, Volume 165, Issue 1, pp 12–22 | Cite as

Chlorophyll biosynthesis from glutamate or 5-aminolevulinate in intact Euglena chloroplasts

  • B. Gomez-Silva
  • M. P. Timko
  • J. A. Schiff


Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO2 fixation were isolated from cells grown on low-vitamin-B12 medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or l-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.

Key words

5-Aminolevulinic acid Chlorophyll synthesis Chloroplast Euglena Glutamic acid Protein synthesis 



5-aminolevulinic acid




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  1. Alhadeff, M., Coronado, R., Figueroa, N., Schiff, J.A. (1983) Regulation of protochlorophyll(ide) levels in dark-grown non-dividing Euglena. 1. Control by light. Photochem. Photobiol. 38, 1.Google Scholar
  2. Battersby, A.R., Fookes, C.J.R., Gustafson-Potter, K.E., Matcham, G.W.J., McDonald, E. (1979) Proof by synthesis that unrearranged hydroxymethylbilane is the product from deaminase and the substrate for cosynthetase in the biosynthesis of uro'gen-III. J. Chem. Soc. Chem. Comm., 1155–1158Google Scholar
  3. Battersby, A.R., Hodgson, G.L., Hunt, E., Saunders, J. (1976) Biosynthesis of porphyrins and related macrocycles VI. Nature of the rearrangement process leading to the natural type III porphyrins. J. Chem. Soc. Perkin. I, 273–282Google Scholar
  4. Bingham, S., Schiff, J.A. (1979a) Events surrounding the early development of Euglena chloroplasts. 15. Origin of plastid thylakoid polypeptides in wild-type and mutant cells. Biochim. Biophys. Acta 547, 512–513Google Scholar
  5. Bingham, S., Schiff, J.A. (1979b) Events surrounding the early development of Euglena chloroplasts. 16. Plastid thylakoid polypeptides during greening. Biochim. Biophys. Acta. 547, 531–543Google Scholar
  6. Bruinsma, J. (1961) A comment on the spectrophotometric determination of chlorophyll. Biochim. Biophys. Acta. 52, 576–578Google Scholar
  7. Carell, E.F., Kahn, J.S. (1964) Synthesis of porphyrins by isolated chloroplasts of Euglena, Arch. Biochem. Biophys. 108, 1–6Google Scholar
  8. Castelfranco, P.A., Beale, S.I. (1981) Chlorophyll biosynthesis. In: The biochemistry of plants. A comprehensive treatise, vol. 8, Photosynthesis, pp. 375–421, Hatch, M.D., Boardman, N.K., eds., Academic Press, New York LondonGoogle Scholar
  9. Castelfranco, P.A., Scharcz, S. (1978) Mg-protoporphyrin-IX and 5-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. Mg-protoporphyrin-IX synthesis. Arch. Biochem. Biophys. 186, 365–375Google Scholar
  10. Chereskin, B.M., Wong, Y-S, Castelfranco, P.A. (1982) In vitro synthesis of the chlorophyll isocyclic ring. Transformation of magnesium-protoporphyrin IX and magnesium-protoporphyrin IX monomethyl ester into magnesium-2, 4-divinyl phoephorphyrin a5. Plant Physiol. 70, 987–993Google Scholar
  11. Chow, P.N.P. (1977) Bleaching of chlorophylls in alcohol extracts with benzoyl peroxide for liquid scintillation counting of 14C-labeled compounds. Anal. Biochem. 80, 507–512Google Scholar
  12. Cunningham, F.X. Jr., Schiff, J.A. (1984) Presence or absence of the light harvesting chlorophyll (LHC) apoprotein in Euglena mutants lacking chlorophyll b (Abstr.) Plant Physiol. 75, Suppl. 154Google Scholar
  13. Daniell, H., Ramanujam, P., Krishnan, M., Gnanam, A., Rebeiz, C.A. (1983) In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation. Biochem. Biophys., Res. Commun. 111, 740–749Google Scholar
  14. Ebbon, J.G., Tait, G.H. (1969) Studies on S-adenosylmethionine-magnesium proporphyrin methyl transferase in Euglena gracilis strain. Z. Biochem. J. 111, 573–582Google Scholar
  15. Edelman, M., Reisfeld, A. (1978) Characterization, translation and control of the 32 000 dalton chloroplast membrane, protein in Spirodela. In: Chloroplast development, pp. 641–652, Akoyunoglou, G., Argyroudi-Akoyunoglou, J.H., eds. Elsevier/ North-Holland, AmsterdamGoogle Scholar
