Incorporation of Oxygen into Glycolate, Glycine, and Serine during Photorespiration in Maize Leaves

Glycolate, glycine, and serine extracted from excised Zea mays L. leaves which had been allowed to photosynthesize in the presence of ¹⁸O₂ were analyzed by gas chromatography-mass spectrometry. In each case, only one of the oxygen atoms of the carboxyl group had become labeled. The maximum enrichment observed in glycine and serine was attained after 5 minutes and 15 minutes of exposure to ¹⁸O₂ at the CO₂ compensation point; the labeling was very high, reaching 70 to 73% of that in the applied O₂. Thus, it appears that all or nearly all of the glycine and serine are synthesized in maize leaves via fixation of O₂. In the presence of CO₂ (380 or 800 microliters per liter), ¹⁸O-labeling was markedly slower.

Glycolate enrichment was variable and much lower than that in glycine and serine. It is possible that there are additional pathways of glycolate synthesis which do not result in the incorporation of ¹⁸O from molecular oxygen. An estimation of the metabolic flow of O₂ through the photorespiratory cycle was made. It appeared that less than 75% of the O₂ taken up by maize leaves is involved in this pathway. Therefore, other processes of O₂ metabolism must occur in the light.

     

Synthesis, Excretion, and Metabolism of Glycolate under Highly Photorespiratory Conditions in Euglena gracilis Z

Glycolate was excreted from the 5% CO₂-grown cells of Euglena gracilis Z when placed in an atmosphere of 100% O₂ under illumination at 20,000 lux. The amount of excreted glycolate reached 30% of the dry weight of the cells during incubation for 12 hours. The content of paramylon, the reserve polysaccharide of E. gracilis, was decreased during the glycolate excretion, and of the depleted paramylon carbon, two-thirds was excreted to the outside of cells and the remaining metabolized to other compounds, both as glycolate. The paramylon carbon entered Calvin cycle probably as triose phosphate or 3-phosphoglycerate, but not as CO₂ after the complete oxidation through the tricarboxylic acid cycle. The glycolate pathway was partially operative and the activity of the pathway was much less than the rate of the synthesis of glycolate in the cells under 100% O₂ and 20,000 lux; this led the cells to excrete glycolate outside the cells. Exogenous glycolate was metabolized only to CO₂ but not to glycine and serine. The physiologic role of the glycolate metabolism and excretion under such conditions is discussed.     

Glycine cleavage and generation of one-carbon units in Euglena gracilis

Euglena gracilis Klebs (strain Z) was maintained in division synchronized autotrophic culture, receiving either air (low CO₂) or 5% CO₂ in     

Purification and Characterization of Serine Hydroxymethyltransferase from Mitochondria of Euglena gracilis z

Mitochondrial serine hydroxymethyltransferase, l-serine: tetrahydrofolate 5,10-hydroxymethyl-transferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with l-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88,000 by gel filtration through Sephadex G-200, and 44,000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5′-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had l-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity.     

Glycolate excretion by air-grown Euglena graeilis z

In contrast to reported fact, the air-grown cells of Euglena gracilis z were found to excrete glycolate into the surrounding medium, when placed in an atmosphere of 100% O₂ under illumination at 20000 lux at the same rate of the 5% CO₂-grown cells, but with a lag phase of about 30 min. The lag was eliminated by lowering intracellular CO₂ concentration in the air-grown cells.This paper is the eighth in a series on the metabolism of glycolate in Euglena gracilis.     

