Correlation of structure with function in Spinacea oleracea chloroplasts

Spinach chloroplasts, isolated in isotonic sucrose medium, were sonically ruptured and separated by differential centrifugation into three main fractions: a green precipitate, a colourless supernatant and a yellow, low density lipid-containing layer. These fractions were analyzed for their physical, chemical and biochemical properties. Comparison of a thin section of an isolated osmium-stained chloroplast with a thin section of osmium-stained green precipitate, by electron microscopy, shows that the particles in the green precipitate correspond both in thickness and lateral dimensions to the lamellar structure of the isolated chloroplast. Heavy metal shadowing of both air-dried and lyophilized green precipitate demonstrates the presence of lamellar structures from 10,000 to 20,000 Å in diameter. The lamellar structure, totaling 160 Å in thickness, is composed of two layers; each layer is made up of granular subunits 100 Å thick. Chlorophyll to nitrogen weight ratios are fairly constant for green lamellar structures from 800 to 20,000 Å in diameter, indicating that chlorophyll is uniformly distributed throughout the lamellar structure of the chloroplast. The similarity of relative photosynthetic pigment concentrations over the visible region, of the Hill reaction activity and of the carbon dioxide fixation capacities for the various sized particles in the green precipitate suggests that the smallest lamellar fragments used in these experiments are large in comparison with the smallest fragment which converts electromagnetic to chemical energy. A model for chloroplast lamellar structure is proposed on the basis of the results obtained. The supernatant, by electron microscopy, is seen to consist principally of oblately spherical particles, 100 Å thick and 200 Å in diameter. These particles are indistinguishable from the main water-soluble protein of the chloroplast with sedimentation coefficient 16 (Fraction I protein). About 90% of the total carboxydismutase activity is associated with the supernatant proteins. In addition, these proteins are able to convert fructose-6-phosphate into other photosynthetic carbon cycle intermediates. This supernatant must be added to the green precipitate in order to obtain maximum carbon dioxide fixation rates. Electron micrographs of unfractionated osmium-fixed chloroplast sonicate show large osmium-stained spherical objects corresponding in size and staining properties to the osmiophyllic granules observed in thin sections