Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis

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Changes in metabolism of inorganic polyphosphate in rat tissues and human cells during development and apoptosis
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  Ž . Biochimica et Biophysica Acta 1335 1997 51–60 Changes in metabolism of inorganic polyphosphate in rat tissues andhuman cells during development and apoptosis Bernd Lorenz  a,b , Jessica Munkner  a , Marco P. Oliveira  c , Anne Kuusksalu  d , Jose M. Leitao  c , ¨ ´ ˜ Werner E.G. Muller  a , Heinz C. Schroder  a, ) ¨ ¨ a  Institut fur Physiologische Chemie, Abteilung Angewandte Molekularbiologie, Uni Õ ersitat, Duesbergweg 6, 55099 Mainz, Germany ¨ ¨ b  Institut fur Biochemie, Uni Õ ersitat, Leipziger Straße 44, 39120 Magdeburg, Germany ¨ ¨ c Unidade de Ciencias e Tecnologias Agrarias, Uni Õ ersidade do Algar  Õ e, Campus de Gambelas, 8000 Faro, Portugal d  Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 0026 Tallinn, Estonia Received 8 August 1996; revised 23 October 1996; accepted 25 October 1996 Abstract Age-dependent studies show that the amount of inorganic polyphosphate in rat brain strongly increases after birth.Maximal levels were found in 12-months old animals. Thereafter, the concentration of total polyphosphate decreases toabout 50%. This decrease in the concentration of total polyphosphate is due to a decrease in the amount of insoluble,long-chain polyphosphates. The amount of soluble, long-chain polyphosphates does not change significantly in the course of ageing. In rat embryos and newborns, mainly soluble polyphosphates could be detected. In rat liver, the age-dependentchanges are less pronounced. The changes in polyphosphate level are accompanied by changes in exopolyphosphataseactivity, which degrades the polymers to orthophosphate; highest enzyme activities were found when the polyphosphatelevel was low. Induction of apoptosis in the human leukemic cell line HL-60 by actinomycin D results in degradation of long polyphosphate chains. The total polyphosphate content does not change significantly in apoptotic cells. Ž . Ž . Keywords:  Polyphosphate; Exopolyphosphatase; Aging; Apoptosis; HL-60 cell; Rat brain ; Rat liver 1. Introduction Ž . Inorganic linear polyphosphates polyP are en-ergy-rich polymers of up to several hundreds of  Ž . orthophosphate  P  residues. They are widely dis- i Abbreviations: EDTA, ethylenediaminetetraacetic acid;  P  , i orthophosphate; polyP , inorganic polyphosphate with a chain n length of   n  residues; SDS, sodium dodecyl sulfate ) Corresponding author. Fax:  q 49 6131-395243; E-mail:hschroed@mzdmza.zdv.uni-mainz.de tributed but have been ignored for a long time. Moststudies on polyP have been performed in lower or- Ž . w x ganisms bacteria, yeasts, and algae 1,2 . There areonly few studies concerning the occurrence of polyP w x in animals 3–8 after the first report from our insti- w x tute more than 30 years ago 9 . PolyP is non-ran-domly distributed within the cell with an enrichment w x in the nucleus 3,10 , the mitochondria and the plasma w x membrane 11 . The biological function of polyP isuncertain and may be different in different compart-ments of the cell. PolyP has been suggested to serve: Ž . Ž . i as a source of energy; ii as an easily available 0304-4165 r 97 r $17.00 Copyright q  1997 Elsevier Science B.V. All rights reserved. Ž . PII   S0304-4165 96 00121-3  ( ) B. Lorenz et al. r  Biochimica et Biophysica Acta 1335 1997 51–60 52 w x  Ž . phosphate reserve 1 ; iii as chelator for divalent Ž  2 q . w x  Ž . cations Ca or toxic metal ions 12 ; iii counter- w x  Ž . ion for basic amino acids in vacuoles 13 ; or iv w x donor of phosphate for certain sugar kinases 14,15 . Ž . More recently, it has been proposed that: v hydroly-sis of polyP may provide a pH-stat mechanism to w x  Ž . counterbalance alkaline stress 16 ; and vi elonga-tion and shortening of polyP chains may be associ- w x ated with the response of cells to osmotic shock 17 .PolyP is able to inhibit the formation of ATP byadenylate kinase in dependence on its chain length,suggesting a potential role in regulation of the intra- w x cellular levels of adenylate nucleotides 8 . The func-tion of nuclear polyP is even less understood.The synthesis of polyP in bacteria is mediated bythe polyphosphate kinase which catalyzes the transfer w x of   P  from ATP to polyP 18 ; cells from higher i organisms may use a different mechanism for the w x formation of high-molecular polyP 6,7 . The degra-dation of polyP is catalyzed