Title page for ETD etd-10102009-020350
|Type of Document
||Hladun, Suzanne Lynn
||Water stress protein is secreted, is subject to rapid proteolysis upon rehydration of dessiccated cells, and may be glycosylated
||Master of Science
|Bevan, David R.
|Mason, Robert W.
|Date of Defense
A novel and highly-abundant 39-kOa acidic water stress protein, Wsp,
has been purified and partially characterized from cells of the desiccationtolerant
cyanobacterium Nostoc commune (Scherer, S. and Potts, M. 1989.
J. BioI. Chem. 264:12546-12553). In the original study, several
inconsistencies were noted and are addressed in this body of work. Firstly, the
microheterogeneity noted on two dimensional electrophoresis gels indicated the
possibility of modifications of the protein or the existence of isoforms of Wsp.
This possibility was not addressed in the original study. On two-dimensional
gels, several proteins were seen to migrate differently in the 4.5 to 5.5 pH
range. These proteins showed a similar change in staining characteristics and
after 24h of rehydration a single darkly staining area was resolved.
Immunocytochemical analysis revealed that a fraction of Wsp is secreted,
and accumulates as a very discrete layer at and around the periphery of the
envelope of both vegetative cells and heterocysts. This introduced the
possibility that Wsp is a glycoprotein with a large proportion of carbohydrate
to protein. However, the original study did not detect carbohydrate attached
to Wsp. This was a direct result of the methodology employed to isolate the
protein for characterization. A high speed centrifugation step in the protocol
of Scherer and Potts (Scherer, S. and Potts, M. 1989. J. BioI. Chern.
264: 12546-12553) selected against isolation of a carbohydrate-bound protein.
Further, only minimal glycosylation of this form of Wsp was detected by
fluorimetric analysis and Concanavalin A binding experiments (Scherer, S. and
Potts, M. 1989. J. BioI. Chem. 264:12546-12553).
In the present study, it is shown that Wsp is intimately associated with
carbohydrate; more specifically I the data suggest that this form of Wsp may be
glycosylated. Wsp is also found to be ubiquitous in materials of Nostoc
commune> collected from the Tropics to the polar regions, some after having
being stored for 17 years in the air-dry state. Through the use of protease
inhibitors, forms of Wsp, of significantly greater molecular mass than those
seen in the primary study, were identified and characterized. This study
indicates that the significant amount of Wsp present in desiccated cells is
turned over rapidly and within minutes of cell rehydration. This turnover is
prevented by a number of different protease inhibitors. Of these, diisopropyl
fluorophosphate (DFP) affords the most efficient protection of Wsp. The
glycosylation status and specific proteolysis of Wsp in Nostoc are discussed.
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