The comparative content of the mitochondrial phosphoglyceroyl-ATP-containing polymer, purinogen, in rat tissues

Philip Freeman, Brinda Patel, John Mowbray *Regulatory Mechanisms Group, Department of Biochemistry and Molecular Biology, University College London, Gower Street,

London WC1E 6BT, UK

Received 22 June 1999; received in revised form 16 February 2000; accepted 21 February 2000

Abstract

Here, we describe an assay for the tissue content of purinogen, a highly phosphorylated labile polymer containing ATP andphosphoglycerate found in the mitochondrial intermembrane space. We report the purinogen content (as adenine nucleotideequivalents) of rat heart and, for the first time, of rat liver, kidney, brain and mixed skeletal muscle. The findings show thatpurinogen contains very significant proportions of cell adenine nucleotides ranging from 25% of the free pool in brain andskeletal muscle to 135% of it in kidney. The evidence that purinogen may form a controlled intracellular reservoir ofinorganic phosphate is briefly discussed. ß 2000 Elsevier Science B.V. All rights reserved.

Keywords: Brain purinogen; Heart purinogen; Kidney purinogen; Liver purinogen; Muscle purinogen; ATP polymer

1. Introduction

Rapid changes in adenine content of perfusedhearts led us to propose that some storage formmust exist [1^3] and in subsequent studies, we locateda very labile rapidly turning-over oligomer of alter-nating units of ATP and 3-phosphoglyceric acid inrat heart [4], kidney [5] and liver [6]. Selective diges-tion and chromatography along with 31P nuclearmagnetic resonance (NMR) and mass spectrometry

studies led us to propose a structure of 3-phospho-[glyceroyl-Q-triphospho-5P-adenosine-3P-3-phospho]n-glyceroyl-Q-triphospho-5P-adenosine (PG-ATP)n forthis compound for which we suggested the name oli-gophosphoglyceroyl-ATP [7]. In subsequent work,we have identi¢ed in liver and heart a (PG-ATP)n-speci¢c 3P-phosphodiesterase which is found only inthe mitochondrial intermembrane space [8] tightlycomplexed with its substrate [9], suggesting that(PG-ATP)n may be exclusively located there too.

Attempts to quantitate (PG-ATP)n, which rapidlyreaches speci¢c radioactivity equilibrium with cell[14C]ATP, led to the recognition that the (PG-ATP)n oligomers appear to be the rapidly labelledend-chains of a more complex polymer whose coreincluded additional nucleotide components [10]. Wehave provisionally called this as yet incompletelycharacterised polymer purinogen and devised a quan-titative assay which involves deliberate destabilisa-

0005-2728 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.PII: S 0 0 0 5 - 2 7 2 8 ( 0 0 ) 0 0 1 0 9 - 2

Abbreviations: (PG-ATP)n or oligo(phosphoglyceroyl-adeno-sine triphosphate), 3-phospho[glyceroyl-Q-triphospho(5P)adenosi-ne(3P)-3-phospho]n-glyceroyl-Q-triphospho(5P)adenosine; PG-ATP,3-phosphoglyceroyl-Q-triphospho(5P)adenosine; PPPAdoPGri, tri-phospho(5P)adenosine(3P)-3-phosphoglycerate

* Corresponding author. Fax: +44-71-387-1831;E-mail : j.mowbray@ucl.ac.uk

BBABIO 44867 11-4-00

Biochimica et Biophysica Acta 1457 (2000) 273^277www.elsevier.com/locate/bba

tion of the precipitated substance and measurementof the breakdown products by high-performance liq-uid chromatography (HPLC). Using this assay, wehave been able to demonstrate the quantitative trans-fer of adenosine units between free ATP and purino-gen in perfused rat hearts [10,11]. However, the ¢nd-ings have shown that purinogen does not act as astorage reservoir for ATP in either moderate or se-vere ischaemia [11]. Conversely, they support a pro-posal that (PG-ATP)n may act as a rapidly exchange-able pool of intracellular inorganic phosphate wheretranssarcolemmal phosphate exchange is relativelyslow [10,11]. Moreover, its properties are consistentwith those expected of a labile sequestered form ofinorganic phosphate which discrepant ¢ndings from31P NMR and extraction studies have suggested mayexist in liver and heart (see [11]).

Here, we describe the technique we have devised toquantify tissue purinogen content, report that it canbe found additionally in brain and skeletal muscleand provide comparative measurements for theamounts present in some rat tissues.

2. Materials and methods

[8-14C]Adenosine (57 Ci/mol) was supplied byAmersham International, Little Chalfont, Bucks,UK. All other materials were as described previously[1,3,5,10,11].

