A three-phase, discontinuous sucrose gradient yielded two distinct fractions of envelope membranes from spinach (Spinacia oleracea L.) chloroplasts. Their buoyant densities were 1.08 g cm-' and 1.11 g cm-3. Electron micrographs showed the lighter and heavier fractions to consist primarily of single and double membranes, respectively. The milligrams of lipidmilligrams of protein ratio for the complete envelope membrane (double membrane fraction) was 1.74. Thin layer chromatograms showed that the lipids of the complete envelope membranes were similar to those found in earlier preparations which consisted of single and double membranes. This isolation procedure is superior to earlier methods in that the percentage of complete envelope membranes is greater and the yield is almost three times as great. Enzymatic and chemical analyses and microscopic examination showed the complete envelope membranes were free of bacterial, fungal, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. The specific activities of nonlatent Mg2'-dependent ATPase (80 ,umoles of phosphate released hr-1 mg protein-') were about 10-fold higher than those values found with earlier preparations consisting of single and double membranes, indicating that the ATPase is largely lost in preparations containing single membranes. These higher values show that the ATPase is located in the double membrane and probably functions in the transport processes of the envelope membrane.Mlethods for the isolation of envelope membranes from chloroplasts have beeen developed recently by Poincelot (14) and others (2, 7). Although these envelope membrane prep-arations are relatively free of lamellar and stromal contamination, they suffer from two disadvantages. First, they have low levels of ATPase activity (approximately 12 ,umoles Pi released hr-' mg protein-1), and second, they consist of a mixture of single and double membrane vesicles (2,7,14).Inasmuch as the Mg2-dependent ATPase has been suggested as an enzymatic marker for chloroplast envelope membranes (2,14 Chloroplasts. Intact chloroplasts were prepared from 10-g batches of freshly harvested leaves by procedures already described (4, 12). Plants, when harvested, were 4 to 6 weeks old.Removal and Isolation of Envelope Membranes. The method is summarized in Figure 1 and is based on the fact that envelope membranes are easily removed from intact chloroplasts by suspending the latter in hypotonic medium. Two pellets of intact chloroplasts (derived from 20 g leaves) were suspended in 6 ml of hypotonic medium (Tricine buffer, pH 7.6, 50 mM). These suspensions of 0.58 to 0.75 mg of Chl/ml were kept at 4 C for 20 min with occasional swirling, whereupon they were pooled and homogenized by making three complete passes in a TenBroeck homogenizer. The homogenized suspension was mixed with 2 ml of 48 % sucrose (w/v) in 50 mm Tricine buffer (pH 7.6) to yield about 8 ml of a suspension containing 12% sucrose (w/v). The suspension was purified on a three-phase, disco...
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