PolyPs complexes

Figure 4.1 Model of the PolyP-PHB channel structure as proposed by Reusch and co-workers (from Reusch, 1999a). The central cylinder represents the PolyP helix with binding sites for Ca2+, with the Ca2+-PolyP complex being surrounded by the PHB helix (Reusch and Sadoff, 1988 Das etal., 1997).

The question of the functions of RNA-PolyP complexes needs further investigation. It is probable that the complexing with PolyP enhances the RNA stability. Some evidence has been obtained that in E. coli PolyPs inhibit RNA degradation by degradosome (Blum et al, 1997). 

Some of the PolyP complexes with proteins are very important in cell regulatory processes. RNA polymerase isolated from the stationary-phase cells of E. coli was found to be closely bound with PolyP (Kusano and Ishihama, 1997). The ATP-dependent protease Lon formed a complex with PolyPs under degradation of ribosomal proteins at amino acids starvation (Kuroda et al, 2001). PolyP is able to compete with DNA for the DNA binding sites at histones (Schroder et al., 1999), while PolyPs can interact with non-histone proteins in the nucleus (Offenbacher and Kline, 1984). 

PolyP complexes with poIy-p -hydroxybut yratc, similar to those in bacteria, were identified in eukaryotic membranes, including animal cells (Reush, 1989, 1999a, 2000). 

To still further determine the composition of the channels, the PHB-Ca2+-PolyP complexes were reconstituted. PHB was recovered from E. coli and carefully purified, and Ca2+-PolyP was prepared from commercial sodium PolyP and calcium chloride. Single-channel currents similar to those described above were obtained by three different experimental procedures, 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). 

One of the characteristics of protein calcium channels is their sensitivity to ablock by transition 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 La3+ (0.1 % of Ca2+) (Das et al., 1997). Evidently, 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). 

As for deaminase, the kinetic analysis suggests a partial mixed-type inhibition mechanism. Both the Ki value of the inhibitor and the breakdown rate of the enzyme-substrate-inhibitor complex are dependent on the chain length of the PolyP, thus suggesting that the breakdown rate of the enzyme-substrate-inhibitor complex is regulated by the binding of Polyphosphate to a specific inhibitory site (Yoshino and Murakami, 1988). More complicated interactions were observed between PolyP and two oxidases, i.e. spermidine oxidase of soybeen seedling and bovine serum amine oxidase. PolyP competitively inhibits the activities of both enzymes, but may serve as an regulator because the amino oxydases are also active with the polyamine-PolyP complexes (Di Paolo et al., 1995). 

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with dense granules. Purified granules contained PolyP complexed with calcium and... 

PHB/polyP complexes were first discovered in the plasma membranes of bacteria by Reusch and Sadoff22-25,85 during spectrofluorometric studies of membrane structure, using the hydrophobic probe, /V-pheny 1-1 -naphthylamine (NPN). When NPN is added to cell suspensions, it partitions into the hydrocarbon region of the cell membranes and... 

The ability of E. coli PHB/polyP complexes to form calcium-selective channels in planar bilayers was investigated in the planar bilayer system described above (Figure 5). E. coli DH5a cells were made genetically competent to increase the concentration of PHB/polyP in the membranes. Then vesicles were prepared from the cell envelopes, and added to the cis side of a planar bilayer formed by synthetic 16 0, 18 1 PC between symmetric bathing solutions of 250 mM CaCl2, 5 mM MgCl2,10 mM Tris Hepes, pH 7.3. The complexes were allowed to insert spontaneously into the bilayer.27 No activity was observed in the absence of an applied... 

The calcium channel activity observed in plasma membrane vesicles of competent E. coli was essentially the same as that for PHB/polyP complexes extracted from plasma membranes into chloroform solution (Figure 11, line 4). The molecu-... 

Figure 12. Representative single-channel current fluctuations of synthetic and E. coli PHB/polyP complexes at various clamping potentials. Left Synthetic PHBua/polyP Right Channels extracted from competent cells of E. coli DH5a. Complexes were incorporated into planar lipid bilayers composed of 16 0, 18 1, PC and cholesterol (5 1 w/w) between aqueous bathing solutions of 200 mM CaCl2, 5 mM MgCh, 10 mM Tris Hepes, pH 7.4 at 22 °C. The bars at the side of each profile indicate the fully closed state of the channel. Clamping potentials with respect to ground are indicated at the left side.28...

