Direct evidence for non-enzymatic fragmentation of chloroplastic glutamine synthetase by a reactive oxygen species

Chloroplastic glutamine synthetase (GS: EC 6.3.1.2), the octamer of the 44 kDa subunit, is rapidly degraded under photo-oxidative stress conditions in leaves, chloroplasts, and chloroplast lysates. Recent studies have suggested that chloroplastic GS might be cleaved by the hydroxyl radical under such conditions (Thoenen & Feller 1998; Australian Journal of Plant Physiology 25, 279-286; Palatnik, Carrillo & Valle 1999, Plant Physiology 121, 471-478). Herein, we present evidence which supports the above hypothesis. When the purified GS from wheat (Triticum aestivum L.) chloroplasts was exposed to the hydroxyl radical-generating system comprising H₂O₂-FeSO₄-ascorbic acid or FeCl₃-ascorbic acid, the GS subunit was degraded into four distinct fragments having apparent molecular masses of 39, 35, 32 and 28 kDa. The apparent molecular masses and isoelectric points of these fragments were identical to those of the respective fragments found in the illuminated lysates of chloroplasts. In addition, the appearance of the GS fragments was completely suppressed in the presence of the scavenger for the hydroxyl radical, n-propyl gallate, in the illuminated lysates of chloroplasts. These results strongly support the hypothesis that the primary cleavage of GS is directly driven by the hydroxyl radical, formed by Fenton reaction under photo-oxidative stress conditions in chloroplasts.