-phosphatase on Ins 1P.

Action of Inositol Polyphosphate 1-Phosphatase on Ins-(1,3,4)P3. Ins(1,3,4-[4-32P])P3 substrate was prepared. Thissubstrate was incubated with inositol polyphosphate 1-phosphatase, and the products were resolved as shown inFig. LA. The product is a 4-labeled inositol bisphosphate thatmigrates faster than Ins(1,4)P2 in a position identical to the3-labeled bisphosphate that resulted from metabolism ofIns(1,3,4-[3-32P])P3. This product must, therefore, be Ins-

Table 1. Heat inactivation of inositol polyphosphate 1-phosphataseIncubation, Inositol, 4-Phosphate release,

min nmol min-1 ml-' nmol min-1ml-10 318 645 285 5.3

15 269 6.230 262 5.9

Hydrolysis of Ins 1P was used to determine inositol monophos-phate phosphatase activity, while release of [32P]P, from Ins(1,4-[4-32P])P2 was used to estimate inositol polyphosphate 1-phosphataseactivity. A pool from the DEAE-Sepharose CL-6B column enrichedin inositol monophosphate phosphatase activity was incubated at70TC. Samples were removed at the indicated times, denaturedprotein was pelleted by centrifugation for 15 min at 10,000 x g, andassays were performed.

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FIG. 1. Hydrolysis of Ins(1,3,4)P3 by partially purified inositolpolyphosphate 1-phosphatase. (A)Ins(1,3,4-[4-32P])P3 (1 nmol) wasincubated for 30 min at 370C with partially purified inositolpolyphosphate 1-phosphatase (6,Ag) in 200 A.l containing 50mMHepes (pH 7.5) and 3mM MgCl2. The reaction mixture waschromatographed on Dowex-formate; after collecting 60 fractions,the gradient was stopped, and the column was step-eluted with 1.5M NH4COOH, pH 3.45. The elution position of a [3H]Ins(1,4)P2standard is indicated. (B) [3H]Ins(1,3,4)P3 (0.1 nmol) was incubatedfor 15 min at 370C with 1.5 ,tg of inositol polyphosphate 1-phosphatase, 50mM Hepes (pH 7.5), and 3mM MgCl2. The reactionmixture was chromatographed on an HPLC SAX column as de-scribed (21). The elution positions of 32P-labeled Pi and Ins(1,4)P2standards are indicated; note that the bisphosphate reaction productelutes after Ins(1,4)P2 in this chromatography system. The positionof unreacted Ins(1,3,4)P3 is also indicated.

(3,4)P2 resulting from removal of the 1-phosphate fromIns(1,3,4)P3, since both the specifically labeled 4-phosphate(Fig. 1) and 3-phosphate groups are retained.

Fig.1B shows the HPLC elution position of [3H]Ins(3,4)P2on a strong anion exchange column. Ins(3,4)P2 elutes afterIns(1,4)P2 on HPLC in contrast to its behavior in Dowex-formate chromatography.

Ins(3,4)P2 is further metabolized to inositol 3-phosphate(Ins 3P) as shown in Fig. 2. In separate incubations contain-ing 2 mM inositol 2-phosphate to inhibit the inositol mono-phosphate phosphatase, only Ins 3P was formed.

Action of InositolPolyphosphate 1-PhosphataseonIns(1,4,5)-P3 and Ins(1,3,4,5)P4. [3H]Ins(1,4,5)P3 (1 nmol, 1500 cpm/nmol) was incubated in 50 ,ul with an amount of inositolpolyphosphate 1-phosphatase capable of hydrolyzing 6 nmolof Ins(1,4)P2. No hydrolysis of the trisphosphate was ob-served under these conditions. In a separate experiment,Ins(1,4-[4-32P])P2 (1 nmol) included in the incubation wasquantitatively converted to Ins 4P while no Ins(1,4,5)P3 washydrolyzed. Similarly, [3H]Ins(1,3,4,5)P4 (0.1 nmol, 10,000cpm/nmol) was incubated in 100 ,ul with inositol polyphos-phate 1-phosphatase, both with and without Ins(1,4-[4-32P])P2 (1 nmol) in the reaction. No hydrolysis ofIns(1,3,4,5)P4 was observed in either condition, though theIns(1,4)P2 was converted to Ins 4P.Metabolism of Ins(1,4)P2 in Crude Brain Homogenate. We

investigated the pathway of Ins(1,4)P2 metabolism in thecrude calf brain supernatant to determine if all Ins(1,4)P2 isconverted to Ins 4P or whether an alternative pathwayinvolving Ins 1P exists. We incubated [3H]Ins(1,4-[4-32P])P2

