©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Inorganic Polyphosphate in Mammalian Cells and Tissues (*)

(Received for publication, November 30, 1994; and in revised form, January 4, 1995)

Krishnanand D. Kumble Arthur Kornberg

From the Department of Biochemistry, Beckman Center, Stanford University School of Medicine, Stanford, California 94305-5307

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (P(i)) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods. Subpicomole amounts of polyP (0.5 pmol/100 µg of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to P(i) by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to 120 µM (in terms of P(i) residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes). PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally. Conversion of P(i) into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h. Synthesis of polyP from P(i) in the medium bypasses intracellular P(i) and ATP pools suggesting the direct involvement of membrane component(s). In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours. The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.


INTRODUCTION

Inorganic polyphosphates (polyP) (^1)are linear polymers of orthophosphate (P(i)) residues linked by high energy phosphoanhydride bonds. PolyP has been found in all organisms ranging from bacteria to mammals(1) . The ubiquitous occurrence of polyP is suggestive of some important physiological role(s) for this polymer. PolyP has been proposed as a (i) source of energy(1, 2) , (ii) phosphate reservoir(1) , (iii) donor for sugar and adenylate kinases(3, 4, 5) , (iv) chelator for divalent cations(6, 7) , (v) buffer against alkaline stress(8) , (vi) regulator of development (9) , and (vii) structural element in competence for DNA entry and transformation(10) .

Most polyP research has dealt with microorganisms, in which it may accumulate in large quantities; in contrast, there are few reports of polyP in higher eukaryotes. The presence of polyP has been demonstrated in human granulocytes (11) and fibroblasts (12) and in rat liver nuclei (13) and mitochondria(14) . However, the lack of definitive and sensitive analytic methods has left their identity and abundance uncertain and their metabolic and functional roles obscure.

In order to establish methods to study the physiological functions of polyP, we devised procedures to estimate subpicomole levels of polyP by employing two novel and specific enzyme assays. One converts polyP to ATP with purified Escherichia coli polyphosphate kinase (PPK) and [^14C]ADP(15) , and the other converts polyP to P(i) with purified recombinant Saccharomyces cerevisiae exopolyphosphatase (rPPX1)(16) . The size distribution of polyP is estimated by urea-PAGE.

In the present report, we determined the level and size of polyP in a variety of rat and mouse tissues and subcellular organelles and in mammalian cell lines labeled with [P]P(i). The dynamic nature of polyP was demonstrated by a rapid rate of synthesis and turnover in some cell lines; our data also indicate a novel route of polyP synthesis from P(i) that bypasses the intracellular pools of P(i) and ATP.


MATERIALS AND METHODS

Reagents

Sources were: ATP, ADP, DNase I, RNase IIIa, and ATP bioluminescence kit from Boehringer Mannheim; polyP (type 65) from Sigma; [^14C]ADP and carrier-free [P]P(i) from Amersham Corp.; polyethyleneimine-cellulose thin layer chromatography plates with fluorescence marker from Merck; all tissue culture media from Life Technologies, Inc.

Tissues and Cell Lines

Fisher 344 rats and BALB/c mice were sacrificed at 4 to 6 weeks of age; organs were frozen in liquid nitrogen. Mouse L, Jurkat, Vero, and 293/E1A cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum; for NIH 3T3 cells, fetal bovine serum was replaced with calf serum. PC12 cells were maintained in Dulbecco's modified Eagle's medium containing 6% fetal bovine serum and 6% equine serum.

[P]P(i) Labeling of Cells

Cells were grown to confluency in 14-cm plastic Petri dishes in the recommended medium. Jurkat cells were grown in suspension to a density of 10^8 cells/ml; the cells were pelleted by centrifugation at 1000 times g for 5 min. The medium was drained off and the cells rinsed once with P(i)-free medium. The cells were then labeled for 60 min with [P]P(i) (0.02 mCi/ml) in P(i)-free medium supplemented with 100 µM KPO(4) (pH 7.0). The cells were rinsed several times with phosphate-buffered saline, harvested, and used for extraction of polyP.

