Journal Article

Glycation by Ascorbic Acid Causes Loss of Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Its Increased Susceptibility to Proteases

Yasuo Yamauchi, Yukinori Ejiri and Kiyoshi Tanaka

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 43, issue 11, pages 1334-1341
Published in print November 2002 | ISSN: 0032-0781
Published online November 2002 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pcf162
Glycation by Ascorbic Acid Causes Loss of Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Its Increased Susceptibility to Proteases

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Glycation is a process whereby sugar molecules form a covalent adduct with protein amino groups. In this study, we used ascorbic acid (AsA) as a glycating agent and purified cucumber (Cucumis sativus L.) ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a model protein in chloroplast tissues, and examined effects of glycation on the activity and susceptibility of Rubisco to proteases. Glycation proceeded via two phases during incubation with AsA and Rubisco in vitro at physiological conditions (10 mM AsA, pH 7.5, 25°C in the presence of atmospheric oxygen). At the early stage of glycation (phase 1), the amount of AsA attaching to Rubisco increased at an almost linear rate (0.5–0.7 mol AsA incorporated (mol Rubisco)–1 d–1). By Western blotting using monoclonal antibodies recognizing glycation adducts, a major glycation adduct, Nε-(carboxymethyl)lysine was detected. At the late stage of glycation (phase 2), incorporation of AsA reached saturation, and a glycation adduct, pentosidine mediating intramolecular cross-linking, was detected corresponding to formation of high molecular weight aggregates cross-linked between subunits. Glycation led to a decrease in Rubisco activity (half-life about 7–8 d). Furthermore, glycated Rubisco of phase 2 drastically increased protease susceptibility in contrast to unchanged susceptibility of glycated Rubisco of phase 1 compared to that of native Rubisco. Results obtained here suggest that AsA is possibly an important factor in the loss of activity and turnover of Rubisco.

Keywords: Keywords: Ascorbic acid — Cucumber (Cucumis sativus L.) — Glycation — Protein modification — Rubisco — Senescence.; Abbreviations: AGE, advanced glycation end product(s); AsA, ascorbic acid; CML, Nε-(carboxymethyl)lysine; DHA, dehydroascorbic acid; HAP, highly aggregated product; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.

Journal Article.  5367 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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