  16. Egan, J.M. Jr., Dorsky, D., Schiff, J.A. (1974) Events surrounding the early development of Euglena chloroplasts. VI. Action spectra for the formation of chlorophyll, lag elimination in chlorophyll synthesis, and appearance of TPN-dependent triose phosphate dehydrogenase and alkaline DNase activities. Plant Physiol 56, 318–323Google Scholar
  17. Egan, J.M., Schiff, J.A. (1974) A reexamination of the action spectrum for chlorophyll synthesis in Euglena gracilis. Plant Sci. Lett. 3, 101–105Google Scholar
  18. Ellsworth, R.K. (1971) Studies on chlorophyllase I. Hydrolytic and esterification activities of chlorophyllase from wheat seedlings. Photosynthetica 5, 226–232Google Scholar
  19. Ellsworth, R.K., Hervish, P.V. (1975) Biosynthesis of protochlorophyllide a from Mg-protoporphyrin IX in vitro. Photosynthetica 9, 125–139Google Scholar
  20. Fischer, H., Filser L., Hagert, W., Moldenhauer, O. (1931) Über neue Entstehungsweisen der Chlorophyllporphyrine und ihre Konstitution. Liebigs. Ann. Chem. 490, 1–38Google Scholar
  21. Foley, T., Beale, S.I. (1982) Aminolevulinic acid formation from 4,56-dioxovaleric acid in extracts of Euglena gracilis. Plant Physiol. 70, 1495–1502Google Scholar
  22. Forsee, W.T., Kahn, J.S. (1972) Carbon dioxide fixation by isolated chloroplasts of Euglena gracilis. 1. Isolation of functionally intact chloroplasts and their characterization. Arch. Biochem. Biophys. 150, 296–301Google Scholar
  23. Fuesler, T.P., Castelfranco, P.A., Wong, Y.-S. (1984) Formation of Mg-containing chlorophyll precursors from protoporphyrin IX, 5-aminolevulinic acid, and glutamate in isolated, photosynthetically competent, developing chloroplasts. Plant Physiol. 74, 928–933Google Scholar
  24. Gomez-Silva, B., Delorme, E., Stern, A.I. Schiff, J.A. (1984) Protein synthesis in organello by purified, intact, functional mitochondria from Euglena gracilis var. bacillaris. (Abstr.) Plant Physiol. 75, Suppl., 196Google Scholar
  25. Gomez-Silva, B., Schiff, J.A. (1981) Light-dependent CO2 fixation (CF) and protein synthesis (PRS) in intact chloroplasts from Euglena gracilis var. bacillaris. (Abstr.) Plant Physiol. 67, Suppl., 32Google Scholar
  26. Gomez-Silva, B., Timko, M.P., Schiff, J.A. (1983) Chlorophyll biosynthesis in isolated developing chloroplasts of Euglena gracilis var. bacillaris (Abstr.) Plant Physiol. 72, Suppl., 37Google Scholar
  27. Gough, S.P., Kannagara, C.G. (1976) Synthesis of aminolevulinic acid by isolated plastids. Carlsberg Res. Commun. 41, 183–190Google Scholar
  28. Gurevitz, M., Kratz, H., Ohad, I. (1977) Polypeptides of chloroplastic and cytoplasmic origin required for development of photosystem. II activity, and chlorophyll-protein complexes, in Euglena gracilis Z. chloroplast membranes. Biochim. Biophys. Acta 461 475–488Google Scholar
  29. Holowinsky, A.W., Schiff, J.A. (1970) Events surrounding the early development of Euglena chloroplasts. I. Induction by preillumination. Plant Physiol. 45, 339–347Google Scholar
  30. Hovenkamp-Obbema, R., Moorman, A., Stegwee, D. (1974) Aminolevulinate dehydratase in greening cells of Euglena gracilis. Z. Pflanzenphysiol 72, 277–286Google Scholar
  31. Jeffrey, S.W. (1968) Quantitative thin-layer chromatography of chlorophylls and carotenoids from marine algae. Biochim. Biophys. Acta 162, 271–285Google Scholar
  32. Kannangara, C.G., Gough, S.P. (1977) Synthesis of delta aminolevulinic acid and chlorophyll by isolated chloroplasts. Carlsberg Res. Commun. 42, 441–457Google Scholar
  33. Kindman, L.A., Cohen, C.E., Zeldin, M.H., Ben-Shaul, Y., Schiff, J.A. (1978) Events surrounding the early development of Euglena chloroplasts. 12. Spectroscopic examination of the protochlorophyll(ide) phototransformation in intact cells. Photochem. Photobiol. 27, 787–794Google Scholar
  34. Manns R.J., Novelli, G.D. (1961) Measurement of the incorporation of radioactive amino acids into proteins by a filter-paper disk method. Arch. Biochem. Biophys. 94, 48–53Google Scholar
  35. Mattheis, J.R., Rebeiz, C.A. (1977a) Chloroplast biogenesis. Net synthesis of protochlorophyllide from magnesium-protoprophyrin monoester by developing chloroplasts. J. Biol. Chem. 252, 4022–4024Google Scholar