2.1. Tissue sampling and extraction

Rats (220^250 g Sprague^Dawley strain bred atUCL) were killed by cervical dislocation. The brainwas immediately removed, rinsed in modi¢ed 4³CKrebs bicarbonate Ringer solution (4.8 mM KCl,1.2 mM KH2PO4, 0.6 mM MgSO4, 1.3 mM CaCl2,120 mM NaCl, 235.3 mM NaHCO3), pH 7, andfreeze-clamped with liquid nitrogen-cooled tongs. Inturn, the heart, liver, kidneys and hind-leg skeletalmuscle were treated in the same way: in total, thetime from tissue removal to freeze-clamping took nomore than 60 s. All subsequent operations were car-ried out with sterile media and apparatus.

The freeze-clamped tissue was then pulverised in amortar pre-cooled with liquid nitrogen and subse-quently homogenised in 3 ml of cold (310³C) 10%

(mass/volume) trichloroacetic acid (TCA)/25% (byvolume) methanol/10 mM MgCl2 using an UltraTurrax homogeniser TP 18 (JankepKunkel). Theprobe was washed with a further 3 ml of TCA/meth-anol/MgCl2 and the combined homogenate centri-fuged in a Sorvall RC-5B at 17 000 rev./min (SM-24 rotor; gav 35 600). The supernatant containingthe soluble purine nucleotides was stored at 370³Cand assayed by HPLC on a Waters Millenium Sys-tem using an APS-Hypersil column (15 cmU4 mm)developed with a linear gradient (5^400 mM) ofKH2PO4 pH 3.0. The column was calibrated usingstandard solutions of the adenine nucleotides.

2.2. Estimation of purinogen

The pelletted TCA/methanol/MgCl2-insoluble ma-terial was washed once with 10 ml cold (310³C)TCA/methanol/MgCl2, once with 10 ml acetoneand three times with 10 ml diethyl ether before beingdried under a stream of N2 gas. At each wash, thepellets were resedimented by centrifugation at 10 000rev./min in a Sorvall RC-5B (SM-24 rotor; 9774 gav).Between 0.1 g and 0.15 g of the weighed dried extractwas resuspended in 1 ml 4 M KOH/10 mM EDTAand placed in a water-¢lled sonic cleaning bath(Cole-Parmer 8850; output 88 W) at room temper-ature and agitated by sonication for 1 min and leftfor 15 min with repeated sonication periods at 5 minand 10 min. The samples were then centrifuged at10 000 rev./min in a Sorvall RC-5B (SM-24 rotor;9774 gav) for 10 min at 4³C. The supernatant wasneutralised with 2 M HCl and the precipitate re-moved by centrifugation for 2 min in an EppendorfZentrifuge 3200 (8000 gav). The pellet recovered fromthe Sorvall was resuspended in 0.5 ml KOH/EDTAand the above procedure repeated. The combinedsupernatants were stored at 370³C. Before HPLCanalysis of the released purine derivatives, 200 Wl ofsupernatant was ¢ltered through a 0.45 Wm Millipore¢lter. The quantitative extraction of purinogen wasfollowed and purinogen breakdown products identi-¢ed using extracts from hearts previously perfusedwith [8-14C]adenosine to label the purinogen [10].For radiochemical estimation, the HPLC column ef-£uent was collected in 750 Wl fractions and 200 Wlsamples of these were added to 4 ml of Ecosint Ascintillation cocktail (National Diagnostics) and as-

BBABIO 44867 11-4-00

P. Freeman et al. / Biochimica et Biophysica Acta 1457 (2000) 273^277274

sayed in a Packard Tri-Carb 900CA liquid scintilla-tion counter.

3. Results and discussion

3.1. Devising a quantitative assay for purinogen

In order to study the metabolic role of thevery labile rapidly turning-over (PG-ATP)n, weneeded a quantitative assay. Since we had shown inboth incorporation experiments [4] and by direct ex-traction and puri¢cation [7] that this compoundrapidly appeared to reach speci¢c activity equilibri-um with [14C]ATP, the obvious solution was to useradioactivity measurements after radiolabelling.However, with perfused hearts subjected to a varietyof metabolic stimuli, we were able to show convinc-ingly that the two pools were not at radioactivityequilibrium and that the stimuli led to changesin the speci¢c activity of the sequestered material[10].

