Characterization of chloroplast photosystems 1 and 2 separated by a non-detergent method

Abstract

Class II spinach chloroplasts were fragmented by passage through the French pressure cell (French press), and the fragments were separated by fractional centrifugation. Fragments sedimenting between 1000 × g and 10000 × g (10K) have a lower chlorophylla/chloropyllb ratio and lower P-700 content than whole chloroplasts. Fragments sedimenting between 40000 × g and 160000 × g (160K) have a much higher chlorophylla/chlorophyllb ratio (6.0) and a much higher P-700 content (1 P-700 per 105 chlorophylls) than whole chloroplasts. The chlorophyll and cytochrome contents of the French press fractions are similar to those found in fractions isolated after digitonin disruption. The 160K fraction performs Photosystem 1 but not Photosystem 2 reactions. The 10K fraction contains both photosystems. Electrophoresis of sodium dodecyl sulfate solubilized 10K and 160K fractions gives further evidence for this distribution of photosystems. Thin sectioning and freeze fracturing show that the 160K fraction originates from stroma lamellae and the end membranes of grana stacks and contains only 110Å particles. The 10K fraction originates from the partition regions of grana stacks and contains both 110 and 175Å particles. This distribution of particles on fracture faces of stroma versus grana lamellae is shown to exist in freeze fractured Class I chloroplasts. These data demonstrate that both digitonin and French press treatments of chloroplasts initially break stroma lamellae and end membranes to yield small vesicles which contain only Photosystem 1.