Degradation of Paramylon by Euglena gracilis

SYNOPSIS. Protozoa of the order Euglenida contain a polysaccharide storage product, paramylon, composed of 1, 3-linked glucose molecules arranged into an extremely resistant granule. An enzyme was purified from the soluble phase of Euglena gracilis which would degrade this polysaccharide to single glucose residues, providing the integrity of the paramylon granule was 1st disrupted by dilute base. This enzyme, a β-1, 3 glucanase, had optimal activity at pH 5.0 and 60 C and bound tightly to base-disrupted paramylon substrate tho not to the intact granules. The specific activity of the enzyme was doubled when cell cultures reached stationary phase, the phase where net carbohydrate utilization began. An ATP-dependent hexokinase reaction was also present in Euglena homogenate. No phosphorylase activity has been found in Euglena. It is suggested, therefore, that Euglena do utilize their paramylon as a carbohydrate reserve and the mechanism of this utilization is by exo-hydrolytic cleavage to free glucose followed by phosphorylation and glycolysis.     

Mercury uptake and removal by Euglena gracilis

The uptake and removal of mercury (added as HgCl2) from the culture medium by Euglena gracilis was studied. In cultures initiated in the light, cells accumulated a small fraction of the added heavy metal (5-13%). Mercury was both biologically and nonbiologically volatilized, and cell growth was partially inhibited; under these conditions the glutathione content was 3.2 nmol/10(6) cells. In contrast, in cultures initiated in the dark, mercury uptake by cells was two to three times higher, biological volatilization remained unchanged and nonbiological volatilization and growth were negligible; the glutathione content diminished to 1.4 nmol/10(6) cells. Biological mercury volatilization depended on cell density and metal concentration, but was light-independent. Thus, volatilization of mercury by Euglena appeared not to be an effective mechanism of resistance, whereas a high intracellular level of glutathione and a low mercury uptake seemed necessary for successful tolerance.     

Evidence of Reentrance of Glycolate Carbon into the Photosynthetic Carbon Reduction Cycle in Photosynthesizing Euglena gracilis Z

Aminooxyacetate induced excretion of glycolate from air-grown cells of Euglena gracilis in both air and 1% CO₂ atmospheres. The rate of the excretion reached 70% of the photosynthetic rate in the air on a carbon basis, and was 10% in 1% CO₂. The compulsory loss of photosynthetically fixed carbon as glycolate at the high rate in air in the presence of aminooxyacetate caused a decrease of the rate of synthesis of paramylon, the reserve polysaccharide. Analyses of the steady levels of photosynthetic intermediates showed that a decrease of the 3-phosphoglycerate level was the cause of the slow rate of paramylon synthesis under these conditions.     

Effects of Glycolate Pathway Intermediates on Glycine Decarboxylation and Serine Synthesis in Pea (Pisum sativum L.)

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O₂ treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O₂-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N₂ supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

Erythromycin-Induced Bleaching of Euglena gracilis

SYNOPSIS. Erythromycin bleaches Euglena gracilis in a manner resembling that of streptomycin. Erythromycin-bleached substrains have been cultivated 16 months in light on erythro-mycin-free media without greening. Bleached substrains were obtained only if erythromycin was added to actively growing cultures: erythromycin did not bleach if added during the stationary phase of growth of green cultures.     

Accumulation of Extracellular Amino Acids by Euglena gracilis

SYNOPSIS. Normal Euglena gracilis , strain z, growing in the light in defined medium (initial nitrogen concentration 140 μ/ml) depletes the medium of all ninhydrin-positive N by the time a cell density of 2 million per ml is reached. A further 2- to 3-fold increase in the cell number takes place in the absence of exogenous N. The N content of an early log phase cell is about 100 picograms but decreases very rapidly as the culture continues to grow, reaching 22 picograms in the stationary phase. When grown in the dark, normal cells take up N somewhat more slowly but the supernatant fluid from saturation cultures is again devoid of N.
At modest cell densities, the permanently bleached strains examined contain less N per cell than do normal strains. The cultures of the bleached strains achieve a maximum density of about 1 to 2 million per ml rather than the 4 to 5 million reached by the normal strain. As a result, supernates from stationary phase cultures of bleached cells still contain a large proportion of the total N supplied.
Paper chromatographic analysis of these supernates reveals several ninhydrin-positive compounds. Most of these have been identified as common amino acids. Some of the properties of two unidentified, ninhydrin-positive compounds are described.