Isolation and Bicarbonate Transport of Chloroplast Envelope Membranes from Species of Differing Net Photosynthetic Efficiency

Abstract

A three-phase discontinuous sucrose gradient yielded two fractions of chloroplast envelope membranes from spinach (Spinacia oleracea L.), sunflower (Helianthus annuus L.), and maize (Zea mays L., mesophyll and undifferentiated chloroplasts). These species were selected to represent plants with fast photorespiration and slow net photosynthesis, fast photorespiration yet fast net photosynthesis, and slow photorespiration and fast net photosynthesis, respectively. Buoyant densities were 1.08 and 1.11 g cm-3. The light fraction contained primarily single (incomplete) membrane vesicles and the heavy fraction double (complete) ones. Enzymic, chemical, and electron microscopic examination of the complete envelope membranes showed a lack of microbial, microsomal, mitochondrial, and lamellar membrane contamination as well as stromal contamination. Envelope membranes for all species examined were found to contain 2 to 4% of the total chloroplast protein and yields of about 0.2 to 0.4 mg of protein were obtained from 40 g leaves. An Mg2+-dependent nonlatent ATPase, a marker enzyme for chloroplast envelope membranes, had the following activities (μmoles of phosphate released/hr-1 mg protein-1): spinach, 77; sunflower, 163; old maize, 126; and young maize, 87. Bicarbonate transport was directly correlated with levels of ATPase activity in spinach and sunflower envelope membranes. Transport of HCO3− with sunflower envelope membranes approached that of young maize.