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Proteolysis of chloroplast thylakoid membranes. I. Selective degradation of thylakoid pigment-protein complexes at the outer membrane surface

1980; Elsevier BV; Volume: 200; Issue: 2 Linguagem: Inglês

10.1016/0003-9861(80)90366-5

1096-0384

David Carter, L. Andrew Staehelin,

Antioxidant Activity and Oxidative Stress

Isolated pea thylakoids were experimentally unstacked in low-salt buffer and incubated with Pronase or trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that brief treatment with a very low concentration (1 μg/ml) of either enzyme had an effect primarily on the light-harvesting chlorophyll ab-protein complexes, which are more sensitive to proteolytic attack than the other proteins of the thylakoid membranes. This mild proteolysis cleaves a ~1000-dalton portion from the predominant 28,000-dalton polypeptide of these complexes. Extensive proteolysis (100 μg Pronase/ml for 15 min) degraded almost all membrane polypeptides not associated with the pigment-protein complexes and degraded the chlorophyll ab-protein complexes further than milder proteolysis. Pronase treatment of thylakoids in the presence of horseradish peroxidase was used to monitor membrane breakage during proteolysis. Treatment with 100 μg Pronase/ ml enabled considerable amounts of peroxidase activity, and presumably, proteolytic enzymes to enter into the intrathylakoid space. This trapping of peroxidase activity was seen only minimally with milder proteolysis (1 μg Pronase/ml). These results suggest that brief exposure to low concentrations of proteolytic enzymes affects only the outer, stromal thylakoid surface, while at higher concentrations, significant proteolysis takes place at both sides of the membrane.