Biochem. J. (1990) 265, 435-452 (Printed in Great Britain) 435 Product-precursor relationships amongst inositol polyphosphates Incorporation of 132PjPi into myo-inositol 1,3,4,6-tetrakisphosphate, myo-inositol 1,3,4,5- tetrakisphosphate, myo-inositol 3,4,5,6-tetrakisphosphate and myo-inositol 1,3,4,5,6- pentakisphosphate in intact avian erythrocytes Leonard R. STEPHENS* and C. Peter DOWNESt Smith Kline and French Research Ltd., The Frythe, Welwyn, Herts. AL6 9AR, U.K. Avian erythrocytes were incubated with myo-[3H]inositol for 6-7 h and with [32P]Pj for the final 50-90 min of this period. An acid extract was prepared from the prelabelled erythrocytes, and the specific radioactivities of the y-phosphate of ATP and of both the myo-inositol moieties (3H, d.p.m./nmol) and the individual phosphate groups (32P, d.p.m./nmol) of [3H]Ins[32P](l ,3,4,6)PJ, [3H]Ins[32P](1,3,4,5)P4, [3H]Ins[32P](3,4,5,6)P4 and [3H]Ins[32P](1,3,4,5,6)P5 were determined. The results provide direct confirmation that one of the cellular InsP4 isomers is Ins(1,3,4,5)P4 which is synthesized by sequential phosphorylation of the 1,4,5 and 3 substitution sites of the myo-Ins moiety, precisely as previously deduced [Batty, Nahorski & Irvine (1985) Biochem. J. 232, 211-215; Irvine, Letcher, Heslop & Berridge (1986) Nature (London) 320, 631-634]. This is compatible with the proposed synthetic route from Ptdlns via PtdIns4P, Ptdlns(4,5)P2 and Ins(1,4,5)P3. The data also suggest that, in avian erythrocytes, the principle precursor of Ins(1,3,4,5,6)Ps is Ins(3,4,5,6)P4. Furthermore, if the y- (and/or fi-) phosphate of ATP is the precursor of the phosphate moieties of Ins(3,4,5,6)P4, then this isomer must be derived from the phosphorylation of Ins(3,4,6)P3. If the y- (and/or fi-) phosphate of ATP similarly acts as the ultimate precursor to all of the phosphates of Ins(l,3,4,6)P4, then, in intact avian erythrocytes, the main precursor of Ins(1,3,4,6)P4 is Ins(1,4,6)P3. This contrasts with the expectation, based on results with cell-free systems, that Ins(1,3,4,6)P4 is synthesized by the direct phosphorylation of Ins(1,3,4)P3. INTRODUCTION Two of the three cellular InsP4 species defined above, Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4, can act as precursors of Three different InsP4 isomers have been identified in Ins(1,3,4,5,6)P5 in cell-free assays (Stephens et al., acid extracts of animal cells: D- or L-Ins(1,3,4,5)P4 (Batty 1 988b,c). The soluble, ATP-dependent, chromato- et al., 1985), Ins(1,3,4,6)P4 (Stephens et al., 1988c) and graphically distinct Ins(1,3,4,6)P4 5-hydroxykinase and Ins(3,4,5,6)P4 (Stephens et al. 1988a). Cellular Ins- Ins(3,4,5,6)P4 1-hydroxykinase activities responsible can (1,3,4,5)P4 is thought, on the basis of results obtained in be detected in homogeneous populations of cells, e.g. cell-free assays, to be synthesized from Ins(1,4,5)P3 [un- avian erythrocytes or primary cultured macrophages ambiguous evidence for the presence of Ins(1,4,5)P3 in (Stephens et al., 1988c). Hence several questions related cells has recently been obtained (Stephens et al., 1989) by to the synthesis of InsP5 have emerged. First, what is the use of an Ins(1,4,5)P3 3-hydroxykinase activity (Irvine metabolic origin of Ins(3,4,5,6)P4 (in the light of the et al., 1986)]. difficulties mentioned above), and secondly, on a more Ins(1,3,4,6)P4 was originally identified as the product general note, are the pathways that can synthesize of the phosphorylation of Ins(1,3,4)P3 in rat liver homo- Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, Ins(3,4,5,6)P4 and Ins- genates (Shears et al., 1987). This activity has since been (1,3,4,5,6)PJ in vitro active in intact cells? If so, what are described in homogenates or lysates derived from a their relative rates? number of other cell types (Balla et al., 1987; Stephens The experiments reported in this paper were aimed at et al., 1988c). The presence of Ins(1,3,4,6)P4 in cells has tackling these problems by analysing the incorporation been assumed to be a result ofthe action of an Ins(1 ,3,4)P3 of [32P]Pi tracer into the individual phosphate moieties of 6-hydroxykinase activity upon cellular Ins(1,3,4)P3 Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins- (Stephens et al., 1988c). (1,3,4,5,6)P5 in intact avian erythrocytes. They confirm The cellular origin of Ins(3,4,5,6)P4 is currently un- expectations