Formation and metabolism of inositol 1, 3, 4, 5-tetrakisphosphate in liver.

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 261, No. 18, Issue of June 25,p. 8100-8103,1986 0 1986 by The American Society of Biological Chemists, Inc.

Printed in U.S.A.

particular importance is the breakdown of PtdIns-4,5-Pz1to Formation and Metabolismof which subsequently causes the release ofCa” Inositol 1,3,4,5-Tetrakisphosphate Ins-1,4,5-P3, from an intracellular vesicular store and initiatesan increase of the cytosolic free Ca2+(4-6). Recently, our understanding in Liver* (Received for publication, March 20,1986)

Carl A. HansenS, Stephanie Mah, and John R. Williamson5 From the Departmentof Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

of inositol polyphosphate metabolism has been complicated by the detection of two novel inositol polyphosphates, Ins1,3,4-P3 (7-9) and Ins-1,3,4,5-P4 (10, ll),in hormone-stimulated tissues. However, the origin of Ins-1,3,4,5-P4 and the relationship of these two inositol polyphosphates to phosphoinositide turnover andto Ins-1,4,5-P3metabolism has not been elucidated. The present study addresses these issues. EXPERIMENTALPROCEDURES

The inositol lipid pools of isolated rat hepatocytes Hepatocyte Isolation and Incubation-Hepatocytes were isolated were labeled with [3H]nyo-inositol,stimulated maxi- from the livers of fed, male Sprague-Dawleyrats weighing 180-220 g mally with vasopressin and the relative contents of as described previously (12). Cellular inositol lipids were labeled by [3H]inositol phosphates were measured by high per- incubating the cells at 25 mg dry weight/ml in Krebs bicarbonate medium supplemented with 1.3 mM CaCI,, 15 mM glucose, 2% (w/v) formance liquid chromatography. Inositol 1,4,5-tris- dialyzed bovine serum albumin, and 2.6 p~ [2-3H]myo-inositol(40 phosphate accumulated rapidly (peak 20 s ) , while ino- pCi/ml) for 90 min a t 37 “C under an atmosphere of Oz/COz (955). sitol 1,3,4-trisphosphateand a novel inositol phosphate Subsequently, the cells were washed twice with isotope free buffer (ascribed to inositol 1,3,4,5-tetrakisphosphate)accu- containing 5 mM myo-inositol. Cells were then resuspended at a concentration of 20 mgdry weight/ml and incubated for an additional mulatedata slower rate over 2 min.Incubationof hepatocytes with 10 mM Li+ prior to vasopressin ad- 20 min before addition of vasopressin. When present, 10 mM LiCl was added at the beginning of the 20-min equilibration period. At dition selectively augmented thelevels of inositol mon- various times after vasopressin addition, 0.5-ml aliquots were ophosphate,inositol1,4-bisphosphate,andinositol quenched with 250 p1 of cold 12% (w/v) perchloric acid containing 3 1,3,4-trisphosphate. A kinase was partially purified mM EDTA and 1 mM diethylenetriaminepentaaceticacid (13), folfrom liver and brain cortex which catalyzed an ATP- lowedby centrifugation and neutralization to pH 7.5 with KOH. dependent phosphorylation of [3H]inositol 1,4,5-tris- Under these conditions, recoveries of [3H]Ins-1,4,5-P3and [3H]Insphosphate to inositol 1,3,4,5-tetrakisphosphate.Incu- 1,3,4,5-P4added to thecell extracts were greater than 85%. High Performance Liquid Chromatography (HPLC)-Inositol bationofpurified[3H]inositol 1,3,4,5-tetrakisphos- polyphosphates were separated and analyzed by a modification of the phate with diluted liver homogenate produced initially HPLC method of Irvine et al. (8) using a Whatman Partisil SAX inositol 1,3,4-trisphosphateand subsequently inositol column (0.46 X 25 cm, 10-pm particle size) and a guard column 1,3-bisphosphate, the formationof which could bein- packed with Whatman Pellicular anion exchange media. Following sample injection, the column was washed for 8 min with water and hibited by Li+. The data demonstrate that the most the inositol polyphosphates subsequently eluted by three successive probable pathway for the formation of inositol 1,3,4,5- convex gradients (Waters Model 660 Solvent Programmer 4, 2, and tetrakisphosphate is by 3-phosphorylation of inositol 1,respectively) of increasing 1.5 M ammonium formate buffer adjusted 1,4,5-trisphosphateby a soluble mammalian kinase. to pH 3.7 with orthophosphoric acid (see Fig. 1).The flow rate was Degradation of both compounds occurs first by a Li+- 1.2 ml/min and the eluant was collected in 1-min fractions over the insensitive 5-phosphatase and subsequently by aLi+- first 10 min and at0.5-min intervals over the next 30 min. Fractions 50% sensitive 4-phosphatase. The prolonged accumulation were counted for 3H radioactivity after addition of0.9mlof methanol in water and 4.6 ml of ACS 11. Recoveries of t3H]Ins-1,4of both inositol 1,4,5-trisphosphate and inositol Pz, [3H]Ins-1,4,5-P3and [3H]Ins-1,3,4,5-P4from the Partisil SAX 1,3,4,5-tetrakisphosphate invasopressin-stimulated column were greater than 90%. hepatocytes suggest that they have separate second Inositol Trisphosphute Kinme-1nsP3 kinase was partially purified messenger roles, perhaps bothrelating to Ca2+-signal- from rat cortex and liver by a modification of the procedure of Chakrabarti and Biswass (14). Tissues were homogenized (MO, w/v) ling events.

