(Received for publication, November 30, 1994; and in revised form, January 4, 1995)
From the
Inorganic polyphosphate (polyP), a linear polymer of hundreds of
orthophosphate (P) 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
by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to
120 µM (in terms of P
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
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
in the medium bypasses intracellular P
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.
Inorganic polyphosphates (polyP) ()are linear
polymers of orthophosphate (P
) 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 [C]ADP(15) , and the other
converts polyP to P
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
. 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
that bypasses the intracellular pools of P
and ATP.
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.
Cultures of mammalian cell lines, upon reaching confluence, were
exposed to [P]P
for 1 h in a medium
to which 100 µM KPO
(pH 7.0) was added.
Incorporation of P
into polyP varied depending on the cell
line (Table 4). Values are based on the specific radioactivity of
P
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.
Figure 1:
Urea-PAGE analysis of
[P]polyP from cell lines. The
[
P]P
-labeled material extracted from
various cell lines was electrophoresed on a 6% urea-PAGE. Chain lengths
of standards are on the right.
Figure 2:
Time course of
[P]P
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
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
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-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
residues. A, NIH 3T3 cells; B, PC12
cells.
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 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 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 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
from the medium, it is clear that there is little or no
mixing with the major pools of P
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
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
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
in the medium to polyP in
the cell involves either an intracellular P
or ATP pool
sequestered from the principal pools or some novel mechanism, such as
fixation of P
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
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 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.