Date of Award

1993

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Biological Sciences

Keywords

Biology, Cell.

Supervisor

Cotter, D.

Rights

info:eu-repo/semantics/openAccess

Abstract

The levels of the disaccharide trehalose, in the dormant stages of various slime molds, were determined. Trehalose comprised 7.1%, 2.4%, 3.2%, and 3.4% of the dry weight of Dictyostelium mucoroides macrocysts, Polysphondylium pallidum microcysts, and Dictyostelium discoideum spores (strains AC4 and SG1), respectively. In the vegetative amoebae of D. discoideum, and P. pallidum, additional roles for this storage carbohydrate were examined. It was found that trehalose could also act as a general stress manager; its levels increasing to those observed in dormant structures, when amoebae were exposed to heat (30$\sp\circ$C), cold (4$\sp\circ$C) and heavy metals. Trehalose-6-phosphate synthetase and trehalase activities remained unchanged in vegetative amoebae during stress and recovery from stress indicating that regulation of trehalose accumulation occurs in a pathway other than a trehalose futile cycle. It was further observed that glycogen levels decreased rapidly during stress regimes. This would result in the accumulation of glucose monomeric units and would drive trehalose synthesis in an equilibrium dependant manner (substrate level). Substrate level regulation of stress-induced trehalose accumulation was supported by the finding that the stress response was unaltered in the presence of the protein synthesis inhibitor, cycloheximide. The inhibitory effects of three trehalose analogs on D. discoideum lysosomal trehalase were examined. Validamycin A, MDL-25, 637, and castanospermine were found to be potent, reversible, inhibitors of the enzyme in vitro, with K$\sb{\rm i}$ values of 10$\sp{-8}$ M, 10$\sp{-7}$ M, and 10$\sp{-4}$ M, respectively. In addition, Validamycin A and MDL 25,637 were shown to be time dependent inhibitors of trehalase. A trehalase-specific affinity resin was developed by covalently coupling validamycin A to Sepharose. The resin was used to purify D. discoideum trehalase to homogeneity in a two step procedure. Due to the high specificity of trehalase for its substrate, Validamycin A-Sepharose has broad applications in the rapid purification of trehalases from a variety of species origins. Source: Dissertation Abstracts International, Volume: 54-09, Section: B, page: 4489. Adviser: D. A. Cotter. Thesis (Ph.D.)--University of Windsor (Canada), 1993.

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