It has recently become evident that receptor-stimulated hydrolysis of phosphoinositides by calcium-mobilizing hormones is critically involved in stimulus-response coupling in many different types of cells (for reviews see Refs. 1-3). Of

in 0.32 M sucrose, 10 mM Hepes/Tris, pH 7.3, 1 mM EGTA, 2 mM MgCl,, and 2 mM dithiothreitol in a Teflon-glass homogenizer at 660 rpm. The homogenate was centrifuged a t 25,000 X g for 10 min and the supernatantwas recentrifuged at 100,000X g for 90 min to remove all membranous components containing phosphatase activity. The supernatant was fractionated with ammonium sulfate and a 2340% ammonium sulfate fraction was dialyzed overnight against 10 mM Tris/HCl, pH 7.0, 2 mM MgCl,, and 2 mM dithiothreitol. The buffer for enzyme activity contained 50 mM Tris/HCl, pH 8.0, 5 mM ATP,

* This work wassupported by National Institutes of Health Grants AM-15120 and AA-05662. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Recipient of a National Research Service Award postdoctoral fellowship. § T o whom correspondence and reprint requests should be addressed.

The abbreviations used are: PtdIns-4,5-Pz, phosphatidylinositol 4,5-bisphosphate; Ins-1,4,5-P3, myo-inositol 1,4,5-trisphosphate; InsIns-1,3,4,5-P4,myo-inositol 1,3,4-P3,myo-inositol1,3,4-trisphosphate, 1,3,4,5-tetrakisphosphate;Ins-2,4,5-P3, myo-inositol 2,4,5-trispbosphate;Ins-1,4-Pz, myo-inositol l,4-bisphosphate; Ins-1,3-Pz, myoinositol 1,3-bisphosphate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HPLC, high performance liquid chromatography; EGTA, [ethylenebis(oxyethylenenitrilo)tetraacetic acid; InsP3, myoinositol trisphosphate.

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Tetrakisphosphate Inositol 5 mM MgC12, 5 mM sodium pyrophosphate and 1 mM dithiothreitol. The reaction was initiated by the addition of 50 p M Ins-1,4,5-P3 and after 10 minwas terminated by addition of perchloric acid as described above. Ins-1,3,4,5-P4 was synthesized from Ins-1,4,5-P3 using a modification of the above assay conditions. The Ins-1,3,4,5-P4produced was purified by HPLC separation using a 5-pm Hibar Lichrosorb NHz column (0.46 X 25 cm) with a linear gradient of water and 1.5 M ammonium bicarbonate, pH 7.7. The ammonium bicarbonate was subsequently removed by lyophilization. Muteriuls-Ins-1,4,5-P3 was obtained from Sigma or Behring Diagnostics; both products contain approximately 20% Ins-2,4,5-P3 as ascertained from 'H NMR spectroscopy? Ins-1,4-P~,[2-3H]myo-inositol and [2-3H]myo-inositol1,4,5-P3 werepurchased from Amersham Corp. [2-3H]myo-inositol1,4-Pz and [2-3H]myo-inositol1,4,5-P3 were gifts from New England Nuclear. [3zP]Inositolpentakisphosphate was prepared from chicken erythrocytes (15). ACS I1 scintillation mixture was from Amersham Corp. All solvents and other chemicals were reagent grade or better.

