Characterization and cloning of cysteine protease that is induced in green leaves of barley

A sodium dodecyl sulfate (SDS)-dependent cysteine protease was investigated in green leaves of barley (Hordeum vulgare L.) by measuring the release of 7-amino-4-methyl-coumarin (AMC) from a synthetic substrate, N-succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA). The enzyme named HvCP3 (Horedeum vulgare cysteine protease activated by 0.1%, w/v SDS) increased well with the development of leaves, but it decreased drastically during senescence. HvCP3 was purified 146-fold with a yield of 7.74% from the crude extracts by four steps of chromatography. The enzyme showed a broad pH optimum at pH 7–8 and the enzyme activity was activated about 10-fold by 0.1% (w/v) SDS. The molecular weight of the native enzyme was estimated to be approximately 50 k. SDS–polyacrylamide gel electrophoresis of the protease suggested that the protein was a complex that consists of 33 k and 18 k subunits. The enzyme activity was specifically inhibited by cysteine protease inhibitors such as 10 μM trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64) and 100 μM leupeptin to 5% and 4%, respectively. A full-length cDNA of HvCP3 was cloned and its sequence displayed high similarity to other plant cysteine proteases of the papain family (C1A). The functional role of this protease as a maintainer in cytosol is also discussed.