Date of Award

1996

Publication Type

Master Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

Chemistry, Biochemistry.

Supervisor

Mutus, B.,

Rights

info:eu-repo/semantics/openAccess

Abstract

The catalytic properties of glutathione reductase for its substrate, glutathione disulphide, were altered following exposure to 100 $\mu$M peroxynitrite. The K$\rm\sb{M}$ increased from 38 $\pm$ 2 to 80 $\pm$ 3 $\mu$M and the V$\sb{\rm max}$ decreased from 328 $\pm$ 16 to 288 $\pm$ 12 $\mu$mol min$\sp{-1}$ mg protein$\sp{-1}.$ Exposure of the enzyme to S-nitrosoglutathione or nitric oxide gas showed no significant effect on the performance of glutathione reductase. The UV-visible spectrum of glutathione reductase exhibited absorbance at approximately 428 nm which is characteristic of nitrotyrosine. The peroxynitrite induced inactivation is not observed in the presence of excess glutathione disulphide. However, excess NADPH offered no protection against peroxynitrite induced inactivation. The observations suggest that approximately 1 tyrosine per subunit of glutathione reductase, at or near the glutathione disulphide binding domain, probably results in the observed catalytic alterations. To test this hypothesis, the two tyrosines closest to the glutathione disulphide binding domain (Tyr$\sb{114}$ and Tyr$\sb{106}$), as indicated by the X-ray crystallographic data (Karplus and Schulz (1989) J. Biol. Chem., 210, 163-180), were each converted to nitrotyrosines by molecular modelling and the structure energy was minimized. These theoretical calculations indicate that the bond lengths between Tyr$\sb{114}$-O and the Gly-N and CysII-N of glutathione disulphide bound to glutathione reductase increased by 3.0 and 4.3 A, respectively, upon nitration. In the case of Tyr$\sb{106}$, this distance also increases by approximately 1.6 A. The loss of these hydrogen bonding contacts is likely to result in the observed catalytic alterations. Cyclosporin A (CyA), was shown to inhibit the enzyme nitric oxide synthase (NOS) derived from rabbit aortic endothelial cells. (Abstract shortened by UMI.)Dept. of Chemistry and Biochemistry. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1996 .F7135. Source: Masters Abstracts International, Volume: 37-01, page: 0262. Adviser: B. Mutus. Thesis (M.Sc.)--University of Windsor (Canada), 1996.

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