Isolation and lipid composition of spinach chloroplast envelope membranes

Abstract

The double outer or envelope membranes, which have a role in CO 2 diffusion and metabolite transport during photosynthesis, were separated from intact spinach chloroplasts by osmotic shock. The envelope membranes were purified by differential centrifugation followed by centrifugation in a discontinuous sucrose gradient. Electron micrographs, enzymatic assays, and chemical analyses showed the envelope membranes were free of stromal, lamellar, mitochondrial, and bacterial contamination. The envelope membrane had a density of 1.10 g cm −3 and a mg lipid/mg protein ratio of 0.78. The lipid composition of the envelope membranes was distinct from that of the lamellar membranes. The major and minor lipids of both membranes were glycolipids and phospholipids, respectively. The envelope membranes contained more monogalactosyldiglyceride, digalactosyldiglyceride, and phosphatidylcholine and less sulfolipid and phosphatidylglycerol than the lamellar membranes. Lipids present exclusively in the envelope membranes were cerebroside (shown for the first time to be a chloroplast lipid), sterylglycoside, acylated sterylglycoside, phosphatidylethanolamine, sterol, and steryl esters. Other lipids identified in the envelope membranes were trigalactosyldiglyceride, phosphatidylinositol, diphosphatidylglycerol, lysophosphatidylcholine, quinone, fatty acids and their methyl esters, and triglycerides. Approximately 90% of the total lipids were accounted for. The large difference in lipid composition between the two kinds of chloroplast membranes probably reflects fundamental differences between their functions in the photosynthetic process.