PURIFICATION AND CHARACTERIZATION OF AN ARGINYL PEPTIDASE FROM THE CHLOROPLAST STROMA OF PEA-SEEDLINGS

Stromal extract of pea (Pisum sativum var. Alaska) chloroplasts was fr actionated by column chromatography, using the cleavage of benzoyl-arg inine p-nitroanilide (BAPNA) as an assay for peptidase activity. We pu rified an arginyl peptidase 168-fold, with a yield of 37%. SDS-PAGE of the purified fraction revealed a major band of 79 kDa and a fainter o ne of 41 kDa. N-terminal amino-acid sequencing identified the smaller protein as aldolase, while the N-terminus of the larger protein was bl ocked. Activity assay identified the larger protein as the active pept idase. Fractionation of the peptidase on a sizing column suggested a s ize of 188 kDa for the native protein. Only Arg-containing peptides we re cleaved by the peptidase, and it was found most sensitive to the in hibitors 3,4-dichloroisocoumarin and antipain. This peptidase could no t cleave stromal or thylakoid proteins, suggesting that its activity i s limited to later stages of the protein degradation process in chloro plasts, in which short peptides are further cleaved to ultimately yiel d free amino acids.