Amino Acid Synthesis in Photosynthesizing Spinach Cells

Abstract

Isolated cells from leaves of Spincia oleracea have been maintaied in a state capable of high rates of pbotosynthetic CO2 fixatin for more than 60 hours. The incorporation of 14CO2 under saturating CO2 conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivty, and pool size have been determined as a function of time for most of the protein amino acids and for y-aminobutyric acid. The measurements of specific radio-activities and of the approaches to "C "saturation" of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulator effects in incorporation of 'C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginie, and histidine. No effect or slght decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, "4C labeling decreased, but specific radioactiity increased. The production of labeled -aminobutyric acid was virtuafly stopped by ammonia.The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. The data on the effects of added ammonia on total labelin, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis.In recent years, the production of photosynthetically active cells from leaf tissue has been reported for a number of plant species, including Papaver somniferum (opium poppy) (22), spinach (31), and cotton (28). Such cells afford possibilities for studying plant metabolism in a simple but intact system. Effects on poppy cell metabolism of ammonia (24), sulfite (23), and 2,4-D (25) have been reported from this laboratory, and others have described effects of ammonia and other nitrogen compounds on metabolism in isolated spinach cells (31,32 incorporation into amino acids has been studied to provide information about amino acid biosynthesis and the provision of carbon skeletons for this purpose. For the amino acids occurring in the free state, values have been obtained for total 14C labeling, specific radioactivities, and, by combination of these data, pool sizes. The methods used have permitted some measurements for all the protein amino acids except cysteine/cystine and methionine, and thus provide a more detailed and complete picture of amino acid formation from CO2 in leaf cells than previously available.Previous work from this and other laboratories has demonstrated that ammoni...

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

“…The ratio of the N-fluxes from alanine into glycine synthesis to that from glutamate is not affected by this correction of the calculations, because the partitioning of alanine and glutamate between the vacuole and the other compartments is the same (27). However, results of '4C labeling experiments with spinach leaves indicate that the active pool of alanine is about 50%Yo of the total pool, whereas the active pool of glutamate might be about 20% of the total pool (15). If alanine and glutamate were partitioned in the oat leaf cells as in spinach leaf cells, this would mean that the ratio of the N-flux from alanine into glycine synthesis to that from glutamate was even higher than 3:1.…”

Regulation of Phosphoenolpyruvate Carboxylase from the Green Alga Selenastrum minutum

“…During NH4' assimilation, the net synthesis of glutamine and glutamate utilizes 2-oxoglutarate in the GS/GOGAT reactions (17). Increased rates of NH4' assimilation have been correlated with elevated rates of dark carbon fixation in cyanobacteria, green algae, and higher plants (4-6, 10,11,21,22,24) and these elevated rates of dark carbon fixation have been attributed to increased activity ofPEPC. However, the factors responsible for increased PEPC activity have not been identified.…”

The Path of Carbon Flow during NO3−-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

“…7C). Increases in the pyr:PEP radiolabel ratio have been interpreted as reflecting increased carbon flux through PEP to pyr since the conversion of PEP to pyr is a rate limiting metabolic step (9,11,12,17,20,21 results documented increased availability of anaplerotic substrates for the tricarboxylic acid cycle during N-induced photosynthetic suppression. Further evidence suggests that tricarboxylic acid cycle activity increased in response to NO3-enrichment.…”