Page 50 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-02
Methods of polyphosphate assay
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transferoftheterminalγ -phosphateofATPtoPolyP(Kornbergetal.,1956)TheEscherichia
coli polyphosphate kinase gene has been cloned, sequenced (Akiyama et al., 1992) and
overexpressed (Crooke et al., 1994). Thus, the recombinant PPK is available for enzymatic
analysis.
Exopolyphosphatase (PPX) catalyses the hydrolysis of the PolyP terminal residues to P i
almost completely (Akiyama et al., 1993). Bacterial PPX does have preference for longer
PolyPs, while the major Saccharomyces cerevisiae exopolyphosphatase PPX1 can act on
PolyP chains of 3 to 1000 residues. Cloning the gene for PPX1 (Wurst et al., 1995) and
overproducing it in E. coli enabled the use of this enzyme as a powerful analytical reagent.
It is ∼ 100-fold more active as the specific exopolyphosphatase of E. coli. In S. cerevisiae,
another highly active exopolyphosphatase was found, which had preference for longer
PolyPs, similar to the bacterial form (Andreeva et al., 2001). Therefore, several enzymes
may be used for the development of enzymatic analysis of PolyPs with different chain
lengths.
Two enzyme-based methods for the estimation of PolyPs in biological samples have been
described (Ault-Rich´e and Kornberg, 1999). The first of these (Rao et al., 1998) requires
32
prior labelling of the culture with P. PolyPs are extracted from the cells by treatment with
a solution containing formic acid, urea, sodium dodecyl sulfate (SDS), EDTA and carrier
PolyP 65 . The suspension is sonicated, and the PolyPs are bound to DE81 ion-exchange filter
discs. The latter are washed, and the PolyPs are eluted with KCl. ATP and other phosphorus-
containing organic compounds are removed from the eluate by using Norit charcoal. The
PolyPs are concentrated by re-adsorption onto DE81 discs. After washing, the discs are
treated with the purified recombinant yeast spPPX1. Decrease in the 32 P content on the
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filters or an increase in the P content released from the filter corresponds to the amount of
PolyP. Limitations of the radioactive method are the requirement for prior culture labelling
and the inconsistent extraction of PolyPs.
There is an example of using the recombinant yeast spPPX1 for PolyP analysis without
the need for prior labelling of the culture (Ruiz et al., 2001a). Aliquots of PolyP extracts
(∼ 1.5 nmol) were incubated for 15 min at 37 C with 60 mM Tris-HCl (pH 7.5), 6 mM
◦
MgCl 2 and 3000–5000 units of purified spPPX1 in a final volume of 0.075 ml. One unit
◦
corresponded to the release of 1 pmol of P i per min at 37 C. The release of P i was monitored
by various well-known chemical methods.
The second modern enzymatic method of PolyP assay (Ault-Rich´e et al., 1998; Ault-
Rich´e and Kornberg, 1999) involves the rapid isolation of PolyP by using powdered glass
or glass filters, followed by its conversion to ATP under the action of PPK and an estimation
of the generated ATP using a luciferin–luciferase system. The cells were lyzed with 4 M
guanidinium isothiocyanate, and PolyP was adsorbed onto glass or a glass filter. SDS was
added during the binding step to prevent protein binding. PolyP was eluted by hot water
for a buffer with low ionic strength. Then, the PolyP was converted to ATP by purified
polyphosphate kinase in the presence of a 10-fold excess of ADP. The ATP generated was
measured by a luminometer.
Therefore, the availability of purified enzymes specific to PolyPs allowed the develop-
ment of rapid, sensitive and definitive assays. It should be noted, however, that PolyPs in
biological samples may not be effectively hydrolysed by exopolyphosphatase or be available
for polyphosphate kinase (Sethuraman et al., 2001).