Page 116 - The Biochemistry of Inorganic Polyphosphates

WU095/Kulaev
WU095-07
Functions of polyphosphate and polyphosphate-dependent enzymes
100 March 9, 2004 15:39 Char Count= 0
2+
and Ba , and blocked by transition metal cations in a concentration-dependent manner.
Recently, both PolyPs and PHBs have been found to be components of ion-conducting
2+
proteins, namely, the human erythrocyte Ca –ATPase pump (Reusch et al., 1997) and
the Streptomyces lividans potassium channel (Reusch, 1999b). The contributions of PolyPs
and PHBs to ion selection and/or transport in these proteins is yet unknown, but their
presence gives rise to the hypothesis that these and other ion transporters are supramolecular
structures, where proteins, PolyPs and PHBs co-operate to form well-regulated and specific
cation transfer systems.
The ability of E. coli PolyP–PHB complexes to form calcium-selective channels in planar
bilayers was investigated first of all (Reusch et al., 1995; Reush, 1999a, 2000). PolyP–PHB
complexes were extracted from cell membranes into chloroform and then pre-mixed with
the phospholipid solution before obtaining the bilayers. Single-channel currents were again
observed with voltage steps of 60 mV or more. When the complexes are extracted from
membranes or cells, the chloroform solutions contain protein and lipopolysaccharides in
addition to PolyP–PHB. To remove these components and to evaluate their influence on
channel activity, the complexes were further purified by size-exclusion column chromatog-
raphy. This eliminated all detectable contaminants and in addition provided an estimate of
the molecular weight of the complexes as 17000 ± 4000. Purified complexes were found to
be more labile, although the single-channel activity they produced closely resembled that
observed for the membrane complexes (Reusch et al., 1995).
2+
To still further determine the composition of the channels, the PHB–Ca –PolyP com-
plexes were reconstituted. PHB was recovered from E. coli and carefully purified, and Ca –
2+
PolyP was prepared from commercial sodium PolyP and calcium chloride. Single-channel
currents similar to those described above were obtained by three different experimental pro-
cedures, as described by Reusch et al. (1995). The chain length of chemically synthesized
PolyP was determined by acrylamide gel electrophoresis to be in the same range (55–65
residues) as in the E. coli complexes (Castuma et al., 1995).
The channels formed in planar bilayers by synthetic complexes were virtually identical
to those formed by PolyP–PHB complexes extracted from E. coli (Reusch et al., 1995;
Reusch, 1999a). The conductances of synthetic and E. coli channels were equivalent. The
channels formed by PolyP–PHB complexes, E. coli or synthetic, show strong selectivity
for divalent over monovalent cations (Reusch et al., 1995).
Oneofthecharacteristicsofproteincalciumchannelsistheirsensitivitytoablockbytran-
sition metal cations. Lanthanum is a particularly potent blocker. It is suggested that permeant
and blocking ions compete for the common binding sites in the channels. The PolyP–PHB
channel complexes are also blocked by transition metal cations in a concentration-dependent
manner. A nearly complete block of single-channel currents was observed in the synthetic
complexes at concentations > 0.1 mM La 3+ (0.1 % of Ca ) (Das et al., 1997). Evidently,
2+
PHB–PolyP complexes are versatile ion carriers whose selectivities may be modulated by
small adjustments of the local pH. The results may be relevant to the physiological function
of PHB–PolyP channels in bacteria and the role of PHBs and PolyPs in the Streptomyces
lividans potassium channel (Das and Reusch, 2001).
The mechanism of ion conduction by PolyP–PHB channel complexes can be rationalized
in terms of the structures and properties of the component polymers (Reusch, 1999a). One
of the notions of how the channel may operate in the cell membrane or planar bilayer is as
follows. Ca –PolyP surrounded by PHB forms a salt bridge extending from the cytoplasm
2+