Extraction of PolyP

The washed, radiolabeled cell pellet was suspended in 500 µl of lysis buffer (50 mM Tris-HCl at pH 7.5, 1 M urea, 0.1% SDS, and 10 mM EDTA) and sonicated in an ice-bath for three 10-s bursts with 30-s cooling intervals. When tissue was the source of polyP, it was suspended in 2.5 volumes of lysis buffer containing 0.25 M sucrose and homogenized using a Polytron (Brinkmann Instruments) for three 10-s bursts with 30-s cooling between bursts. The lysate was incubated with proteinase K, 750 µg/ml, at 37 °C for 2 h. The sample was extracted with phenol/chloroform (1:1, w/v equilibrated with Tris-HCl, pH 7.5); the phases were separated by centrifugation at 14,000 times g for 10 min. The aqueous phase was transferred to another tube and the phenol layer was back-extracted twice with 50 mM Tris-HCl, pH 7.5, 10 mM EDTA. The pooled aqueous phase was extracted with chloroform. PolyP (Sigma type 65; 125 nmol as P(i) residues) was added as carrier when extractions were performed with radiolabeled cells. The polyP was precipitated with barium acetate at a final concentration of 0.1 M at pH 4.5 for 4 h at 4 °C. The precipitate was collected by centrifugation at 14,000 times g for 30 min. The precipitate was solubilized using Dowex AG-50W (sodium form) and the sample was incubated with DNase I and RNase A each at 350 µg/ml, in 5 mM MgCl(2) for 1 h at 37 °C. The sample was deproteinized by extracting with phenol/chloroform and chloroform as described above. The polyP was concentrated under vacuum and analyzed. The efficiency of extraction as determined by recovery of exogenously added [P]polyP (synthesized from purified E. coli PPK and [P]ATP) was 90-95%.

Assay for PolyP

Assay using PPK

PolyP extracted from unlabeled sources was estimated using purified E. coli PPK. This enzyme catalyzes the reversible transfer of phosphate from polyP to ADP. With ADP in excess, PPK catalyzes the complete consumption of polyP, yielding an equivalent amount of ATP. The reaction mixture (10 µl) contained 50 mM HEPES-KOH (pH 7.2), 40 mM (NH(4))(2)SO(4), 4 mM MgCl(2), 100 µM [^14C]ADP, 2000 units of PPK, and the sample containing polyP. The reaction was incubated at 37 °C for 45 min and terminated by chilling on ice and the addition of ATP and ADP to 1 mM each. Then, 4 ml was spotted onto polyethyleneimine cellulose-TLC, and the chromatograms were developed in 0.4 M LiCl and 1 M HCOOH. Spots corresponding to ATP and ADP were cut out after visualization by UV-shadowing, and the amount of [^14C]ATP was determined by liquid scintillation.

Assay Using rPPX1

PolyP extracted from labeled cells was estimated using purified rPPX1 in a reaction mixture containing (in a final volume of 10 µl) 100 mM Tris-HCl (pH 7.5), 50 mM ammonium acetate, 5 mM magnesium acetate, and 1000 units of rPPX1. The assay was at 37 °C for 30 min, followed by polyethyleneimine cellulose-TLC as described. PolyP disappearance was estimated by removal of radioactivity from the origin.

Electrophoretic Analysis of PolyP

Urea-polyacrylamide gels were prepared by mixing 10.51 g of urea, 3.75 ml of acrylamide solution (38 g of acrylamide and 2 g of bisacrylamide dissolved in deionized water to a final volume of 100 ml), and 2.5 ml of 0.9 M Tris borate (pH 8.3) and 27 mM EDTA, to a final volume of 25 ml. Ammonium persulfate (15 µg) and TEMED (25 µl) were added; the gel was poured (14 times 23 times 0.75 mm) and allowed to polymerize. Gels of other strengths were made with appropriate volumes of the stock acrylamide solution. The gel was pre-electrophoresed at 300 V for 1 h. PolyP was mixed with 0.25 volume of 5times sample buffer (50% sucrose, 0.125% bromphenol blue, and 450 mM Tris borate at pH 8.3, 13.5 mM EDTA) and loaded on the gel. Electrophoresis was at 300 V until the dye was 7 to 8 cm from the top of the gel. Gels were stained with 0.05% toluidine blue in 25% methanol and 5% glycerol for 20 min followed by destaining in the same solvent without toluidine blue. Gels containing radioactivity were analyzed on a PhosphorImager (Molecular Dynamics) and autoradiography on Kodak X-Omat AR film. For determination of chain lengths, marker polyP of various sizes was electrophoresed along with the samples and the markers localized by toluidine blue staining. Centimeters migrated versus chain length was plotted, the mobility of the sample was measured, and its chain length was determined by extrapolating from the plot.

