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...

Thioredoxins are small redox proteins, alternating between the S-S (oxidized) and SH (reduced) states, that function in a number of important biochemical processes, including DNA synthesis, DNA replication, and enzyme regulation. Reduced ferredoxin is known to serve as the source of reducing power for the reduction of thioredoxins only in photosynthetic cells that evolve oxygen. In all other organisms, the source of hydrogen (electrons) for thioredoxin reduction is considered to be NADPH. We now report evidence that Clostridium pasteurianum, an anaerobic bacterium normally living in the soil unexposed to light, resembles photosynthetic cells in that it uses reduced ferredoxin as the reductant for thioredoxin. Moreover, the transfer of electrons from reduced ferredoxin to thioredoxin is catalyzed by a flavoprotein enzyme that has not been detected in other organisms. Our results reveal the existence of a pathway for the reduction of thioredoxin in which ferredoxin, reduced fermentatively either by molecular hydrogen or by a carbon substrate, provides the reducing power for the flavoprotein enzyme ferredoxin-thioredoxin reductase, which in turn reduces thioredoxin.

Addition of ammonia to a suspension of photosynthesizing isolated mesophyll cells from P. somniferum quantitatively alters the pattern of carbon metabolism by increasing rates of certain key ratelimiting steps leading to amino-acid synthesis and by decreasing rates of rate-limiting steps in alternative biosynthetic pathways. Of particular importance is the stimulation of reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase. The increased rates of these two reactions, which result in an increased flow of carbon into the tricarboxylic-acid cycle, correlate with a rapid rise in glutamine (via glutamine synthetase) which draws carbon off the tricarboxylic-acid cycle as α-ketoglutarate. Increased flux of carbon in this direction appears to come mainly at the expense of sucrose synthesis. The net effect of addition of ammonia to mesophyll cells is thus a redistribution of newly fixed carbon away from carbohydrates and into amino acids.

Most chloroplast proteins are encoded by nuclear genes and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported posttranshationally into the chloroplasts, where they are proteolytically processed, and sorted to their proper locations. The first step of this import process is thought to be the binding of precursors to putative receptors on the outer envelope membrane of chloroplasts. We have investigated the interaction of the precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with its putative receptor by using a heterobifunctional, photoactivatable cross-linker. The resulting cross-linked conjugate has a molecular weight of 86,000, and is present on the surface of chloroplasts as determined by its sensitivity to digestion with protease. Control experiments demonstrated that the label in the conjugate is derived from small subunit precursor and that the conjugate is formed only when modified precursor is reacted in the presence of chloroplasts. Based on these results, we postulate that a protein on the surface of chloroplasts is part of the receptor which interacts with the small subunit precursor.Most proteins found in chloroplasts and mitochondria are nuclear encoded and synthesized on cytoplasmic ribosomes. The majority of these proteins are made as higher mol wt precursors which are then imported into the respective organelle, proteolytically processed to the mature size and sorted to their proper location (cf 9 and 21 for recent reviews). The first step of this import process is thought to be the recognition and binding of the precursor protein to a specific receptor on the surface of the proper organelle. The most extensive evidence for the involvement ofspecific receptors comes from the study of mitochondrial protein import (9). One type of evidence for the involvement of mitochondrial surface proteins comes from the inhibition of protein import by pretreatment of mitochondria with proteases (19,23,24 Despite the studies described above, we still know very little about the putative receptor proteins or their role in the import process. For example, a receptor protein has not been identified in either mitochondria or chloroplasts. The lack of rigorous proof for the presence of receptors, combined with the observation that precursors can interact with lipids (20), has led to the suggestion that precursor proteins may interact directly with lipid bilayer membranes without the involvement of proteinaceous receptors (20,22). Thus, it becomes important to determine whether proteinaceous receptors exist, to identify the proteins which function as receptors, and to investigate their involvement in the import process. It should be possible to examine precursor/ receptor interactions and to identify the receptor proteins using some of the techniques that have been applied to other receptor/ ligand interactions, such as the well characterized interactions between polypeptide hormones and their receptors. One approach which has been successfull...