Isolation and Properties of the Envelope of Spinach Chloroplasts

Abstract

A method has been described for isolating separately the envelope and thylakoids of chloroplasts. The method makes use of the fact that gentle swelling of intact chloroplasts causes breakage and total detachment of the envelope. The envelope was purified by a sucrose density gradient procedure. The thylakoids and the envelope appeared to be very different in structure. All the activities associated with thylakoids (ferredoxin: NADP+ oxidoreductase, latent Ca2+-dependent ATPase) and the stroma (fructose 1,6-diphosphatase, P-glycolate phosphatase, carbonic anhydrase) were entirely absent in the envelope. NAD(P)H:cytochrome c oxidoreductase activity was found to be negligible in the envelope. Hence, this fraction was essentially free of microsomal and mitochondrial contaminations. Phosphatidylcholine comprised 75% of the total phospholipids and violaxanthin 50% of the total carotenoids in the envelope. Chlorophylls a and b were barely detectable in the envelope. A Mg2+-dependent ATPase, insensitive to N,N′-dicyclohexylcarbodiimide was associated with the envelope.