SYNTHESIS OF I-125 UBIQUITIN CONJUGATES IN EXTRACTS OF LEMNA-MINOR

Low levels of ubiquitin conjugating activity are typically detected in the green tissues of plants, an observation that may at least partial ly explain why no method to purify multi-ubiquitinated proteins from p hotosynthetic cells has been reported in the literature, The present p aper provides a contribution to improve the available methodology for the isolation of an efficient ubiquitin conjugating system from photos ynthetic cells. We have selected Lemna minor L. as a plant system and have developed a simple and rapid methodology to synthesize and purify high molecular mass ubiquitin-protein conjugates, formed with endogen ous substrates and exogenous I-125-ubiquitin, using small amounts (< 2 g) of green tissue, It is demonstrated that L. minor possesses an ATP -dependent activity capable of forming ubiquitin conjugates with endog enous proteins in vitro, Anion exchange chromatography on diethylamino ethyl-cellulose provides a simple and rapid technique to remove endoge nous ubiquitin and to concentrate and partially purify the enzyme syst em responsible for ubiquitin conjugating activity, This enriched fract ion has therefore been utilized to synthesize high molecular mass I-12 5-ubiquitin conjugates formed with L. minor proteins, These conjugates were subsequently purified by directly loading the reaction mixture o n a Sephacryl S-300 gel filtration column, with no requirement for add itional concentration or purification steps. This methodology is highl y reproducible.