Page 73 - The Biochemistry of Inorganic Polyphosphates

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the total intracellular PolyP were made by Harold and Miller (1961) on the mycelium
of Neurospora crassa. They determined the distribution of PolyPs and some other com-
pounds in various cell structures of this organism after disrupting of the mycelia in a Nos-
sal’s apparatus in 0.05 M tris(hydroxymethyl)amino methane (TRIS), pH 7.0, containing
0.25 M sucrose, and separation of cell structures by differential centrifugation (Table 5.1). It
should be noted that, under mechanical disruption of the cells, a rapid degradation of PolyP
may occur, and there is a possibility of secondary sorption and desorption of PolyP during
sub-fractionation. Harold and Miller reported the occurrence of both of these processes.
For example, acid-soluble PolyP degraded to P i in the course of this work. Furthermore,
Harold (1962a) showed that the presence of a large amount of acid-insoluble PolyP in the
cell wall material from N. crassa could be due to secondary sorption by polysaccharides,
such as chitin, which form a part of the cell wall of this organism.
Much more promising were the investigations which had no recourse to mechanical
disruption of the cell. Weimberg and Orton (1965) treated the cells of Saccharomyces mellis
with an enzyme preparation from the snail Helix pomatia. This treatment causes lysis of
the fungal polysaccharide cell wall, resulting in the formation of spheroplasts devoid of cell
walls. It was shown that approximately one fourth to one third of the total cellular PolyP was
removed from the cells and degraded to P i during the spheroplasts formation (Weimberg
and Orton, 1965; Weimberg, 1970). It was concluded that these PolyPs were localized in
the immediate vicinity of the external cytoplasmic membrane (Weimberg and Orton, 1965).
Souzu (1967a,b) showed that yeast cells during freezing and thawing underwent distur-
bance of the cytoplasmic membrane, accompanied by rapid hydrolysis of cellular PolyP
to P i . This was interpreted as evidence of localization of a significant portion of PolyP in
yeast in the region of the cytoplasmic membrane. The localization of a portion of PolyP in
yeast cells on the cell surfaces was proposed by Van Steveninck and Booij (1964), obtaining
some evidence for PolyP participation in glucose transport.
Later, PolyP was revealed outside the plasma membrane of the yeast Kluyveromyces
marxianus by fluorescence of 4 6-diamidino-2-phenylindole (Tijssen et al., 1982), by lead

staining (Voˇr´ıˇsek et al., 1982) and X-ray microanalysis (Tijssen and Van Steveninck, 1985),
by a decrease in the 31 P NMR signal under UO 2 2+ binding (Tijssen and Van Steveninck,
1984), by osmotic shock treatment (Tijssen et al., 1983), and by 9-aminoacrydine binding
(Vagabov et al., 1990a). The cell envelope of yeast can contain about 20 % (and even more)
of the total PolyP content of yeast cells (Vagabov, 1988; Ivanov et al., 1996).
Different cellular localization of various PolyP fractions on the basis of examination of
their functions and metabolism was first proposed for Chlorella (Miyach, 1961; Miyachi
and Miyachi, 1961; Miyachi and Tamiya, 1961; Kanai et al., 1963; Miyachi et al., 1964).
In particular, it was shown that this alga possesses PolyP fraction A, which is a constituent
of volutin granules and closely involved in nuclear metabolism, and another PolyP fraction
C, which is sited adjacent to the chloroplasts and involved in photosynthetic processes.
Fractions B and D appeared to be localized in other cell structures.
The investigation of intracellular localization of PolyP was carried out for the fungi
Neurospora crassa and Endomyces magnusii (Kulaev et al., 1966a, 1967a,b, 1970a,b;
Krasheninnikov et al., 1967, 1968; Kulaev and Afanas’eva, 1969, 1970; Skryabin et al.,
1973). While mechanical disruption of cells was shown to be unsuitable for obtaining sub-
cellular fractions, protoplast isolation was used in these studies. Although the total amount
of phosphorus present in the intact cells and in the protoplasts was the same, nevertheless the