Specific Radioactivity Determinations of Intracellular P(i) and ATP

Confluent cells were labeled as described, washed, and resuspended in 0.5 ml of 8% chilled perchloric acid. The mixture was kept on ice for 15 min and neutralized with 1 M KOH. The mixture was centrifuged at 12,000 times g in a Microfuge, and the supernatant was used for analyses. P(i) was estimated by the method of Chen et al.(17) . Specific radioactivity of P(i) was determined by a modified, acid-molybdate extraction (11) . ATP was estimated by the luciferase assay performed according to the manufacturer's instructions (Boehringer Mannheim). Specific radioactivity of ATP was determined by liquid scintillation after cutting the spot corresponding to ATP following polyethyleneimine cellulose-TLC.

Other Methods

PPK and rPPX1 were purified as described(15, 16) . The protein concentration was determined by the method of Bradford (18) with bovine serum albumin as the standard. Rat liver organelles were isolated according to Fleischer and Kervina(19) .


RESULTS

PolyP Abundance in Rodent Tissues

PolyP was present in all tissues examined and at similar levels (Table 1); values for rat and mouse tissues were also similar. PolyP in rat brain examined during various stages of development showed no marked differences in levels (Table 2) or in size distribution. Only long chain polyP of about 800 residues was observed (see below).





The distribution of chain lengths was similar in all the tissues examined except for brain. PolyP with chain lengths from 50 to 800 residues was detected in heart, kidneys, lungs, and liver on 4% urea-PAGE after staining with toluidine blue. By contrast, polyP obtained from brain had an average size of about 800 residues; short-chain polyP was less than 2% of the total.

Distribution of PolyP in Subcellular Organelles

PolyP in subcellular organelles, isolated from rat liver by the method of Fleischer and Kervina(19) , showed relatively higher levels in the nuclei and plasma membranes compared to the cytosol, mitochondria, and microsome fractions (Table 3). The polyP to protein ratio in nuclei was 490 pmol/mg; that of plasma membrane was 230.



Incorporation of [P]P(i) into PolyP in Mammalian Cell Lines

Because of the low levels of polyP in mammalian cells, previous measurements based on barium precipitation and acid lability gave equivocal results. We have used two specific enzyme assays to measure [P]polyP extracted from labeled cells. One assay uses rPPX1, an effective exopolyphosphatase that hydrolyzes polyP of lengths ranging from 3 to 750 completely to P(i) and one residue of PP(i)(16) . The other assay uses E. coli polyP kinase (PPK) to transfer a phosphate from polyP to ADP to form ATP; with ADP in excess, PPK converts near 90% of polyP to ATP using [^14C]ADP(20) . When cells were labeled with [P]P(i) at specific activities of 500 cpm/pmol, the [P]polyP synthesized can be estimated using either PPK or rPPX1. Agreement between the two assays was within 5%. Amounts ranging from 0.5 pmol to 60 pmol of polyP (as P(i) equivalents) can be estimated using either enzyme. These enzymes do not hydrolyze ATP, PP(i), or nucleic acids. Among several procedures for extraction of polyP, the one described under ``Experimental Procedures'' recovered 90 to 95% of exogenously added [P]polyP.

Cultures of mammalian cell lines, upon reaching confluence, were exposed to [P]P(i) for 1 h in a medium to which 100 µM KPO(4) (pH 7.0) was added. Incorporation of P(i) into polyP varied depending on the cell line (Table 4). Values are based on the specific radioactivity of P(i) in the medium and represent the average of at least three independent labeling experiments. Mouse fibroblasts (NIH 3T3), human lymphoma cells (Jurkat), and transformed Monkey kidney cells (Vero) synthesized 2- to 3-fold more polyP per h than did human embryonal kidney cells (293/E1A) or mouse mammary gland cells (Mouse L). PC12, a cell line derived from pheochromocytoma of the rat adrenals, had the lowest synthesis of polyP.