Attempts to base an assay on the speci¢c phospho-diesterase which we had found to be located in themitochondrial intermembrane space [8] foundered,not only because we were unsuccessful in removingits tightly bound substrate while keeping it native,but also because, probably as a result of this tightassociation with protein, we were unable to resolu-bilise completely the radioactivity in the TCA/meth-anol-precipitated material in order to make it avail-able as substrate. We therefore devised a procedurewhich depended on conditions which promoted thebreakdown of purinogen but not that of more stablepolynucleotide species also present in washed TCA/methanol extracts: the breakdown products couldthen be separated by HPLC and measured by theirabsorbance at 259 nm as ATP equivalents (see [10]).Initial extraction using alkali/EDTA only solubilisedabout 50% of the precipitated radioactivity, mostlyas ATP, ADP and triphospho(5P)adenosine(3P)-3-phosphoglycerate (PPPAdoGri) (the product of(PG-ATP)n cleavage at its labile mixed anhydridebond): repeated extraction using sonication to aiddispersal of the insoluble pellet solubilised most ofthe remaining radioactivity but provided a much in-creased quantity of PPPAdoGri of much lower spe-ci¢c radioactivity (see [10]). It yielded moreover sev-

eral labelled peaks eluting before PPPAdoGri in theHPLC. These `unknowns' have as yet not been de-¢nitively identi¢ed but appear to be oligomers whichbreak down further on storage in the absence ofMg2� to give products that co-chromatograph withATP, adenosine, inosine and glyceric acid (M. Sarci-na, unpublished ¢ndings). While these unknownscould derive from a separate rapidly labelled labileprecipitable species, these ¢ndings suggest that theyare closely associated with (PG-ATP)n although theirphosphate:purine ratio is somewhat lower at around2:1. For these reasons, we have suggested that themore highly labelled (PG-ATP)n may form the end-chains of a more complex polymer of adenine nucle-otide, phosphate and glycerate for which we havesuggested the name purinogen [10].

Fig. 1 gives an example of the HPLC pro¢le ofpurinogen breakdown products obtained from a ratheart which had previously been perfused for 10 minwith [8-14C]adenosine to label the tissue adenine nu-cleotides [4]. Using the extraction technique de-

Fig. 1. The TCA/methanol/MgCl2-insoluble extract from aheart was incubated with KOH/EDTA as detailed in Section2.2. Fifty Wl of the solubilised ¢ltrate was analysed by HPLCon APS-Hypersil as detailed in Section 2.1. The triangles de¢nethe beginning and end of each peak whose computed area isbounded by the solid and dotted lines. The known peaks wereidenti¢ed by co-chromatography of standards. Those productsrapidly labelled by prior perfusion with [8-14C]adenosine [10]are shaded: purinogen content is estimated as the sum of thesepeaks using the molar absorbance of ATP as standard.

BBABIO 44867 11-4-00

P. Freeman et al. / Biochimica et Biophysica Acta 1457 (2000) 273^277 275

scribed here (Section 2.2), solubilisation of 98^100%of the precipitated radioactivity was obtained. Fig. 1shows too that the breakdown products also containspecies absorbing at 259 nm which are unlabelled byour perfusion regime and are therefore not rapidlyexchanging with free [14C]ATP as is purinogen. Wehave therefore chosen to ignore them and de¢ne pu-rinogen content as the sum of the absorbances of thelabelled species. The validity of this de¢nition is sup-ported by the observation of the quantitative transferof ATP equivalents between purinogen and solubleadenine nucleotides in perfused hearts subjected totwo di¡erent conditions which might be expected toalter the demand for intracellular inorganic phos-phate [11].

3.2. The purinogen content in rat tissues

A number of rat tissues were rapidly removed,freeze-clamped and the extracts assayed for purino-gen and the soluble adenine nucleotides as describedabove and in Section 2. As might be expected, thereis some variation in the proportions of the purinogenbreakdown products from di¡erent preparations; thesum of the adenine nucleotides represented between19 and 32%, PPPAdoGri 16 and 27% and the un-knowns 43 and 55% of the total. However, theHPLC traces all conformed to the typical patternshown in Fig. 1 and were thus readily identi¢ed bycomparison with those from labelled hearts. Radio-active perfusion studies similar to those with heartshad previously shown the presence of purinogen inkidney [5] and liver [6] but not its quantity. Theseextracts showed that purinogen was also readily de-tectable in brain and skeletal muscle and Table 1 setsout the amounts measured in tissues from four rats

relative to the tissue protein [12] and DNA [13] con-tent.

The use of a number of tissues from the sameanimal and the consequent time di¡erence in freeze-clamping between di¡erent tissues, however slicklythe sampling was organised, raises the prospect ofnucleotide metabolism in the brief ischemic interval.Previous studies with ischemic hearts, livers and kid-neys [5,6,10] have shown that in brief ischemic peri-ods, there is some net conversion of ATP to ADPand AMP but no change in the sum of these nucleo-tides. In the experiments reported here, with one ex-ception, ATP represented between 57% and 77% ofthe adenine nucleotide content (data not given): theexception was brain which, although sampled ¢rst,had equal amounts of ATP and AMP at 40% ofthe total. In the case of purinogen, we have shownwith perfused hearts that neither severe reversible (20min) or irreversible (40 min) ischemia causes anychange in the measured purinogen content fromthat in controls [11] and in line with this, the heartpurinogen content in Table 1 compares well withprevious estimates [10,11] using a mean protein con-tent of 140 mg/g wet tissue. It seems reasonabletherefore, at least initially, to expect that a brief pe-riod of ischemia will have no e¡ect on the purinogencontent of other rat tissues.

