Fixation in Spinacia oleracea

Smnmary. Washed whole chloroplasts of Spinacia oleracea isolated and assayed in a tris (hydroxymethyl aminomethane)-HCl bu'ifered sucrose solution exhibited low dark CO.2 fixing activity, whereas washed whole chloroplasts isolated in the same buffer but assayed in that ibuffer without sucrose exh;ibited much greater dark CO2 'fixing activity. The lowered activity could be attributed to the impermeability of the chiloroplast membiane to ribose-5-phosphate or adenosine triphosphate. The preservation of the integrity of the chloroplast menibrane, as reflected by its impermeability to either or both of the abovementioned compounds, was measured by the fixation of 1"CO2 into acid-stable products in the presence of riibose-5-phosphate and adenosine triphosphate by the whole chloroplast as compared with (fixation by the chloroplast extract. An effect (i.e., apparent resistance to the passage of ribose-5-phosphate or adenosine-5-triphosphate into the chloroplast) similar to, but iless pronounced than, that produced by the presence of sucrose in the isolation medium was observed upon the addition of MnCi, or CaCl2 to the buffered sucrose isolation medium. The addition of KCl enhanced slightly the effect produced by addition of sucrose alone to the isolation medium. The ipresence of Mg!'Cl2 in the isolation medium, however, either caused the chloroplasts to become leaky or more fragifle since more of the activity of the car-boxylative phase enzymes appeared in the cytoplasm. When a mixture of all ocf the metal ions was added to the buflfered sucrose sus'pending medium, the chloroplasts exhibited the same response observed with MgGC2 alone. The addition of ethylene diaminetetraacetate or d'ithiothreitol appeared to alter the permeability of the chloroplast membrane nonspecifically when the assay was conducted in the absence of sucrose. Specific act'ivities (umoles 'CO2 fixed/mg chlorophyll X hr) as high as 329.6 have 'been observed for dark fixation 'by chloropl'asts. The iphosphoenolpyruvate carboxylase activity in the chl,oroplasts was only one-seventh that of ribulose diphosphate carboxylase. The phosphoenolpyruvate carboxylase activity in the cytoplasm was 5 times that of the chloroplasts.