Pathway of P(i) Incorporation into PolyP

Measurement of polyP synthesis based on the specific radioactivity of P(i) in the medium is uncertain because of the possibility of dilution with intracellular pools of P(i), ATP, and other intermediates. The intracellular specific radioactivities of P(i) and ATP were nearly similar and from 9- to 50-fold lower than that of P(i) in the medium (Table 5). When the extent of polyP synthesis was estimated on the basis of intracellular specific radioactivities of either P(i) or ATP, the values calculated (Table 6) were 2- to 12-fold higher in view of the levels of total polyP estimated from measurements from unlabeled cells (Table 5). Values of polyP synthesis calculated on the basis of the specific radioactivities of P(i) in the medium (Table 4) were more in line with those expected from the known content of polyP and thus indicative of a pathway of P(i) incorporation into polyP that bypasses the intracellular pools of P(i) and ATP.





Chain Lengths of PolyP in Cell Lines

[P]polyP extracted from cell lines was electrophoresed on 6% urea-PAGE to determine the size distribution (Fig. 1). There were two size classes of polyP in labeled mammalian cells: long chains of 500 to 800 residues and short chains of 5 to 15 residues. The relative amounts of the two species varied depending upon the cell line. Hydrolysis of the [P]P(i)-labeled material upon rPPX1 treatment before electrophoresis confirmed that the isolated material was polyP (data not shown).


Figure 1: Urea-PAGE analysis of [P]polyP from cell lines. The [P]P(i)-labeled material extracted from various cell lines was electrophoresed on a 6% urea-PAGE. Chain lengths of standards are on the right.



Kinetics of PolyP Synthesis and Turnover in Cell Lines

[P]PolyP levels increased linearly with time in NIH 3T3 cells at a rate of 0.5 pmol of polyP (in terms of P(i) residues) synthesized per min per mg of protein (Fig. 2A). PolyP synthesis in PC12 cells after an initial lag increased rapidly up to 60 min and then declined to 50% of the maximal levels at 2 h (Fig. 2B).


Figure 2: Time course of [P]P(i) incorporation into polyP in cell lines. Confluent cells in 14-cm plates were labeled for various periods of time, and the [P]polyP was extracted and quantified using rPPX1. PolyP is expressed as picomoles of P(i) residues incorporated per mg of protein. A, NIH 3T3 cells; B, PC12 cells.



To study the turnover of polyP, confluent cells were labeled with [P]P(i) for 1 h and then chased with unlabeled medium. In PC12 cells, about 70% of the label in polyP was removed within 1 h (Fig. 3B); whereas in NIH 3T3 cells there was no significant change after 4 h (Fig. 3A).


Figure 3: Turnover of polyP in cell lines. Confluent cells in 14-cm plates were labeled for 60 min and the medium was drained off. Cells were washed with P(i)-free Dulbecco's modified Eagle's medium followed by addition of unlabeled medium. At the indicated times, the cells were harvested and washed, and the polyP was extracted. The amount of [P]polyP was estimated using rPPX1 and is expressed in terms of P(i) residues. A, NIH 3T3 cells; B, PC12 cells.




DISCUSSION

There have been several reports of inorganic polyP in animal cells and tissues(11, 12, 13, 14, 21) , but it is difficult to assess their validity and possible significance for lack of sensitivity and reliability of the methods employed. Suggestions for a polyP role in DNA and RNA synthesis by interactions with non-histone nuclear proteins (22) and with chromatin (23, 24, 25) have not been supported by further studies. Nor have any enzymes been identified and characterized for the synthesis and utilization of polyP.

In an effort to understand the metabolism and functions of polyP in all biologic forms, we have improved the assays for polyP with two definitive and sensitive enzymatic reagents. Conversion of polyP to ATP by a pure polyP kinase and hydrolysis of polyP to P(i) by a pure exopolyphosphatase each give similar values. Still some uncertainties exist about how to obtain extracts from cells and tissues that ensure the dissociation of polyP from its aggregates and complexes. Also, fractionation of the extracts to prepare polyP as a substrate for the enzyme assays remains rather cumbersome and may entail losses.

