A PURIFIED ZINC PROTEASE OF PEA-CHLOROPLASTS, EP1, DEGRADES THE LARGESUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE

A previously reported endopeptidase (EP1) from pea chloroplasts was pu rified over 11,000-fold using a four-step protocol involving ultrafilt ration, sucrose gradient centrifugation, isoelectric focusing, and hig h performance liquid chromatography gel filtration. The enzyme was det ermined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chlor oplasts was confirmed by demonstrating its insensitivity to thermolysi n when the envelope was intact. A contaminating serine protease that a ttacks EPI was found. the contaminating protease was inhibited by 4-(2 -aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, wh ereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygena se under physiological conditions.