Jongyun Heo

Publication Date

1997

Document Type

Dissertation/Thesis

First Advisor

Holbrook, Gabriel P.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Biological Sciences

LCSH

Photobiochemistry; Organophosphorus compounds

Abstract

This research addresses two separate questions related to the light and dark regulation of Rubisco. The first part of this study concerns the regulation of a specific phosphatase for 2-carboxy-D-arabinitol 1-phosphate (CA1P): a known inhibitor of Rubisco synthesized in darkened chloroplasts of numerous plant species. CA1P phosphatase is a component of the multiple mechanisms responsible for the regulation of Rubisco activity. Previous studies indicate that this phosphatase may be regulated by the change in chloroplast redox conditions associated with light/dark transitions experienced by leaves. We observed that glutathione, which occurs naturally in leaves as a redox buffer, was maximally capable of a 35-fold stimulation of CA1P phosphatase activity. A shift in the reduction status of glutathione, { [glutathione (GSH) ] / ( [glutathione (GSH)] + [glutathione disulfide (GSSG)])}, from 60% to 100% GSH caused a > 20-fold stimulation of the phosphatase activity. Further experiments with a range of sulfhydryl reagents indicated that CA1P phosphatase contains two physically distinct sets of thiol groups critical to its redox regulation. Accordingly, we suggest a model for reversible activation where GSH reduces an accessible intramolecular dithiol on the exterior of the minimally active oxidized enzyme to an activated open form containing two sulfhydryls. ABSTRACT The second part of this study addressed light/dark regulation of Rubisco in the 2+ photosynthetic bacterium Rhobacter sphaeroides. C02/Mg activated Rubisco activity in dark-harvested toluene-permeabilized R. sphaeroides cells was substantially less than in light-harvested cells. This was observed in cells grown either photoheterotrophically or photolithoautotrophically. The inhibition was reversed by treatment with alkaline phosphatase, restoring Rubisco activity to levels observed with the light-harvested control sample. We conclude that a phosphorylated inhibitor accumulates in darkened cells. Chromatographic analysis yielded a fraction which inhibited Rubisco and eluted in a position corresponding to 6-phosphogluconolactone but clearly differing from CA1P. During in vitro assays of Rubisco in toluene-permeabilized R. sphaeroides cells, the degree of inhibition by 6-phosphogluconate was strongly dependent upon the concentration of HC03’: an effect not observed with spinach Rubisco. However, at low concentration (0.2 mM), the isolated bacterial inhibitor preparation did inhibit purified spinach Rubisco activity while authentic 6-phosphogluconate did not. The isolated bacterial inhibitor may therefore contain small quantities of an additional unidentified inhibitor. We propose that Rubisco activity in R. sphaeroides cells may be regulated by the synthesis and degradation of 6- phosphogluconate, but the additional presence of an unidentified inhibitor cannot be excluded.

Comments

Includes bibliographical references (pages [102]-112)

Extent

ix, 146 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

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