The Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase is Fragmented into 37-kDa and 16-kDa Polypeptides by Active Oxygen in the Lysates of Chloroplasts from Primary Leaves of Wheat

Lysates of chloroplasts isolated from wheat (Triticum aestivumL. cv. Aoba) leaves were incubated on ice (pH 5.7) for 0 to60 min in light (15 µmol quanta m^–2 s^–1),and degradation of the large subunit (LSU) of ribulose-l,5-bis-phosphatecarboxylase/oxygenase (Rubisco: EC 4.1.1.39 EC] ) was analyzed byapplying immunoblotting with site-specific antibodies againstthe N-terminal, internal, and C-terminal amino acid sequencesof the LSU of wheat Rubisco. The most dominant product of thebreakdown of the LSU and that which was first to appear wasan apparent molecular mass of 37-kDa fragment containing theN-terminal region of the LSU. A 16-kDa fragment containing theC-terminal region of the LSU was concomitantly seen. This fragmentationof the LSU was inhibited in the presence of EDTA or 1,10-phenanthroline.The addition of active oxygen scavengers, catalase (for H₂O₂)and n-propyl gallate (for hydroxyl radical) to the lysates alsoinhibited the fragmentation. When the purified Rubisco fromwheat leaves was exposed to a hydroxyl radical-generating systemcomprising H₂O₂, FeSO₄ and ascorbic acid, the LSU was degradedin the same manner as observed in the chloroplast lysates. Theresults suggest that the large subunit of Rubisco was directlydegraded to the 37-kDa fragment containing the N-terminal regionand the 16-kDa fragment containing the C-terminal region ofthe LSU by active oxygen, probably the hydroxyl radical, generatedin the lysates of chloroplasts. (Received October 28, 1996; Accepted February 7, 1997)