that cellular Ins(1,3,4,5)P4 is synthesized defined. Experiments designed to detect phosphorylation from Ins(1,4,5)P3. However, the data also suggest that, in of a cell-derived [3H]InsP3 to [3H]Ins(3,4,5,6)P4 in vitro intact cells, (1) Ins(1,3,4,6)P4 is principally derived from have repeatedly failed because insufficient product is- Ins(1,4,6)P3, (2) Ins(3,4,5,6)P4is derived from Ins(3,4,6)P3 formed to assign its structure (L. Stephens, unpublished and (3) the major precursor of Ins(1,3,4,5,6)P5 is Ins- work). (3,4,5,6)P4. Abbreviations used: SAX, strong-anion-exchange; WAX, weak anion-exchange; BSA, bovine serum albumin. * To whom correspondence and reprint requests should be sent, at present address: Department of Biochemistry, A.F.R.C. Institute of Animal Physiology, Babraham, Cambridge CB2 4AT, U.K. t Present address: Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee DDI 4HN, U.K. Vol. 265 436 L. R. Stephens and C. P. Downes MATERIALS AND METHODS Dual-label liquid scintillation counting Erythrocytes from 5-day-old chicks were washed twice Aqueous samples containing 3H and/or 32P radio- either in a Hepes-based medium [prepared as described activity were counted in a Beckman benchtop LS 1801 previously (King et al., 1987) except that it contained liquid scintillation counter utilizing the associated dual- 0.38 mM-Na2HPO4], or in a NaCl-based medium (com- label, decay and quench-correction software. A range of position identical to that described above, except that the chloroform-quenched 3H and 32P standards were counted total Hepes concentration was reduced to 25 mm and (under conditions identical with those used to count all replaced with 140 mM-NaCl) before being resuspended experimental samples; see below) to calibrate the scin- finally in the medium in which they had been washed at tillation counter. All radioactive solutions to be counted a density of 4 ml of packed erythrocytes/10 ml of total were dissolved in a scintillation fluid mixture of the incubation volume. The erythrocyte suspensions were following composition: 10 ml of Insta-gel (Packard), shaken in a thermostated water bath at 37 'C. Radio- 2 ml of CH30H/water (1:1, v/v) and 0.4 ml of h.p.l.c. active tracers that were to be introduced into the cell column eluant (0-1000% B). The salt content of the suspension were freeze-dried and dissolved in the in- h.p.l.c. eluant significantly altered the quench environ- cubation medium or in the final cell suspension. ment ofthe samples, but this was corrected appropriately by the quench-correction procedure. (The system was tested by mixing defined aliquots of 3H and 32p with a Extraction and purification of 13HjInsI32PIP species series of fractions containing various percentages of B, Incubations were terminated by removing aliquots of then dissolving them in the scintillation fluid cocktail the cell suspension (typically 250-500,tl). The cells were defined above and counting them for radioactivity with lysed and then acid-precipitated as described previously the calibrated counter described.) The values of the (Stephens et al., 1988a). The acid extracts were treated background 3H and 32p that were subtracted from with charcoal, neutralized (with KOH/Hepes), applied to radioactive peak totals were defined by blank vials run a Partisil 10 strong-anion-exchange (SAX) column (90- with every experiment. 95 0 of exogenous [3H]InsP4 and [3H]InsP5 standards added to the original lysates were recovered at this point), and eluted as described in Stephens et al. (1988a). Measurement of Ins Fractions containing the Ins[32P]P4s were pooled, as were The concentrations and specific radioactivities of those containing Ins[32P]P5. The pooled fractions were h.p.l.c.-purified [3H]inositol phosphates were determined desalted (Stephens et al., 1988c) and redissolved in 1 ml by assaying the Ins liberated after dephosphorylation of 5.0 mM-EDTA (pH 7.0 with NaOH). These Partisil (see below), essentially as described previously (Mac- 10-SAX h.p.l.c.-purified [3H]Ins[32P]P4 and [3H]Ins[32P]P5 Gregor & Matschinsky, 1984) with certain procedural fractions were further purified by chromatography on a modifications as noted below. Inositol phosphates were Partisphere weak-anion-exchange (WAX) h.p.l.c. dephosphorylated with alkaline phosphatase (Sigma, column. [3H]Ins[32P](1,3,4,6)P4, [3H]Ins[32P](I,3,4,5)P4 type P-5521).