The levels of polyP, expressed as P(i) equivalents, were determined in murine tissues (Table 1) and in several cell lines ( Table 4and Table 5). The values were near 100 µM in mouse brain, heart, and lungs, which is roughly 1% that of DNA, were expressed as nucleotides. The amounts were in the same general range (26 to 54 µM) for rat tissues.

Among the subcellular organelles isolated from rat liver, nuclei and plasma membranes contained several times the concentrations found in cytosol, mitochondria, and microsomes (Table 3). Earlier reports on polyP synthesis by rat liver nuclei (13) gave values of 1 mg of polyP/g of tissue/h with chain lengths ranging from 2 to 500 residues. Lynn and Brown (14) studied synthesis of polyP by rat liver mitochondria and estimated the size to be around 1000 residues. These chain length estimations were based on migration on paper chromatograms and could be erroneous since the method usually assumes an arbitrary length for nonmigrating species.

With regard to the biosynthesis of polyP, little is known beyond the capacity of granulocytes (11) and lysosomes (12) to take up P(i) in indeterminate amounts. No enzyme comparable to PPK has been described as yet. In our own studies of the uptake by several cell lines of P(i) from the medium, it is clear that there is little or no mixing with the major pools of P(i) or ATP inside the cell ( Table 5and Table 6). For example, the amount of polyP synthesized in 1 h by mouse fibroblasts calculated from the specific radioactivity of intracellular P(i) and ATP, respectively, would be 3100 and 2090 pmol per mg of protein per h, whereas only one-tenth as much is actually present in these cells. Calculations based on the specific radioactivity of P(i) in the medium would indicate that about 20% of the cellular level of polyP was synthesized in 1 h. Similar results with the other cell lines also indicate that the pathway from P(i) in the medium to polyP in the cell involves either an intracellular P(i) or ATP pool sequestered from the principal pools or some novel mechanism, such as fixation of P(i) in polyP driven by a protonmotive force. Cowling and Birnboim (11) have reported that depletion of cellular ATP levels in granulocytes resulted in an increase in the amount of [P]P(i) incorporated into polyP and suggest a mechanism not requiring ATP.

The polyP metabolic patterns of the cell lines examined differed sharply. Measured by the uptake of P(i) into polyP (Fig. 2), confluent PC12 cells synthesized half or more of their cell content of polyP in 1 h, as compared with embryonal kidney cells which synthesized only about 5% of the polyP complement. Measured after a 1-h chase (Fig. 3), the turnover of polyP was 70% in PC12 cells, but hardly evident in fibroblasts. The rapid turnover in PC12 cells is remarkable in that the cells were confluent, having attained that stage with a generation time of 48-72 h. How metabolic patterns of polyP are influenced by stages in the cell cycle is an intriguing question. In the one instance tested, in which T-cells (Jurkat) were activated, no significant differences in polyP synthesis were observed (data not shown).

Based on several parameters, the functions of polyP must be various and also differ among the cells and tissues. Principal among these parameters are: 1) the size of the polyP chain which may range from the short (5 to 15 residues) to the long (500 to 800 residues), (Fig. 1), 2) the subcellular location (Table 3), and 3) the metabolic pattern ( Fig. 2and Fig. 3). In addition, it can be assumed that the polyP functions are influenced by their counterions, whether they be mono-, di-, or polyvalent (e.g. polyamines), and whether polyP is complexed to a protein, and if so, which. The ubiquity of polyP in cells and tissues and its diverse physical and metabolic features plead for attention and an effort to reveal the functions of this neglected polymer.


FOOTNOTES

*
This work was supported by grants from the Human Frontier Science Program and the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: polyP, polyphosphate; PAGE, polyacrylamide gel electrophoresis; TEMED, N,N,N`,N`, tetramethylethylenediamine; PPK, polyphosphate kinase; rPPX1, recombinant S. cerevisiae exopolyphosphatase.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.