Changes in Steady-State Photosynthetic 14C-Incorporation into cellular Metabolites of Chlorellax pyrenoidosa during Transitions from High to Low Irradiance

Changes in the levels of ¹⁴C-labelled metabolites were monitoredin Chlorella pyrenoidosa cells during a transition from highto low irradiance, i.e., from 700 to 430 µmol quanta (400–700nm) m^–2 s^–1. Chlorella cells assimilated ¹⁴CO₂ photosynthetically(steady-state ¹⁴C-labelling) for 12 min at the high irradianceand then 10 min at the low irradiance. With the transition tolow light, the level of ¹⁴C-labelled ribulose 1,5- bisphosphate(RuBP) did not decrease, even though the rate of total ¹⁴C-incorporationdecreased by 80%. The data suggest that RuBP carboxylase deactivatesrapidly (within 1 or 2 min) on exposure to low light, causingRuBP pool sizes to be maintained (or even increased) in spiteof a decreased rate of RuBP regeneration. There was also evidenceof light modulation of other enzymes, including some enzymesinvolved in sucrose synthesis. The rate of sucrose synthesisdecreased with decrease in light intensity while the level ofuridine diphosphoglucose increased, but within a few minutes,both returned to their former levels. ¹Present address: Chemical Biodynamics, Lawrence Berkeley Laboratory,Building 3, 1 Cyclotron Road, Berkeley, CA 94720, U.S.A. (Received March 8, 1986; Accepted June 25, 1986)     

Pyruvate orthophosphate dikinase in wheat leaves

Pyruvate orthophosphate dikinase (PPDK) was found in wheat (Triticum aestivum L. cv Cheyenne [CI 8885]) leaves both by activity assays and by the protein blot method. The specific activity of the wheat enzyme is comparable to that of PPDK from maize leaves. Of the total soluble protein in wheat leaves, about 0.05% was PPDK, comparable to the amount in the immature wheat seed and about 1/70th the amount found in mesophyll cells of maize. Immunoprecipitation of wheat PPDK with maize enzyme antiserum indicates partial identity, and the apparent subunit molecular weight is the same based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Antimycin A Stimulation of Rate-limiting Steps of Photosynthesis in Isolated Spinach Chloroplasts

Changes in levels of metabolites in isolated spinach (Spinacia oleracea) chloroplasts seen upon addition of antimycin A suggest that the activities of enzymes mediating several regulated reactions are affected. Apparently, the presence of added antimycin A does not increase the level of CO₂ in the chloroplasts, nor does it stimulate CO₂ fixation by increasing the level of the carboxylation substrate, ribulose-1,5-diphosphate. Rather, it appears that antimycin A increases CO₂ fixation rate by indirectly stimulating the enzyme, ribulose-1,5-diphosphate carboxylase (E.C. 4.1.1.39), which mediates the carboxylation of ribulose-1,5-diphosphate to give 3-phosphoglycerate. Another rate-limiting enzyme of the reductive pentose phosphate cycle, hexose diphosphatase (E.C. 3.1.3.11), seems also to be stimulated. The synthesis of polysaccharides (mostly starch) seems also to be stimulated. These results are interpreted as indicating that antimycin A addition enhances the general activation of those enzymes which already are activated during photosynthesis but are inactive in the dark. The ratio of adenosine triphosphate-adenosine diphosphate under conditions of photosynthesis was only moderately decreased in the presence of antimycin A, perhaps accounting in part for an observed increase in accumulation of 3-phosphoglycerate as compared with dihydroxyacetone phosphate. No significant effect on movement of metabolites from the chloroplast to the medium was seen.     

Light-Dark Transients in Levels of Intermediate Compounds during Photosynthesis in Air-Adapted Chlorella

The labeling of intermediate compounds and photosynthetic cofactors during photosynthesis and periods of darkness by Chlorella pyrenoidosa in the presence of ³²P-labeled phosphate and ¹⁴CO₂ have heen investigated. Algae adapted to photosynthesis in air were used, and the level of carbon dioxide was maintained at approximately 0.04 % and at constant specific radioactivity during the course of the experiments. The transient changes which occur in the levels of labeled fructose-1,6-diphosphate and in sedoheptulose-1,7-diphosphate, and in the corresponding monophosphates when the light is turned off suggest a light activation of the diphosphatase enzymes which decays after about 2 minutes of darkness. It is suggested that a light-dark switch in enzymic activities permits photosynthesis and glycolysis to occur in light and dark respectively with the same enzymic apparatus. The greatly diminished rate of disappearanec of the carboxylation substrate, ribulose-1,5-diphosphate, after about 2 minutes suggests that there is also a light activation of the carboxylation reaction in vivo. Large transient changes in the level of pyrophosphate between light and dark indicate that there may be an unstable cofactor which decomposes to give pyrophosphate during or alter killing of the algal cells. The possibility that this cofactor is involved in an activation of Carbon dioxide for the carboxylation reaction in vivo is suggested. Light-dark transient changes in labeling of other compounds of the photosynthetic carbon reduction cycle and related compounds were determined, and possible significance of these changes is discussed.(PDF DAMAGED)     

Transport of proteins into chloroplasts. Delineation of envelope "transit" and thylakoid "transfer" signals within the pre-sequences of three imported thylakoid lumen proteins.