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FIG. 2. Inositol monophosphate product of Ins(3,4)P2 metabo-lism by crude calf brain supernatant. [3H]Ins(3,4)P2 was prepared byincubation of [3H]Ins(1,3,4)P3 with inositol polyphosphate 1-phos-phatase and isolated by Dowex-formate chromatography using alinear gradient (200 ml) of 0.05-1 M NH4COOH (pH 3.45). [3H]Ins-(3,4)P2 (0.03 nmol) was incubated 20 min at 370C with 25 ,g of crudecalf brain supernatant, 50mM Mes (pH 6.5), and 3 mM MgCl2 in 0.1ml, and the products were separated by Dowex-formate chromatog-raphy. The elution positions of standards are indicated.

with crude calf brain supernatant using conditions in which-50% of the substrate was hydrolyzed as shown in Fig. 3.dl-Ins 1P was included in this reaction to saturate the inositolmonophosphate phosphatase and thus trap any inositolmonophosphate intermediate of Ins(1,4)P2 metabolism. The32P/3H ratio in the inositol monophosphate peak is identicalto that of the unreacted Ins(1,4)P2, indicating that allIns(1,4)P2 breakdown occurred via 1-phosphate removal.Moreover, no peak of 32p inorganic phosphate was observed,

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FIG. 3. Inositol monophosphate product of Ins(1,4)P2 metabo-lism by crude calf brain supernatant. A mixture of [3H]Ins(1,4)P2 andIns(1,4-[4-32P])P2 (2.5 nmol total) was incubated 10 min at 37°C with25 ,g of crude calf brain supernatant, 50 mM Hepes (pH 7.5), and 3mM MgCl2 in 50 ,ul. dl-Ins 1P (1.2 mM) was included to saturate theinositol monophosphate phosphatase and thus trap the inositolmonophosphate product of Ins(1,4)P2 metabolism. The reactionmixture was chromatographed on Dowex-formate.

which demonstrates that no phosphate was removed from the4-position of Ins(1,4)P2. This experiment was repeated witha sample of unwashed membrane pellet from the 48,400 x gcentrifugation (21 Ag ofprotein, resuspended by sonication in50 mM Hepes, pH 7.5) to investigate whether a membrane-associated 4-phosphatase exists. Again, the 32P/3H ratio inthe inositol monophosphate peak was identical to that of theunreacted Ins(1,4)P2 and no peak of [32p]p, was observed. Weconclude that under these experimental conditions allIns(1,4)P2 is hydrolyzed to Ins 4P.

DISCUSSIONThis study describes a pathway for the metabolism ofIns(1,3,4)P3 and presents information on the breakdown ofIns(1,4)P2. Our current understanding of inositol phosphatemetabolism is illustrated in Fig. 4. We demonstrated that thefirst product of Ins(1,3,4)P3 hydrolysis in calf brain isIns(3,4)P2, an inositol bisphosphate isomer. Ins(3,4)P2 elutesafter Ins(1,4)P2 on HPLC using a SAX column (Fig. 2B). Thiselution position is consistent with that of the inositolbisphosphates resulting from Ins(1,3,4)P3 metabolism thatwere described in rat liver (27) and rat parotid glands (16).Hansen et al. (27) suggested that the new inositol bisphos-phate was Ins(1,3)P2, which is inconsistent with our results.4:Preliminary evidence from our laboratory suggests thathuman platelets also hydrolyze Ins(1,3,4)P3 to yield Ins(3,4)-P2. Removal of the 1-phosphate from Ins(1,3,4)P3 may thusbe a general initial step in the pathway of Ins(1,3,4)P3metabolism.Our experiments with [3H]Ins(3,4)P2 showing conversion

to inositol 3-phosphate (see Fig. 2) suggest that Ins(3,4)P2 isfurther hydrolyzed to Ins 3P by a 4-phosphatase.We find that Ins(1,4)P2 metabolism in calf brain yields Ins

