Characterization of a Photosynthesizing Reconstituted Spinach Chloroplast Preparation

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Abstract
A particulate preparation (MgP) capable of photosynthetic CO2 assimilation without the addition of stromal protein was obtained by rupturing whole spinach (Spinacia oleracea var. America) chloroplasts in 15 millimolar MgCl2 buffered with Tricine at pH 8.5. This CO2 assimilation was dependent upon light, inorganic phosphate, ferredoxin, ADP, NAD or NADP, and primer. Excepting glycolate, the products of CO2 fixation by MgP were similar to those found with whole chloroplasts. Glycerate-3-phosphate (PGA), fructose-1, 6-bisphosphate (FBP), and ribose-5-phosphate (R5P) but not fructose-6-P (F6P) functioned as primers. Concentrations of PGA and FBP but not of R5P higher than 2 millimolar were inhibitory to CO2 fixation. A lag of CO2 fixation was observed with PGA and FBP but not with R5P. This lag as well as inhibition by NADP, ADP, and ATP in the FBP-primed preparation was eliminated by an equimolar mixture of FBP plus F6P indicating FBPase as the sensitive site. NADP, ADP, and ATP also blocked CO2 fixation by the PGA-fortified preparation but inhibition was even more sensitive than that observed when FBP was added. Inhibition by AMP in the PGA and FBP-primed preparations was not affected by the addition of F6P. When R5P was the starting primer, inhibition of CO2 fixation was relatively insensitive to the adenylates and NADP. In contrast to the parent whole chloroplast, CO2 fixation by MgP was insensitive to high (5 millimolar) inorganic phosphate. Depending upon the ferredoxin concentration, NAD was as effective as NADP in supporting CO2 fixation.

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