title: Glycation by ascorbic acid causes loss of activity of ribulose-1,5-bisphosphate carboxylase/oxygenase and its increased susceptibility to proteases.
abstract: 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 degrees 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, N( epsilon )-(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.
authors.affiliation: Laboratory of Plant Biotechnology, Faculty of Agriculture, Tottori University, Koyama, Tottori, 680-8553 Japan. yamauchi@muses.tottori-u.ac.jp
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Yamauchi
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Ejiri
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology*; D015153:Blotting, Western; D002250:Carbon Radioisotopes; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006146:Guanidines / Q000494:pharmacology; D007120:Immunochemistry; D008239:Lysine / Q000494:pharmacology; D012273:Ribulose-Bisphosphate Carboxylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002250:Carbon Radioisotopes; D017127:Glycation End Products, Advanced; D006146:Guanidines; D010450:Endopeptidases; D002918:Chymotrypsin; D012273:Ribulose-Bisphosphate Carboxylase; D005978:Glutathione; D008239:Lysine; D001205:Ascorbic Acid; C004479:pimagedine