Chemistry Reference
In-Depth Information
from the siliceous valve (hypotheca or epitheca) formed within the shaped vesicle.
However, some information on the chemical environment present in the SDV is known as a
result of the use of a fluorescent, cationic, lipophilic dye rhodamine 123 that can
accumulate within the SDV that indicates precipitation under acidic conditions around pH
5 (Vrieling et al. 1999). The molecules, other than silica within the SDV are unknown.
Armed with the knowledge that the formation of species-specific biominerals other
than silica requires the services of biopolymers including proteins and carbohydrates a
search has been carried out to find similar regulatory molecules for silicifying organisms
including diatoms. For the production of silicified structures, the “control” molecules
need to regulate nucleation (location and time of deposition in relation to the organism/
cell cycle), growth and cessation of growth. Early observations came from the study of
silicifying diatoms where a marked increase in protein concentration in the cell wall was
found during silica deposition with carbohydrate incorporation only occurring after
silicification (Coombs and Volcani 1968). Nakajima and Volcani also identified the
presence of three unusual amino acids, namely 3,4-dihydroxyproline,
-
hydroxylysine and its phosphorylated derivative from the cell walls of different diatoms
including
Cylindrotheca fusiformis
(Nakajima and Volcani 1969, 1970). They were not
able to proceed further with their investigations as they could not isolate the proteins
containing these species. Other experiments designed to chemically inhibit protein
synthesis during valve development produced morphological abnormalities, suggesting
the requirement for
de novo
protein synthesis in biosilicification (Blank and Sullivan
1983). Although the SDV cannot be isolated, researchers have found ways to explore the
identity of biopolymers associated with the silica phase. Protein containing extracts have
been isolated from various freshwater and marine diatoms and their amino acid
composition determined (Hecky et al. 1973; Swift and Wheeler 1992). Treatment of
“cleaned” diatoms with buffered HF to dissolve the silica phase released material
enriched in serine/threonine (ca. 25 mol%), glycine (ca. 25 mol%) and acidic residues
(ca. 20 mol%) (Swift and Wheeler 1992). (It is perhaps notable that none of these
researchers identified the unusual amino acids found by Nakajima and Volcani in their
extracts). The model proposed by Hecky et al. (1973) envisaged that the serine/threonine-
enriched protein would form the inner surface of the silicalemma and, as such, would
present a layer of hydroxyls, facing into the SDV, onto which orthosilicic acid molecules
could condense. This initial layer of geometrically constrained orthosilicic acid molecules
would promote condensation with other orthosilicic acid molecules. Carbohydrates were
also found in extracts from the diatoms and this was variously assigned a range of roles
including action as a physical buffer between the organism and the aquatic environment,
providing resistance to chemical and bacterial degradation and functioning as an ion
exchange medium (Hecky et al. 1973).
More recently extremely sophisticated structural studies have been performed on a
range of isolates from the diatom cell wall of
Cylindrotheca fusiformis
and three classes of
proteins involved with the diatom cell wall identified. Of these groups of molecules, the
frustulins with molecular weights ~75
ε
-N-trimethyl-
δ
200 kDa are mainly involved in forming a
protective coat around the diatom and will not be discussed further (Kröger et al. 1994;
Kröger and Sumper 2000). The second family of proteins are known as HEPs (hydrogen
fluoride extractable proteins) or pleuralins (Kröger et al. 1997). The apparent molecular
weights of these molecules are 150
−
200 kDa and they are highly acidic molecules with a
structure comprising a signal peptide, a proline-rich (> 65%) region, a C-terminal domain
and repeats of proline (> 22%)-serine (> 11%)-cysteine (> 11%) aspartate (> 9%) rich
domains. The molecules have been sequenced and antibodies raised against the PSCD-
domains so that experiments to investigate the cellular localization of these molecules
could be performed. Immunoelectronmicrosopy studies showed that for the specific
−
