Synthesis of 125I-ubiquitin conjugates in extracts of Lemna minor
1996
Ramos, P.C.R. | Ferreira, R.M.B. | Ricardo, C.P.P.
Low levels of ubiquitin conjugating activity are typically detected in the green tissues of plants, an observation that may at least partially explain why no method to purify multi-ubiquitinated proteins from photosynthetic cells has been reported in the literature. The present paper provides a contribution to improve the available methodology for the isolation of an efficient ubiquitin conjugating system from photosynthetic 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 endogenous substrates and exogenous 125I-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 endogenous proteins in vitro. Anion exchange chromatography on diethylaminoethyl-cellulose provides a simple and rapid technique to remove endogenous ubiquitin and to concentrate and partially purify the enzyme system responsible for ubiquitin conjugating activity. This enriched fraction has therefore been utilized to synthesize high molecular mass 125I-ubiquitin conjugates formed with L. minor proteins. These conjugates were subsequently purified by directly loading the reaction mixture on a Sephacryl S-300 gel filtration column, with no requirement for additional concentration or purification steps. This methodology is highly reproducible.
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