Protein Ca2+ channels are highly permeant to Ca2+, Sr2+, and Ba2+. In the presence of these divalent cations, they select strongly against monovalent cations, such as K+ and Na+84 however, the channels become highly permeable to these monovalent cations when all divalent cations are removed. These characteristics are shared by the Ca2+channels formed in bilayers by E. coli or synthetic PHB128/polyP complexes. The channels are selective for divalent over monovalent cations, permeant to Ca2+, Sr24, and Ba2+, and become permeable to monovalent cations when divalent cations are absent.27,28... 

Synthetic OHB19/23/polyP complexes demonstrated weak but explicit selectivity for divalent over monovalent ions79 when incorporated in planar bilayers composed of synthetic di22 l PC, cholesterol (5 1 w/w) between unequal solutions of Ca2+ and Na+ at pH 7.4 (Figure 15C). The reversal potential was -20 mV the Nernst equilibrium potentials were the same as for the synthetic complexes above. The data indicate selectivity for Ca2+ over Na+ of about 4 1, a significant improvement over channels formed by the oligomers alone (see Figure 8C), but still much poorer discrimination than the >90 1 selectivity demonstrated by the natural and synthetic... 

Figure 16. A. Block of E. coli PHB/polyP channels by transition metal cation, La3+. Representative single-channel current steps at 120 mV for PHB/polyP complexes, extracted from E. coli, purified by size-exclusion chromatography, and incorporated into bilayers of 16 0,18 1, PC between symmetric solutions of 100 mM CaCh, 2 mM MgCh,10 mM Tris Hepes, pH 7.3, and with LaCl3 added to the cis side as stated.27 (a) No LaCb (b) 0.20 mM LaCb (c) 0.55 mM LaCb. B. Block of synthetic PHBi2s/polyP channels by transition metal cation, La3+. The bilayer composed of 16 0,18 1, PC and cholesterol (5 1 w/w) was formed between aqueous bathing solutions of 200 mM CaCb, 5 mM MgCb, 10 mM Tris Hepes, pH 7.4. After incorporation of the channel, activities were recorded for 5 min at a clamping potential of-80 mV. Then, LaCb was added to the trans compartment to achieve the indicated concentrations. The bath was stirred and activities were recorded after 1 min of addition of La+3. Data points represent mean values of the amplitude histograms error bars show the standard deviation from the mean.28...

The physiological role of PHB/polyP complexes has not been established, but bacterial cells like eukaryotic cells maintain low internal Ca2+,101-103,112 and there is increasing evidence pointing to calcium involvement in a number of important cellular functions, such as chemotaxis, cell division, heat shock, pathogenicity, and differentiation.104-107 Systems for calcium export have been identified.108-109 Mechanisms for calcium entry are less well known. An L-type channel was reported in Bacillus subtilis,n0 lu and Jones et al. and Holland et al.103,112 recently demonstrated the presence of a putative Ca2+ influx channel in stationary phase E. coli,... 

In summary, PHB/polyP complexes share many of the notable characteristics reported for proteinaceous Ca2+ channels i.e. selectivity for divalent over monovalent cations permeance to Ca2+, Sr2+, and Ba2+ block by transition metal cations voltage activation and voltage dependence. Moreover, the structures postulated for the complexes bear a striking resemblance to the single-file, multiple-site Ca2+ channel structures described by Hagiwara and Byerly, Hess and Tsien, McCleskey and Aimers, and Tsien et al.113-116... 

PHB/polyP complexes were first discovered in genetically competent bacteria.22 Although the complexes are present in the plasma membranes of log-phase cells of diverse organisms, their concentrations are low under optimal growth conditions. 

The recent discoveries of PHB and polyP in a human calcium pump and bacterial potassium channel suggest that the naked PHB/polyP complexes found in bacteria are progenitors of protein ion transporters. The process by which protein channels and pumps may have evolved from PHB/polyP complexes is unknown however, one may surmise that over time proteins surrounded the complexes to support and regulate their activity. At first, the association may have been nonco-valent, but subsequently PHB may have become tethered to the protein by a covalent bond. By this view, many of the channels and pumps of prokaryotes and eukaryotes may be supramolecular structures in which protein, polyP, and PHB join together for efficient regulation of transmembrane ion transport. 

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