4P as the sole monophosphate product (see Fig. 3). Thisfinding contradicts the conclusions of several other investi-gators. Storey et al. (28) claim that Ins(1,4)P2 is acted uponby both a 1- and a 4-phosphatase in rat liver. They supportthis hypothesis with the observation that Ins(1,4-[4-32P])P2hydrolysis yielded both a labeled inositol monophosphate(Ins 4P) and labeled Pi. We included Ins IP in our incubationsto suppress inositol monophosphate phosphatase activity anddetected no release of labeled Pi from Ins(1,4-[4-32P]P2. Sinceno such inhibitors were included in the former study, it ispossible that the [32P]P, measured by Storey et al. (28)resulted from inositol monophosphate phosphatase actionupon the Ins 4[32p]p intermediate rather than direct action ofa 4-phosphatase upon Ins(1,4-[4-32P])P2. Berridge et al. (29)detected a 4-phosphate specific bisphosphatase in blowflysalivary glands, though these data were not specificallyreported. It is unclear whether this represents a differenceamong species or whether the investigators were unable toresolve Ins 4P and Pi on the Dowex chromatography systemused. Finally, Ackermann et al. (26) incubated 700 ,uMIns(1,4)P2 with crude rat brain cytosol and detected forma-tion of both Ins 4P and Ins 1P, though Ins 4P was the majorproduct. The difference between this result and the datapresented here might result from action of a 4-phosphatasewith low affinity for Ins(1,4)P2 that we were unable to detectat low substrate concentrations.An interesting feature of the pathway described here is that

Ins(1,4,5)P3 recycles to inositol via Ins 4P rather than Ins 1P.Sherman et al. (30) directly measured the levels of Ins 1P andIns 4P in rat cerebral cortex using gas chromatography. Inboth control and lithium-treated animals, Ins 1P was thepredominant inositol monophosphate. In lithium-treated

tUnder some conditions we find evidence for a 4-phosphatase thatutilizes Ins(1,3,4)P3 to form Ins(1,3)P2 (unpublished results).

Biochemistry: Inhorn et al.

Pi Ins(3,4)R Ins(1,4)P2i I2f-

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Proc. Natl. Acad. Sci. USA 84 (1987)

/mPKC -activationPA= 1,2 dioclglycerol -rochidonte

icosanoid

c1:2P 11:2,4P2--- -CII24.5P3

14oPdI 3$4-7m1ACO mobilization

3P --- I3,0P2 - [1,3.4P3

FIG. 4. Scheme for inositol phospholipid breakdown and resynthesis. I, inositol; P, phosphate; numbers, phosphate position on the inositolring. Note that the noncyclic inositol phosphates are metabolized to inositol via inositol 4-phosphate and Ins 3P, while Ins 1P is derived fromcyclic inositol phosphate metabolism and direct phosphatidylinositol breakdown.

rats, 92% of the inositol monophosphate was Ins 1P, while8% was Ins 4P. Similarly, Siess (31) found that Ins 1P was themost abundant inositol monophosphate in thrombin-stimu-lated platelets using an HPLC system that separates Ins 1Pfrom Ins 4P. The current results suggest that the presence ofIns 1P in cells results from either direct action of phospho-lipase C upon phosphatidylinositol or from hydrolysis ofcyclic inositol 1:2 phosphate to Ins 1P (see Fig. 4).We have partially purified an enzyme that removes the

1-phosphate from both Ins(1,3,4)P3 and Ins(1,4)P2. Ourpreliminary work suggests that this enzyme does not utilizeIns 1P as a substrate, so we have tentatively named theenzyme inositol polyphosphate 1-phosphatase. We find noactivity of this enzyme upon Ins(1,4,5)P3 or Ins(1,3,4,5)P4.Our preliminary data show that 5 mM Li' causes >50%

inhibition of the partially purified inositol polyphosphate1-phosphatase activity. This result is consistent with thefindings of several investigators who noted increases inIns(1,3,4)P3 and Ins(1,4)P2 after Li' treatment (27, 32, 33). Inaddition, Storey et al. (28) found that Li' inhibitedbisphosphatase activity in rat liver cytosol. An enzymeactivity similar to the inositol polyphosphate 1-phosphatasedescribed in this paper may thus account for the inositolphosphate metabolism reported by those investigators.We thank Dan Lips, Hans Deckmyn, Teresa Bross, and Brian

Whiteley for advice and assistance; Susan Brodack and CecilBuchanan for substrate preparation; Lois Isenberg for expert typing;and Tom Connolly for invaluable discussions throughout the courseof this work. This research was supported by Grants HLBI 14147(Specialized Center for Research in Thrombosis), HL 16634, andTraining Grant T32 HLBI 07088 from the National Institutes ofHealth, and by National Institutes of Health Research Service AwardGM-07200, Medical Scientist, from the National Institute of GeneralMedical Sciences.

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