Page 100 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-06
Enzymes of polyphosphate biosynthesis and degradation
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yeast exopolyphosphatase with respect to its Mg 2+ requirement, optimal pH and sensitivity
to cations, amino acids and heparin (Rodrigues et al., 2002). In contrast to the yeast enzyme
and other known exopolyphosphatases, it hydrolysed PolyP 3 with a higher rate and affinity.
This processive enzyme did not hydrolyse pyrophosphate, ATP or p-nitrophenylphosphate.
Immunofluorescence microscopy using affinity-purified antibodies against the recombinant
enzyme indicated its acidocalcisomal and cytosolic localization (Rodrigues et al., 2002).
Exopolyphosphatases purified from Neurospora crassa (Umnov et al., 1974) and En-
domyces magnusii (Afanas’eva and Kulaev, 1973) are close to the yeast cytosol ex-
opolyphosphatase through its molecular mass and divalent cations requirements. The fact
that it actually did not hydrolyse PolyP 3 may be due to its low affinity to this substrate,
which was used in a 10-fold lower concentration than K m for the yeast enzyme (Umnov
et al., 1974). Two other exopolyphosphatase activities were observed in the ‘slime’ variant
of N. crassa which cannot synthesize cell walls (Trilisenko et al., 1985a,b). One of these
was K - and Mg -dependent, hydrolysing high-molecular-weight polyPs, while the other
2+
+
was K - and Mg -independent, hydrolysing low-molecular-weight polyPs. The study of a
2+
+
number of exopolyphosphatases from the lower eucaryotes is important to clarify the PolyP
functions in each individual compartment of these microorganisms.
As regards animals, the first exopolyphosphatases were purified from the marine sponge
Tethya lyncurum (Lorenz et al., 1995). Two exopolyphosphatases were identified in this
simple metazoa. Exopolyphosphatase I had a molecular mass of 45 kDa, a pH optimum of
5.0, and did not required divalent cations for its activity, while exopolyphosphatase II had
a molecular mass of 70 kDa, a pH optimum of 7.5, and displayed optimal activity in the
presence of Mg 2+ (Lorenz et al., 1995).
Exopolyphosphatase activity is also present in human osteoblasts (Leyhausen et al.,
1998). The specific activity of the enzyme in osteoblasts was much higher than those in
other mammalian cells and tissues tested (Schr¨oder et al., 2000) (Table 6.7.). More than
50 % of the exopolyphosphatase activity in osteoblast cells was ‘membrane-bound’. Ex-
opolyphosphatase activity has also been found extracellularly, e.g. in synovial fluid (Sch¨oder
et al., 1999), as well as in human blood plasma and serum (Schr¨oder et al., 1999, 2000)
(Table 6.7).

Table 6.7 Exopolyphosphatase activities with PolyP 35 as the
substrate in different cells, tissues and extracellular fluids from
mammals (Schr¨oder et al., 2000).

Exopolyphosphatase activity
Cell/tissue (nmol of P i per h per mg of protein)

Rat liver 48
Rat brain 54
Human plasma 5.5
Human serum 5.4
Human osteoblasts 210
Human HL-60 cells 25
Human peripheral blood 15
mononuclear cells