Isolation of Envelope Membranes from Bundle Sheath Chloroplasts of Maize

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Abstract
Bundle sheath strands were isolated from maize (Zea mays L.) leaves treated with preparations of cellulase, hemicellulase and pectinase. A 3-phase discontinuous gradient yielded 2 fractions of envelope membranes from bundle sheath chloroplasts. Buoyant densities were 1.06 and 1.09 g cm-3. The lighter fraction contained membrane vesicles under light microscopy, but centrifugation produced a pellet that was too small and unstable for purposes of EM. The heavier fraction contained single and double membrane vesicles and was studied further. Enzymic, chemical, light microscopic and EM examination showed less than 2% contamination by stromal contents, no contamination by microbial, microsomal, or mitochondrial membranes and possible low levels of lamellar membrane contamination. Yields of 0.5 mg of envelope membrane protein were obtained from 56 g leaf sections. The Mg2+-dependent nonlatent ATPase activity, a marker enzyme for chloroplast envelope membranes, was 40 .mu.mol Pi released/h-1 .cntdot. mg protein-1, a value similar to that obtained with pure mesophyll chloroplast envelope membranes from other plants.