It has been demonstrated previously that the synthesis of amino acids from photosynthetically fixed carbon in leaves of Capsicumn annuum L. cv. California Wonder occurs in the middle of the photoperiod. This paper reports experiments which identify control points regulating the carbon flow in these leaves.Estimations have been made of the levels of intermediates between 3-phosphoglycerate and pyruvate and between 3-phosphoglycerate and fructose 6-phosphate in leaves at different times in the photoperiod. Application of the Chance crossover analysis indicates that during periods of amino acid synthesis, pyruvate kinase is activated, possibly by ammonium ions. Fructose diphosphate aldolase could possibly be an additional control point, showing activation when amino acid synthesis has ceased. There was no indication of diurnal periodicity in the activity of fructose diphosphate aldolase.A previous study (21) indicated that a major factor in diurnal periodicity in the nature of photosynthetic products was a concomitant periodicity in the availability of reduced nitrogen in the leaf. The investigation has been continued by measuring the levels of carbon intermediates in the leaves of Capsicuim annuumn. ' The crossover analysis (7) was originally developed for a study of the mitochondrial electron transport pathway but has been used successfully in analysis of control points in glycolytic carbon metabolism in yeast cells (10). in aging carrot root tissue (1), and in senescing leaves (16). The method has been used here to identify control points in the steps between 3PGA2 and pyruvate and between 3PGA and F6P, in photosynthesizing leaves during different phases of amino acid synthesis in the first 7 hr of the photoperiod. Aldolase Assay. Aldolase (EC 4. 1 .2.7) was assayed in leaf homogenates prepared in 50 mm tris-H2SO4 buffer pH 8.0 containing 10 mM MgCl2 and 1 mM dithiothreitol. After filtration through Miracloth and centrifugation, the supernatant was used for spectrophotometric estimation of aldolase activity. The reaction mixture consisted of 150 ,umoles of trischloride, pH 7.5 0.24 ,tmole of NADH; 32 IU of a-glycerophosphate dehydrogenase; 60 IU of triose-P isomerase and supernatant to make a total volume of 1.21 ml. The reaction was started by adding 1 pmole of FDP. Controls minus FDP or a-glycerophosphate dehydrogenase were run in parallel.
MATERIALS AND METHODSExtraction of Intermediates. The extraction process of Macnicol (15) was used. Two leaves for each sample were ground at 4 C with 4 ml of 5% (w/v) trichloroacetic acid containing 0.05% (w/ v) 8-hydroxyquinoline. After centrifugation the pellet was re-extracted. The combined extracts were washed 6 times with 4.5 ml of cold diethyl ether (-20 C). The combined ether washes were then extracted with three lots of 8 ml of 25 mm triethanolamine chloride, pH 7.5, and the aqueous phases were added to the original ether-extracted aqueous phase. Residual ether was removed from the aqueous solution of intermediates by bubbling with N2, this was followed b...