RESULTS

Formation of Inositol Polyphosphates in Hepatocytes-Batty et al. (10) recently reported the formation of Ins-1,3,4,5-P4 following muscarinic stimulation of rat cortical slices. Analysis of vasopressin-stimulated [3H]inositol prelabeled hepatocytes by anion exchange HPLC also revealed the presence of an inositol polyphosphate more polar than Ins-1,4,5-P3 (Fig. 1).This inositol polyphosphate eluted a t 1.5 M ammonium formate, intermediatebetween Ins-1,4,5-P3 (0.85 M) and inositol pentakisphosphate (2.0 M) (not shown), suggesting that thiscompound was the same as that identified by Batty et al. (lo), namely Ins-1,3,4,5-P4. As shown in Fig. 1, two peaks containing [3H]inositol eluted prior to the InsP3 peaks. The first peak coeluted with standard [3H]Ins-1,4-P2and with the only InsP2 peak formed by hydrolysis of standard [3H] Ins-1,4,5-P3 and, therefore, can be unequivocally identified as Ins-1,4-P2. The second, more slowlyeluting InsP2peak, which accumulated to a much less extent than Ins-1,4-P2,is tentatively ascribed to Ins-1,3-Pz. The kinetics of the accumulation of Ins-1,3,4,5-P4 in relation to thetwo InsP3 isomers following maximal stimulation of [3H]inositol-labeledhepatocytes with vasopressin are illustrated in Fig. 2. The time for half-maximal Ins-1,3,4,5-P4 production was intermediate between that for Ins-1,4,5-P3 and Ins-1,3,4-P3.By 20 s after vasopressin addition, Ins-1,4,5P3 formation had reached its peak and Ins-1,3,4,5-P4 had attained 60% of its maximum level, whereas Ins-1,3,4-P3 had

8101

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2 3 Minutes after Vasopressin

4

5

FIG. 2. Effect of vasopressin (20 nM) onaccumulationof inositol phosphates in hepatocytes. Values shown are mean f S.E. for three separate experiments. Control values remained unchanged over 15 min and were: 113 f 24 cpm for Ins-1,4,5-P3,59 f 9 cpm for Ins-1,3,4-P3, and 55 f 9 cprn for Ins-1,3,4,5-P4. lOmM Lithium Srnin=3,820cpm

.

i /" 5rni". 2l.Ooocpm I"Sll,41P*

)-1".ll.3PIP,

Minutes ofler vasopressin

FIG. 3. Effect of Li+ on vasopressin-stimulated accumulations of inositol phosphates in hepatocytes. Lithium (10 mM) was present for 20 min prior to addition of 20 nMof vasopressin. Values for control and Li+-treated cells (10 mg dry weight) were: Ins1,4-Pz, 625 f 78 and 606 f 85 cpm; Ins-1,3-Pz, 238 f 83 and 200 +. 47 cpm; Ins-1,3,4-P3, 66 f 20 and 55 f 19 cpm; Ins-1,4,5-P3, 240 rl: 54 and 195 f 43 cpm; and Ins-1,3,4,5-P4, 71 +. 27 and 82 f 8 cpm. These values were subtracted from the values observed after vasopressin addition.

reached only 18%of its peak production, which occurred after about 2 min. The slower rise of Ins-1,3,4-P3 relative to Ins1,3,4,5-P4is consistent with it being formed by the action of a 5-phosphatase on Ins-1,3,4,5-P4 (10). In the steady state (5 to 15 min), the accumulations of Ins-1,4,5-P3 and Ins-1,3,4P3 were similar, while that of Ins-1,3,4,5-P4 was only onethird as great. In studies of hormone-activated inositol lipid metabolism, tissues have frequently been incubated with Li+, which inhibits the degradation of inositol mono- and bisphosphates (16, 17). The effect of 10 mMLi' on the vasopressin-induced accumulation of inositol phosphates in hepatocytes is illusMinutes of elution trated in Fig. 3. In agreement with previous measurements of FIG. 1. Elution profile of inositol phosphates by HPLC. The total InsP2 and InsPs levels in vasopressin-stimulated hepasample analyzed was a neutralized perchloric acid extract of hepatocytes (10 mg dry weight of cells) prelabeled with [3H]myo-inositol tocytes (18), therewas little effect of Li+ on theinitial rate of formation of the inositol phosphates or on control levels in and stimulated with 5 nM of vasopressin for 5 min. the absence of hormone stimulation. There was no effect of S. Cerdan and J. R. Williamson, unpublished observations. Li+ on the accumulation of Ins-1,3,4,5-P4. In addition to a