The targeting of cytosolically synthesized proteins into the thylakoid lumen is mediated by an aminoterminal pre-sequence consisting of an "envelope transit" and a "thylakoid transfer" signal in tandem. We have investigated the structural characteristics of several thylakoid transfer signals by determining the intermediate sites at which the stromal processing peptidase cleaves to remove the transit sequences. Using this approach we have found that the thylakoid transfer signals of Silene pratensis plastocyanin, 23-kDa oxygen-evolving complex protein from wheat, and 33-kDa oxygen-evolving complex protein from wheat, are 25, 39, and 48 residues in length, respectively. All of the transfer signals contain hydrophobic core sequences and a "-3,-1" motif reminiscent of those found in signal sequences, but the amino-terminal regions of the transfer signals of the 23- and 33-kDa proteins are both longer and more highly charged. The net charge of each amino-terminal region of the transfer sequences is +1, including the amino-terminal amino group. In each case, the stromal processing peptidase cleaves immediately after a positively charged residue, but otherwise the cleavage sites exhibit no common elements of either primary or secondary structure.     

Botryococcus braunii carbon/nitrogen metabolism as affected by ammonia addition

Carbon metabolism in photosynthesizing and respiring cells of Botryococcus braunii was radically changed by the presence of 1 mM NH₄Cl in the medium, when the so-called resting state previously had been subjected to a nitrogen-deficient medium. Ammonia addition to the algae photosynthesizing with ¹⁴C-labelled HCO ₃ ^- almost completely inhibited the synthesis of ¹⁴C-labelled botryococcenes and other hexane-extractable compounds, and also inhibited the formation of insoluble compounds; however, it resulted in a large increase in the synthesis of alanine, glutamine, other amino acids, and especially of 5-aminolevulinic acid. Total CO₂ fixation decreased about 60% and O₂ evolution decreased more than 50%.CO₂ fixation in the dark with ammonia present led to labelled products derived from phosphoenolpyruvate carboxylation, such as glutamine, glutamate, and malate. Respiratory uptake of O₂ increased by about 70%.The inhibition of terpenoid synthesis and increased synthesis of C₅ amino acids by Botryococcus upon ammonia addition indicates 1) a diversion of acetyl coenzyme A from synthetic pathways leading to terpenoids and 2) increased operation of pathways leading to the synthesis of amino acids, especially 5-aminolevulinic acid, a precursor to chlorophyll biosynthesis.This work was supported in part by the Office of Energy Research, Office of Basic Energy Sciences, Biological Energy Research Division of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098, in part by a grant from SOHIO, and, in part, by a grant from the Japan-U.S. Cooperative Science Program (The Japan Society for the Promotion of Science, National Science Foundation, Division of International Programs)     

Carbon allocation in wild-type and Glc+ Rhodobacter sphaeroides under photoheterotrophic conditions

The photosynthetic bacterium Rhodobacter sphaeroides is capable of producing H2 via nitrogenase when grown photoheterotrophically in the absence of N2. By using 14C-labeled malate, it was found that greater than 95% of this substrate was catabolized completely to CO2 during H2 production. About 60% of this catabolism was associated with H2 biosynthesis, while almost 40% provided reductant for other cellular purposes. Thus, only a small fraction of malate provided carbon skeletons. The addition of ammonium, which inhibited nitrogenase activity, increased substrate conversion into carbon skeletons threefold. Catabolism of malate occurred primarily via the tricarboxylic acid cycle, but gluconeogenesis was also observed. The wild-type organism grew poorly on glucose, accumulated gluconate and 2-keto-3-deoxygluconate, and did not produce H2. More than 50% of metabolized glucose appeared in carbon skeletons or in storage compounds. A glucose-utilizing mutant was five times more effective in utilizing this substrate. This mutant produced H2 from glucose, using 74% of metabolized substrate for this purpose. Glucose converted to storage products or to other carbon skeletons was reduced to 8%. Fixation of CO2 competed directly with H2 production for reducing equivalents and ATP. Refixation of CO2 released from these substrates under H2-producing conditions was, at most, 10 to 12%. Addition of ammonium increased refixation of respired CO2 to 83%. Patterns of carbon flow of fixation products were associated with the particular strains and culture conditions.