by several endo- and w x exopolyphosphatases 8,19–23 .The cellular content and size of polyP may varyconsiderably depending on the availability of phos-phate and the period of growth cycle. PolyP is accu-mulated when cells are transferred from aphosphate-depleted medium to a phosphate-rich w x medium 1,24 . In addition, the polyP content maychange during development, as shown in  Physarum w x w x  polycephalum  25 and  Ephydatia muelleri  26 .In the present work the age-dependent changes inpolyP content and breakdown were studied in differ-ent tissues of rat. High amounts of polyP were foundin the nuclei of rat brains and livers. Nuclear polyPhas been proposed to play a role in regulation of chromatin activity by the dissociation of histones w x w x 27,28 or interaction with non-histone proteins 5 .Here we show that polyP metabolism markedlychanges in human leukemic HL-60 cells during apop-tosis, a physiological control mechanism character-ized by degradation of DNA to oligonucleosomal w x fragments 29,30 . 2. Materials and methods 2.1. Materials w 32 x  Ž Monosodium P phosphate specific activity, 600 . Ci r mmol was obtained from ICN Biomedicals Ž . Meckenheim, Germany ; polyP , polyP , polyP types 3 4 Ž 15, 35, 65, and 75 q  sodium salts; with average . chain lengths 15, 34, 65, and 91 , actinomycin D, and Ž  w x 3- 4,5-dimethylthiazol-2-yl -2,5-diphenyltetrazolium Ž . bromide MTT were purchased from Sigma Chemi- Ž . cal Co. St. Louis, MO ; leupeptin, pepstatin, deoxy- Ž . ribonuclease I DNase I; bovine pancreas and ribo- Ž . nuclease A RNase A; bovine pancreas were from Ž . Boehringer Mannheim Mannheim, Germany . w 32 x P PolyP was synthesized as described previ- w x  Ž ously 23,31 . The specific radioactivity was 0.6– .  7 1.0  P 10 dpm r m mol of   P  . i 2.2. Animals Ž Wistar rats inbred strain; from our own breeding . Ž facilities of different age embryo: 18 days old;newborn: 5 days old; young adult: 3 months old;middle-aged adult: 12 months old; and old: 28 months . old were used. Brain and liver were rapidly takenfrom the animals and frozen in liquid nitrogen. 2.3. Cell culture Human promyelocytic leukemia HL-60 cells were Ž grown in RPMI 1640 medium Gibco, Karlsruhe, . Germany supplemented with 10% fetal calf serum at37 8 C in 5% CO r air. Cells were routinely seeded at 2 a concentration of 1 P 10 5 cells r ml.Apoptosis was induced by treatment of exponen- Ž . tially growing proliferating or non-proliferating HL- Ž . 60 cells 30-ml cultures with 0.5  m g r ml of actino-mycin D for 0–19 h. After harvesting the cells werewashed in medium without serum, and the wet massof the pellet and the amount of protein and ex-tractable polyP, and the percentage of fragmentatedDNA were determined. 2.4. Preparation of nuclei Nuclei were isolated from rat liver and brain using w x a modification 32 of the method described by Blo- w x bel and Potter 33 . 2.5. Extraction and determination of polyP content  PolyP was extracted from HL-60 cells or frozen rat w x tissues as described 17,34 . Step 1 extracts containacid-soluble short-chain polyP, while step 2 extractsrepresent the easily extractable, ‘soluble’ portion of long-chain polyP and step 3 extracts the remaining  ( ) B. Lorenz et al. r  Biochimica et Biophysica Acta 1335 1997 51–60  53 w x ‘insoluble’ portion of long-chain polyP 34 . Residualamounts of DNA and RNA were removed from theextracts obtained in the steps 2 and 3 by treatment Ž . with RNase A and DNase I 250  m g r ml each in thepresence of 1 mM MgCl for 1 h at room tempera- 2 ture; reactions were stopped by addition of 2 mM Ž . EDTA final . Protein was then removed by one Ž . extraction with phenol r chloroform 1:1, v r v , fol-lowed by three successive extractions with chloro-form.The content of long-chain polyP of the step 2 andstep 3 extracts was estimated by measuring themetachromatic effect produced by polyP on the ab- w x sorption spectrum of toluidine blue, as described 17 .Samples containing polyP were added to the dye Ž solution final concentration: 6 mg r l; dissolved in 40 . mM acetic acid and the absorbance values at 530 nmand at 630 nm were determined, using a BeckmanDU-64 spectrophotometer. PolyP samples of un-known concentration were diluted or concentrateduntil the ratios of the absorbance values at 530 nmand at 630 nm were within the range 0.5–2.0. Previ-ously we showed that the change in the absorbanceratio 530 nm r 630 nm is within this range roughly Ž proportional to the polyP concentration concentra- . tion range, 25–75  m M; expressed in  P  if the chain i w x length of the polymer is  G 15  P  residues 17 . A i Ž calibration curve was obtained using polyP Sigma 35 . type 35 as a standard. PolyP of less than 15  P i residues induces metachromasy to a lesser extent or Ž does not react metachromatically polyP F 5  P i .  