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Inositol Tetrakisphsphate in Liver

large increase of the inositol monophosphate pool (not shown), the major effect of Li+ was to increase the vasopressin-induced accumulations of Ins-1,4-P2 and Ins-1,3,4-P3 by 5- and %fold, respectively, after 5 min. Although Fig. 3 indicates that there were small changes in the accumulations of Ins-1,3-P2 and Ins-1,4,5-P3, further experiments showed that they were of borderline statistical significance. These data suggest that a 4-phosphatase, which hydrolyzes Ins-1,4P2 and Ins-1,3,4-P3 to Ins-1-P and Ins-1,3-P2, respectively, is inhibited by Li+ (cf. Refs. 9 and 17). On the other hand, the 5-phosphatase (17, 19-21), which apparently can use both Ins-1,4,5-P3and Ins-1,3,4,5-P4as substrate,is Li+-insensitive. The accumulation of Ins-1,4-P2 over the first 10 s of vasopressin stimulation was a t least as fast as that of Ins-1,4,5P3. In the absence of Li+, the net accumulation of Ins-1,4-P2 ceased after about 30 s (mean cpm of 4100 f 150 in four experiments) and thereafter remained approximately constant for at least 15 min, whereas in the presence of Li+ it continued to accumulate. These data suggest that Ins-1,4-P2 is formed primarily from the breakdown of phosphatidylinositol4-phosphate ratherthan from either of the InsPsisomers. Formation of h-1,3,4,5-P4by Imp3 Kinase-Since Ins1,3,4,5-P4 is rapidly formed in hepatocytes following vasopressin stimulation, the question arises as to its origin. Batty et al. (10) discuss the possibility that the Ins-1,3,4,5-P4that was formed in carbachol-stimulated brain slices may have arisen from the hydrolysis of phosphatidylinositol 3,4,5-trisphosphate but could provide no evidence in favor of this pathway. Alternatively, the presence of inositol polyphosphate kinases has been well documented in avianerythrocytes (14) and plant tissues(22). A search for InsP3 kinase activity in rat tissues showed that it was present inrat liver and brain cortex homogenates. Addition of [3H]Ins-1,4,5-P3to either homogenate, supplemented with MgATP, resulted in the appearance of a 3H-containing peak having the identical retentiontime (33.5 min, cf. Fig. 1) as the Ins-1,3,4,5-P4 peak observed in vasopressin-stimulated hepatocytes. Rat cortex InsP3 kinase had a specific activity of0.12 nmol/min/mg protein, whereas, in liver, the specific activity of the kinase could not be accurately determined due to high Ins-1,4,5-P3 phosphatase activity. Following partial purification of the InsP3 kinase, the remaining 5-phosphatase activity was minimizedby addition of 5 mM sodium pyrophosphate. The resultant specific activity of InsPB kinase from brain cortex was 2.0 nmol/min/mg protein, while that from liver was 0.8 nmol/min/mg protein. Fig. 4 illustrates the conversion of Ins-1,4,5-P3 to Ins1,3,4,5-P4using the crude liver InsP3 kinase. The 5-phosphatase activity was not entirely suppressed, as shown by the formation of Ins-1,4-P2and Ins-1,3,4-P3from Ins-1,4,5-P3 and Ins-1,3,4,5-P4,respectively. Small amounts of Ins-l,3-P2 and Ins-1-P were also formed (not shown). Similar results were obtained with the InsPskinase preparation from brain, which had a higher ratio of kinase to phosphatase activities. [3H] Glycerophosphoinositol 4,5-bisphosphate (elution time 21 min) was also found to be converted to a more polar compound that eluted from the HPLC column 2 min before Ins-1,3,4,5P4 and which is presumably glycerophosphoinositol 3,4,5trisphosphate. On the other hand, no conversion of [3H]Ins1,4-P2to [3H]inositol-containingpeakswith a longer retention time was observed. Present information suggests, therefore, that the mammalian InsP3 kinase is specific for phosphorylating the 3 position of the myo-inositol ring and requires vicinal phosphates inthe 4 and 5 positions. Further phosphorylation of Ins-1,3,4,5-P4 to inositol pentakisphosphate has not been observed with either the liver or brain InsP3 kinase

0

O

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IO

15 20 Minutes

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FIG. 4. Phosphorylation of Ins-1,4,5-P3 to Ins-1,3,4,5-P4 by InsP3 kinase.The reaction medium (0.7 ml) contained 50 mM Tris, pH 8.0, 5 mM ATP, 5 mM M$+, 5 mM sodium pyrophosphate, 1mM dithiothreitol, 50 p M [3H]Ins-1,4,5-P3(1000 cpm/nmol), and 2 mg of protein/ml of partially purified InsPBkinase from liver. Aliquots (0.1 ml) were removed at the times shown, deproteinized with perchloric acid, and assayed by HPLC.

