Tp. Bushnell et al., A PURIFIED ZINC PROTEASE OF PEA-CHLOROPLASTS, EP1, DEGRADES THE LARGESUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE, Plant physiology, 103(2), 1993, pp. 585-591
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.