Leaves of 10 rndomly selected plants representing eight dicotyledonous families were exposed to 14CO2 for a 10-minute period in the light.Citrate and alanine were isolated, purified isotopicafy, and degraded to obtain the 14C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.Since Calvin and co-workers originally elucidated the pathway of photosynthesis in higher plants and demonstrated that PGA2 is the primary product of CO2 fixation, the conclusion has prevailed that acetyl-CoA arises from PGA via the glycolytic sequence and is thus the precursor of the tricarboxylic acid cycle (1,2,5,7,9,13,15,16,24,27). Data supporting this reasonable assumption include inhibitor studies, tracer studies, the presence of mitochondrial citric acid cycle activity, and the presence of the requisite glycolytic enzymes. However, a very essential criterion for establishing the precursor-product relationship between chloroplastic PGA and mitochondrial acetyl-CoA during photosynthesis was not investigated until recently when a method for obtaining the complete stereochemical distribution of isotope in citrate was developed (18). The method was applied in a study of the higher plant, Vicia faba (19,20), and it was found that during photosynthesis in "4CO2 by leaves of this plant that the acetate moiety of citrate, which is classically derived from pyruvate, does not, in fact, possess the requisite "4C-isotope distribution characteristic of a Calvin cycle product.While the acetate carbon atoms of citrate were unequally labeled (Cl > C2) after short term photosynthesis in "4CO2, the corresponding a-and ,3-carbon atoms of alanine were equally labeled (Ca = C,8). The latter distribution follows the universal patterns described for both alanine and PGA in higher plants (3,6,14,19 blocks the labeling of the methyl carbon atom of the acetate fragment of citrate (Cl >>> C2); the labeled distribution in alanine is unaffected (Ca = C,8) (20). In the same study, the assimilation of infiltrated [3H]formate was found to be lightdependent; this substrate labeled the cited methyl carbon atom of citrate, but failed to label the corresponding carbon atom in alanine. These data fail to support the classical precursor-product relationship between PGA and acetyl-CoA (see Scheme I). Finally, serine, rather than PGA, possessed exactly the requisite isotope distribution (after photosynthesis in "4CO2 by V. faba leaves) to be the precursor of the acetyl-CoA pool which enters the citric acid cycle (21).These data minimally s...
The cyaneDle from the photosynthetic biflagelate protist Cyaophora paradoxa has been studied in terms of its There is considerable interest in the photosynthetic biflagellate protist Cyanophora paradoxa. This organism was first isolated by Korschikoff (10) and subsequent studies showed that the host cell contained two to four internal structures (4) which had the properties of a cyanobacterium and were designated as cyanelles. The cyanelles resemble unicellular cyanobacteria structurally and contain a pigment system consisting of Chl a, li-carotene, zeaxanthin, C-phycocyanin, and small amounts of allophycocyanin (2). Electron micrographs of the organism reveal a structure expected of a unicellular cyanobacterium, except for the apparent absence of a cell wall (4). The cyanelles do have a rudimentary cell wall, however, since they are surrounded by a thin layer which can be solubilized by lysozyme (15). All indications are that the cyanelle is derived from a cyanobacterium which has been incorporated into a host protist to form an endosymbiotic relationship.Because of its unique environment and apparent retention of the photosynthetic capabilities of cyanobacteria, it is important to study the photosynthetic properties of the cyanelle and compare these with known cyanobacteria. Herdman and Stanier (5) have
Two dual label methods were used to investipte kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.139). In addition to using 1-'4C,5-3HIRuBP (method 1), we describe here the detailed assay with '4CO2 and 15-3HIRuBP (method 2), which generates 3H,'4C,3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (vs/v.) With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. okracea was 69 t 3.occurring flora, kinetic variance of the enzyme was indicated in studies of Anabaena variabilis (2), and Rhodospirillum rubrum (19). Badger (2) concluded that evolutionary pressures have increased the affinity of CO2 for the enzyme in higher plants, albeit diminishing turnover number. To probe the issue ofkinetic variability, we developed the concepts of dual label analysis (9) for the simultaneous kinetic analysis ofthe two enzyme activities of RuBisCo. Using a modification (6) of these methods, Jordan and Ogren (7) report a trend in the relative carboxylation/ oxygenation ratio of RuBisCo which increases in the evolutionary progression from photosynthetic bacteria to higher plants. An
Evidence is presented to support the hypothesis that serine, rather than 3-phosphoglycerate of the Calvin cycle, is a precursor of the tricarboxylic acid cycle during photosynthesis by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C(1) > C(2) > C(3) isotope distribution for that metabolite following the fixation of (14)CO(2). This labeling pattern, while incompatible with an origin either in the Calvin cycle or the glycolate pathway, satisfies a critical criterion for the 3-carbon precursor of the anomalously labeled organic acids. The predominant carboxyl carbon atom labeling of serine reflects either a mixing of two pools of that metabolite, ie., C(1) = C(2) > C(3) and C(1) > C(2) = C(3), or a higher order of complexity in its synthesis. An anomalous C(1) = C(2) > C(3) < C(4) distribution for aspartate, however, suggests an origin by the carboxylation of a 3-carbon intermediate related to serine which has a C(1) = C(2) > C(3) distribution. The latter distribution has been proposed for the serine intermediate of the postulated formate pathway. This pathway is described by the generalized metabolic sequence: CO(2) --> formate --> serine --> organic acids. Corresponding carbon atom distributions for citrate (C(1) > C(2)), aspartate (C(2) > C(3)), and serine (C(2) > C(3)) belie a precursor-product relationship with alanine (C(2) = C(3)), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate.
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