Minutes

FIG.5. Hydrolysisof Ins-1,3,4,5-P4by liver homogenate. A 10% (w/v) rat liver homogenate prepared in 0.25 M sucrose and 20 mM Tris/Hepes, pH 7.2, was diluted 10-fold into 0.7 ml of buffer containing 110 mM KCl, 10 mM NaC1,l mMKH&'O4,3mMMgC12, 20 mM K+-Hepes, and 1 p~ [3H]Ins-1,3,4,5-P4(lo5 cpm/nmol). Miquots (0.1 ml) were removed at the times shown, deproteinized with perchloric acid, and assayed by HPLC.

preparation. Conversion of milligram quantities of Ins-1,4,5P3 to Ins-1,3,4,5-P4 can be obtained with an overall yield of about 70%. 'H and 31P NMR spectroscopy of this material arecurrently being performed to provide an unequivocal confirmation of its structure (cf. Ref. 23). Degradation of Ins-1,3,4,5-P4 by Liver Homogenate Phosphatases-The degradative pathway for Ins-1,3,4,5-P4metabolism wasinvestigated by addition of 1PM of [3H]Ins-1,3,4,5P4 to a liver homogenate (Fig. 5 ) . The only Imp3 product formed was the Ins-1,3,4-P3 isomer, while the only InsP2 product detected was in a peak tentatively identified as the Ins-1,3-P2 isomer. Fig. 5B shows that the accumulation of Ins-1,3,4-P3 was enhanced by the presence of 25 mM Li+ in the reaction buffer, while the accumulation of Ins-1,3-P2 was diminished. On the other hand, the overall rate of disappear-

Tetrakisphosphate Inositol ance of Ins-1,3,4,5-P4 was not appreciably affected by the presence of Li+. Small amounts of inositol monophosphate accumulated in the presence and absence of Li', but its isomeric configuration has not been determined. These experiments support the suggestion of Batty et al. (10) that Ins1,3,4,5-P4is degraded to Ins-1,3,4-P3 by a Li+-insensitive 5phosphatase and show that hydrolysis of Ins-1,3,4-P3 occurs by a Li+-sensitive phosphatase to an Impz product that is different from the Ins-1,4-Pzisomer of Ins-1,4,5-P3hydrolysis. DISCUSSION

The present study provides the first evidence that Ins1,3,4,5-P4 is produced in hormone-stimulated hepatocytes. Improvements in theprocedure for prelabeling the polyphosphoinositide pools with [3H]myo-inositol compared with a previous study (18),together withassay of 3H-labeled inositol phosphatesintissueextracts by high performance liquid chromatography, has allowed these compounds to be measured with much greater precision than hitherto. For instance, peak increases of Ins-1,4,5-P3 and Ins-1,3,4,5-P4 levels approximately 10- and20-fold, respectively, above control values are observed with maximum vasopressin stimulation. Although the specific activity of the inositol lipids and inositol phosphates have not been measured in this study,it is probable that to a first approximation [3H]inositol radioactivity in the separated inositol phosphates is proportional to mass. Until an independent confirmation is available (e.g.by NMR), identification of the [3H]inositol phosphatepeakseluting before and afterIns-1,4,5-P3in the ammonium formate/ phosphoric acid HPLC system as Ins-1,3,4-P3and Ins-1,3,4,5P4, respectively, is based on the periodate, borohydride, and alkaline phosphatase treatmentsdescribed by Irvine et al. (7) and Batty et al. (10). The kinetics of accumulation of the various inositol phosphates in vasopressin-stimulated hepatocytes, together with in vitro studies on the synthesis (Fig. 4) and degradation (Fig. 5) of Ins-1,3,4,5-P4, strongly suggest that itis formed from Ins-1,4,5-P3 by a soluble ATP-dependent kinase and is degraded to Ins-1,3,4-P3. The hydrolysis is probably catalyzed by the same low K,, membrane bound 5phosphatase that degrades Ins-1,4,5-P3 to Ins-1,4-Pz (17, 19, 20). A soluble, Li+-sensitive 4-phosphataseappears to be responsible for the furtherdegradation of both Ins-1,4-Pz and Ins-1,3,4-P3. The most intriguing aspects of the new ramification introduced into the field of inositol lipid metabolism by the demonstration of Ins-1,3,4,5-P4formation in hormone-stimulated cells concerns its possible biological function. The existence of a metabolic branch pointat the level of the Ca2+-mobilizing second messenger Ins-1,4,5-P3, with one path leading to hydrolysis and inactivation and the other to synthesis of Ins1,3,4,5-P4,suggests not only that therelative flux through the two pathways is likely to be regulated but also that Ins1,3,4,5-P4may have a separate second messenger role. Studies