There is no obvious correlation between the con-tents of free nucleotides and those in purinogen inthe di¡erent tissues. The ¢ndings do show, however,that substantial proportions of tissue nucleotides arebound up in the polymer. In brain and mixed skeletalmuscle, the purinogen nucleotide content amounts toa quarter of that in free adenine nucleotide, while inheart it is 60% and in liver equal to that of the freeadenine nucleotides. Relatively the greatest propor-

Table 1The contents of purinogen and of free adenine nucleotides in some rat tissues

Purinogen (nmol/mg protein) 4 AN Purinogen (nmol/mg DNA) 4 AN

Liver 14.4 þ 1.5 14.9 þ 3.5 1 204 þ 74 1 209 þ 281Heart 10.96 þ 0.55 18.9 þ 1.2 647 þ 33 1 115 þ 73Brain 5.69 þ 0.78 23.5 þ 5.1 677 þ 62 2 769 þ 439Kidney 19.97 þ 0.99 14.5 þ 1.7 951 þ 110 726 þ 62Skeletal muscle 11.2 þ 1.8 46.2 þ 2.8 3 571 þ 574 16 600 þ 1 100

4 AN denotes the sum of the contents of ATP+ADP+AMP in the soluble tissue extract.

BBABIO 44867 11-4-00

P. Freeman et al. / Biochimica et Biophysica Acta 1457 (2000) 273^277276

tion is in kidney which has 35% more nucleotide inpurinogen than in the free state. For comparison, theadenine nucleotide content of rat heart total RNA isabout 30% of that in free nucleotides [1,14].

From these ¢ndings, the potential for rapidly turn-ing-over purinogen to augment the adenine nucleo-tide pool would appear to be considerable: and itsfailure to do so in severely ischemic rat hearts whenthere is substantial conversion of ATP to oxidisedpurines [11] is all the more surprising. By contrast,we have found that removing inorganic phosphatefrom heart perfusion bu¡er and providing glucoseto a heart previously perfused without nutrient todeplete endogenous supplies of glycogen and fattyacid caused net transfer of nucleotide between freeATP and purinogen [10,11]. These treatments mightbe expected to alter the demand for intracellular in-organic phosphate and have led to the suggestion[11] that the four phosphates per purine base presentin (PG-ATP)n may provide a controlled reservoir ofintracellular phosphate, which a number of 31PNMR studies have suggested may exist (see [11] forfurther discussion). Further investigation is needed toexamine this proposition; but it could be consistentwith the presence of a relatively large purinogen con-tent in kidney which has a major role in the controland transport of body inorganic phosphate.

References

[1] D.J. Bates, D. Perrett, J. Mowbray, Biochem. J. 176 (1978)485^493.

[2] J. Mowbray, D.J. Bates, D. Perret, FEBS Lett. 131 (1981)55^59.

[3] J. Mowbray, D. Perrett, Int. J. Biochem. 16 (1984) 889^894.[4] J. Mowbray, W.L. Hutchinson, G.R. Tibbs, Biochem. J. 223

(1984) 627^632.[5] W.L. Hutchinson, P.J. Ratcli¡e, J. Mowbray, Biochem. J.

240 (1986) 597^599.[6] B. Patel, C.D. Gove, W.L. Hutchinson, J. Mowbray, Bio-

chim. Biophys. Acta 1074 (1991) 178^181.[7] W.L. Hutchinson, P.G. Morris, J. Mowbray, Biochem. J.

234 (1986) 623^627.[8] B. Patel, A. Costi, D.L. Hardy, J. Mowbray, Biochem. J. 274

(1991) 275^279.[9] J. Mowbray, B. Patel, Adv. Enzym. Regul. 33 (1993) 221^

234.[10] B. Patel, M. Sarcina, J. Mowbray, Eur. J. Biochem. 220

(1994) 663^669.[11] B. Patel, Y. Berhane, A. Petrovic, J. Mowbray, Eur. J. Bio-

chem. 254 (1998) 75^80.[12] O.H. Lowry, N.J. Roseborough, A.L. Farr, R.J. Randall,

J. Biol. Chem. 193 (1951) 265^275.[13] B.R. Switzer, G.K. Summer, Clin. Chim. Acta 32 (1971)

203^206.[14] C. Mackie, T.H.E. Bryant, J. Mowbray, Biochem. J. 177

(1979) 977^979.

BBABIO 44867 11-4-00

P. Freeman et al. / Biochimica et Biophysica Acta 1457 (2000) 273^277 277