w x residues 17 , possibly resulting in an underestima-tion of polyP content. However, the step 2 and step 3extracts contain only very low amounts of polyP of  Ž . - 15  P  residues see also Fig. 5 and Section 3 . i Residual polyP not extracted by steps 1–3 wasdetermined by the method of Malpartida and Serrano w x w x 35 , after acid hydrolysis at 100 8 C 36 . Determina-tions of the amount of acid hydrolyzable phosphateremaining in the residual pellets revealed that ; 95%of the total polyP has been extracted by steps 1–3 Ž . results not shown . 2.6. Extraction and assay for exopolyphosphataseacti Õ ity Frozen rat tissues were thawed and homogenized Ž . in 50 mM Tris-HCl buffer pH 7.5 containing 10mM MgCl , 0.5 mM EDTA, 150 mM NaCl, 100 2 Ž m M leupeptin, and 100 nM pepstatin 2 ml of buffer . per gram tissue; 4 8 C . The supernatant obtained after Ž . centrifugation 15000 = g , 5 min was used for de-termination of exopolyphosphatase activity. The reac- Ž tion mixture consisted of 50 mM Tris-HCl buffer pH . 7.5, containing 150 mM NaCl and 5 mM MgCl in a 2 Ž final volume of 100  m l. PolyP Sigma type 35, 0.3 35 . m mol of   P  residues r assay was used as substrate. i Incubations were performed at 37 8 C for various timeperiods. The  P  formed during the reaction was i determined spectrophotometrically as previously de- w x scribed 26 , by adding 300  m l of distilled water, 1ml of a solution containing 0.5% ammonium molyb-date, 0.35 M sulfuric acid and 0.5% SDS, and 10  m lof 10% ascorbic acid. Measurements of absorbancewere performed after 5 min.In some assays, unlabelled polyP was replaced by w 32 x  Ž . P polyP 1.6 nmol of   P  residues r assay . The i degradation products were analyzed either by gel Ž . electrophoresis see below or by chromatography oncellulose thin layer plates which were developed with Ž . isobutyric acid r NH OH r water 25:3:12 containing 4 w x 0.8 mM EDTA 16 . [ 32 ]2.7. Isolation of P polyP from HL-60 cells Ž  5 . HL-60 cells 5 P 10 cells r ml were incubated in 32 Ž  10 . medium supplemented with  P  1 P 10 dpm r ml i for the indicated time periods. Cells were then har-vested by centrifugation, washed in 10 mM Tris-HCl Ž . buffer pH 7.2, containing 140 mM NaCl and then Ž resuspended in 10 mM Tris-HCl buffer pH 7.2, .  5 containing 1 mM EDTA at 5 P 10 cells r ml. After Ž . addition of 0.1 volume of 10% w r v SDS the cells Ž were disrupted by sonication 3 P 10 min with 30 s . chilling periods , then extracted twice withphenol r chloroform and once with chloroform. ThepolyP was then precipitated by addition of 0.01 vol-ume of 3 M sodium acetate plus 3 vols. of ethanol.The pellet obtained after standing for 30 min at Ž . y 80 8 C and centrifugation 14000 rpm, 4 8 C was Ž resuspended in 10 mM Tris-HCl buffer pH 7.2, . containing 1 mM MgCl supplemented with 250 2 m g r ml RNase A and 250  m g r ml DNase I. Afterincubation for 1 h at room temperature the sampleswere extracted with phenol r chloroform and chloro-form, and polyP was isolated by precipitation with  ( ) B. Lorenz et al. r  Biochimica et Biophysica Acta 1335 1997 51–60 54 Ž . sodium acetate and ethanol as above . The pellet wasdissolved in distilled water. 2.8. Determination of chain length of polyP The size of the polyP was determined by elec-trophoresis on 7 M urea r 16.5% polyacrylamide gels w x w 32 x as described 23,37 . P PolyP was detected byautoradiography and unlabelled polyP by stainingwith  o -toluidine blue O. PolyP standards of defined w x chain lengths 37 were run in parallel. 2.9. DNA fragmentation DNA fragmentation was determined by elec-trophoresis on 1% horizontal agarose gels as de- w x scribed 38 . The gels were stained with ethidiumbromide, and the ladders were visualized by UVfluorescence. In addition, for quantitative analysis of DNA fragmentation the sedimentation technique was w x applied 29,39 . 2.10. Cell  Õ iability The viability of total cells was determined using w x the MTT colorimetric assay 40 , followed by evalua- Ž . tion with an ELISA reader Bio-Rad, model 3550 . 2.11. Analytical methods Protein was determined as described by Bradford w x 41 with bovine serum albumin as a standard, and w x DNA by the procedure of Kissane and Robbins 42 . 