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8103

in thislaboratory concerning a possible second messenger role for Ins-1,3,4,5-P4have shown that at concentrations of 5 PM it neither elicits a release of Ca2+from the saponin-permeabilized hepatocytes nor affects the sensitivity of Ins-1,4,5-P3 as a Ca2+-releasingagent in this system. Preliminary experiments with rat liver plasma membrane vesicles3indicate that, unlike Ins-1,4,5-P3,Ins-1,3,4,5-P4promotes a rapid release of the sequestered Ca" and hence may serve to integrate plasma membrane Ca" fluxes with intracellular Ca2+ mobilization during the sequence of events that couples hormone-receptor stimulation with the cellular response. REFERENCES 1. Berridge, M. J., and Irvine, R. F. (1984) Nature 312,315-321 2. Williamson, J. R., Cooper, R. H., Joseph, S. K., and Thomas, A. P. (1985) Am. J. Physiol. 2 4 8 , C203-C216 3. Hokin, L. E. (1985) Annu. Reu. Bioehem. 54,205-235 4. Streb, H., Irvine, R. F., Berridge, M. J., and Schulz, I. (1983) Nature 3 0 6 , 6 7 4 9 5. Joseph, S. K., Thomas, A. P., Williams, R. J., Irvine, R. F., and Williamson, J. R. (1984) J. Biol. Chem. 259,3077-3081 6. Burgess, G. M., Godfrey, P. P., McKinney, J. S., Berridge, M. J., and Putney, J. W., Jr. (1984) Nature 309,63-66 7. Irvine, R. F., Letcher, A. J., Lander, D. J., and Downes, C. P. (1984) Biochem. J. 223,237-243 8. Irvine, R. F., Anggard, E. E., Letcher, A. J., and Downes, C. P. (1985) Bwchem. J. 229,505-511 9. Burgess, G. M., McKinney, J. S., Irvine, R. F., and Putney, J.W. (1985) Biochem. J. 232,237-248 10. Batty, I. R., Nahorski, S. R., and Irvine, R. F. (1985) Biochem. J. 232,211-215 11. Heslop, J. P., Irvine, R. F., Tashjian, A. H., and Berridge, M. J. (1985) J. Exp. Bwl. 119,395-401 12. Meijer, A. J., Gimpel, J. A., Deleeuw, G. A., Tager, J. M., and Williamson, J. R. (1975) J.Bwl. Chem. 2 5 0 , 7728-7738 13. Cosgrove, D. G. (1980) in Studies in Organic Chemistry 4, Inositol Phosphates-Their Chemistry, Biochemistry and Physiology,p. 21, Elsevier, Amsterdam 14. Chakrabarti, S., and Biswas, B. B. (1981) Indian J. Biochem. Biophys. 18,398-401 15. Isaacks, R. E., Harkness, D. R., Froeman, G. A., and Sussman, S. A. (1976) Comp. Biochem. Physiol. 53A, 95-99 16. Hallcher, L. M., and Sherman, W. R. (1980) J.Biol. Chem. 2 5 5 , 10896-10901 17. Storey, D. J., Shears, S. B., Kirk, C. J., and Michell, R.M. (1984) Nature 312,374-376 18. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984) J. Biol. Chem 259,5574-5584 19. Seyfred, M. A., Farrell, L. E., and Wells, W. W. (1984) J. Biol. Chem. 259,13204-13208 20. Joseph, S. K., and Williams, R. J. (1985) FEBS Lett, 180, 150154 21. Connolly, T. M., Bross, T. E., and Majerus, P. W. (1985) J. Biol. Chem. 260,7868-7874 22. Chakrabarti, S., and Biswas, B. B. (1981) Phytochemistry ( O x f ) 20,1815-1817 23. Lindon, J. C., Baker, D. J., Farrant, R. D., and Williams, J. M. (1986) Biochem. J. 233,275-277

C. Hansen, S. Mah, and J. R. Williamson, manuscript in preparation.