ABSTRACIOne endopeptidase (EPI) and at least three aminopeptidases (API, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EPI requires added Mg2" or Ca2" for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly residues. Its molecular mass is 93 kilodaltons, estimated on a gel filtration column. Aminopeptidase activities were detected with the aid of different amino acyl-f-naphthylamides as substrates. They were resolved into at least three individual proteins by gel filtration and DEAE-cellulose chromatography, having apparent molecular masses of 269,000 (API), 84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a unique specificity for substrates, with API hydrolyzing only the Prolyl-B-naphthylamide. None of the APs require added divalent cations for activity, but the possibility of a tightly bound metal function was suggested in AP2 and AP3 (not API) from effects of inhibitors. A probable sulfhydryl residue function was indicated for all three, from inhibition by p-hydroxymercuribenzoate and Zn2". All these peptidases had pH optima at 7.7.Specific protein turnover or selective protein degradation has been found to be an essential part of chloroplast development. In many cases there are reasons to think that the responsible proteases are located inside the chloroplasts. Proteolytic activity was postulated to be responsible for prolamellar body degradation when etiolated leaves are put in the light (5); for the removal of damaged QB apoprotein, and therefore for its high turnover rate (10, 15); for accelerated turnover of chloroplast-encoded 48 and 34.5 kD polypeptides in thylakoids lacking PSII (1 1); and for rapid degradation of NADPH:Pchlide reductase in barley etioplasts once the plants are put into the light (9). During leaf senescence, extensive protein degradations occurring in apparently intact chloroplasts are explained by an internal set of proteases (21). Chloroplast localization of peptidases was noted in wheat by Waters et al. (22). The best defined internal protease is the one in pea chloroplasts which processes ribulose 1,5-bis P carboxylase small subunit precursors imported from the cytosol; this was purified to a considerable degree ( 19).A thylakoid-located, ATP-dependent proteolytic activity was found responsible for removal of a significant fraction of newly translated proteins in pea chloroplasts, perhaps those which are mature but unassembled into larger complexes (12,14). On the other hand, an ATP-independent but Mg2+-dependent proteolytic activity seemed to be responsible for removal of newly translated proteins containing abnormal amino acids, and of prematurely terminated proteins (13). In an effort to locate the specific enzyme responsible for one or another of these activities, we have fo...

Proteins newly formed from labeled amino acids by isolated intact pea chloroplasts are not entirely stable. Between 20 and 35% of the labeled protein is degraded over a 20–30 min incubation period in pulse‐chase experiments. Protein degration is prevented when chloroplast ATP level drops, as in the dark without added ATP. Degration is stimulated by adding ATP directly or by generating it in photophosphorylation. Susceptible new proteins are not stabilized against further additions of ATP, during incubation under ATP‐deficient conditions.