3. Results 3.1. Age-dependent changes of polyP in rat tissue The changes in polyP metabolism in rat brain andliver in the course of ageing and development weredetermined by measuring the amount of the polymerin the soluble and insoluble polyP fraction applyingthe metachromatic reaction.The total polyP content in adult rat brain wasfound to be about 2-fold higher than in adult rat liver Ž . Figs. 1 and 2 . Age-dependent studies revealed thatthe amount of polyP in embryonic tissue was low Fig. 1. Age-dependent changes in polyP content of rat brain. Ž PolyP was determined in step 2 extract soluble polyP; dark grey . Ž . bars and step 3 extract insoluble polyP; light grey bars from ratbrain as described in Section 2. Each value represents the mean Ž . Ž " S.D. of the assays performed on five E, embryo; N, new- . born; Y, young adult; and M, middle-aged adult rats or four Ž . animals O, old rats . Ž . compared to adult tissue Fig. 1 . The polyP in ratembryo consists essentially of soluble polyP chains.The amount of this polymer strongly decreased in Ž . newborn rat brain Fig. 1 . Thereafter it stronglyincreased. This increase in polyP in rat brain ismainly caused by an increase in insoluble, long-chain Ž . polyP Fig. 1 . The amount of polyP remained essen-tially unchanged when young adult and medium-agedadult animals were compared. In old rat brain the Fig. 2. Age-dependent changes in polyP content of rat liver. Forfurther details, see legend to Fig. 1.  ( ) B. Lorenz et al. r  Biochimica et Biophysica Acta 1335 1997 51–60  55 amount of total polyP markedly decreased by about Ž . 50% Fig. 1 . This decrease is mainly due to adecrease in the amount of insoluble long-chain polyP;the amount of soluble long-chain polyP remainedessentially constant.These age-dependent and development-dependent Ž . changes were less pronounced in rat liver Fig. 2 . Incontrast to embryonic brain, significant amounts of long-chain polyP could be detected in liver from ratembryo. The amount of insoluble long-chain polyPbut less the amount of soluble long-chain polyPdecreased in newborn liver. Thereafter it increases.Only a slight decrease was found in liver of old rats Ž . Fig. 2 .Determination of polyP in nuclei from rat brain Ž . and liver adult tissue indicate that the polyP ismainly present in these organelles. The concentra-tions of polyP in nuclei from rat brain amounted to Ž . Ž . 45  m M soluble polyP and 119 m M insoluble polyPand those in nuclei from rat liver were 36  m M Ž . Ž . soluble polyP and 71  m M insoluble polyP . 3.2. Changes in exopolyphosphatase acti Õ ity in rat tissue Extracts from both rat brain and liver were foundto contain a polyP-degrading activity when incubated Fig. 3. Age-dependent changes in exopolyphosphatase activity inextracts from rat brain and liver. Exopolyphosphatase assays Ž . were performed in total cell extracts from brain dark grey bars Ž . and liver light grey bars , using polyP as substrate. Results are 35 Ž . Ž means  " S.D. of the assays performed on five E, embryo; N, . newborn; Y, young adult; and M, middle-aged adult rats or four Ž . animals O, old rats .Fig. 4. Analysis of the product formed by polyP-degrading w 32 x activity in rat brain extract. P PolyP was incubated with theenzyme present in rat brain extract under standard assay condi-tions. Reaction was terminated by ethanol precipitation and both Ž . the supernatant containing  P  lane a and the pellet containing i Ž . undegraded polyP lane b were subjected to analysis by thin-layer w 32 x  Ž chromatography. Lane c, unreacted P polyP zero time con- . trol . O, srcin. Ž . with polyP as substrate Fig. 3 . Degradation of  35 polyP resulted in the production of   P  as revealed by i w 32 x analysis of the degradation products of P polyP by Ž . thin-layer chromatography Fig. 4 . This result indi-cates that the enzyme is an exopolyphosphatase. Sim- Ž . ilar results were obtained when shorter polyP and 15 Ž . Ž longer polymers polyP were used as substrate not 91 . shown .As shown in Fig. 3, the age-dependent changes inpolyP content in rat brain and liver were accompa-nied by changes in exopolyphosphatase activity.Measurements of degradation of polyP substrate 35 revealed that the level of enzyme activity strongly Ž . increased in newborn rats both brain and liver .Thereafter it markedly decreased. Therefore the activ-ity of the polyP-degrading activity is reversely pro-portional to the polyP content. 3.3. Changes in size and content of polyP duringapoptosis HL-60 cells underwent apoptosis when incubatedwith actinomycin D. The typical stepladder-like gel w x pattern of multiples of   ; 180 base pairs 29 was
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