Title page for ETD etd-10102009-020350


Type of Document Master's Thesis
Author Hladun, Suzanne Lynn
URN etd-10102009-020350
Title Water stress protein is secreted, is subject to rapid proteolysis upon rehydration of dessiccated cells, and may be glycosylated
Degree Master of Science
Department Biochemistry
Advisory Committee
Advisor Name Title
Potts, Malcolm Committee Chair
Bevan, David R. Committee Member
Mason, Robert W. Committee Member
Keywords
  • Glycosylation
Date of Defense 1992-12-05
Availability restricted
Abstract
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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