  36. Mattheis, J.R., Rebeiz, C.A. (1977b) Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts. J. Biol. Chem. 252, 8347–8349Google Scholar
  37. Mattheis, J.R., Rebeiz, C.A. (1978) Chloroplast biogenesis. XXIII. The conversion of exogenous protochlorophyllide into phototransformable protochlorophyllide in vitro. Photochem. Photobiol. 28, 55–60Google Scholar
  38. Miller, M.E. Price, C.A. (1982) Protein synthesis by developing chloroplasts isolated from Euglena gracilis. FEBS Lett. 147, 156–160Google Scholar
  39. Ortiz, W., Reardon, E.M., Price, C.A. (1980) Preparation of chloroplasts from Euglena highly active in protein synthesis. Plant Physiol. 66, 291–294Google Scholar
  40. Pennington, F.C., Strain, H.H., Svec, W.A., Katz, J.J. (1964) Preparation and properties of protochlorophyll a, methyl pyrochlorophyllide a, and methyl pyropheophorbide a derived from chlorophyll by decarbomethoxylation. J. Am. Chem. Soc. 86, 1418–1426Google Scholar
  41. Price, C.A., Reardon, E.M. (1982) Isolation of chloroplasts for protein synthesis from spinach and Euglena gracilis by centrifugation in silica sols. In: Methods in chloroplast molecular biology, pp. 183–209, Edelman M., Hallick, R.B., Chua, N.-H., eds, Elsevier, AmsterdamGoogle Scholar
  42. Rebeiz, C.A., Wu, S.M., Kihadja, M., Daniell, H., Perkins, E.J. (1983) Chlorophyll a biosynthetic routes and chlorophyll a chemical heterogeneity in plants. Mol. Cell. Biochem. 57, 97–125Google Scholar
  43. Salvador, G.F. (1978) La synthèse d'acide 5-aminolevulinique par des chloroplastes isolés d'Euglena gracilis. C.R. Acad. Sci. Paris Ser. D 286, 49–52Google Scholar
  44. Salisbury, J.L., Vasconcelos, A.C., Floyd, G. (1975) Isolation of intact chloroplasts of Euglena gracilis by isopycnic sedimentation in gradients of silica. Plant Physiol. 56, 399–403Google Scholar
  45. Santel, H.J., Apel, K. (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADP: protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 95–103Google Scholar
  46. Sato, T. (1968) A modified method for lead staining of thin sections. J. Electron Microsc. 17, 158–159Google Scholar
  47. Schiff, J.A. (1972) A green safelight for the study of chloroplast development and other photomorphogenetic phenomena. Methods Enzymol. 24, 321–322Google Scholar
  48. Schiff, J.A., Schwartzbach, S.D. (1982) Photocontrol of chloroplast development in Euglena. In: The biology of Euglena, vol. 3: Physiology, pp. 313–352, Buetow, D.E., ed. Academic Press, New YorkGoogle Scholar
  49. Schoch, S., Scheer, H., Schiff, J.A., Rüdiger, W., Siegelman, H.W. (1981) Pyropheophytin a accompanies pheophytin a in darkened light grown cells of Euglena. Z. Naturforsch. Teil C. 36, 827–833Google Scholar
  50. Schurman, P., Ortiz, W. (1982) Photosynthetic activity of isolated chloroplasts from Euglena gracilis. Planta 154, 70–75Google Scholar
  51. Schwartzbach, S.D., Schiff, J.A., Klein, S. (1976) Events surrounding the early development of Euglena chloroplasts. 9. Biosynthetic events required for lag elimination in chlorophyll synthesis in Euglena. Planta 131, 1–9Google Scholar
  52. Shigeoka, S., Yokota, A., Nakano, Y., Kitaoka, S. (1980) Isolation of physiologically intact chloroplasts from Euglena gracilis Z. Bull. Univ. Osaka Pref. B 32, 37–41Google Scholar
  53. Smith, J.H.C., Benitez, A. (1955) Chlorophylls: Analysis in plant materials. In: Modern methods in plant analysis, vol. 4, pp. 142–196, Peach, K., Tracey, M., eds. Springer, BerlinGoogle Scholar
  54. Stella, A.M., Battle, A.M. del C. (1978) Porphyrin biosynthesisimmobilized enzymes and ligands. VIII. Studies on the purification of 5-aminolevulinate dehydratase from Euglena gracilis. Plant Sci. Lett. 11, 87–92Google Scholar
  55. Stern, A.I., Schiff, J.A., Epstein, H.T. (1964) Studies of chloroplast development in Euglena. V. Pigment biosynthesis, photosynthetic oxygen evolution, and carbon dioxide fixation during chloroplast development. Plant Physiol. 39, 220–226Google Scholar
  56. Tokunaga, M., Nakano, Y., Kitaoka, S. (1976) Preparation of physiologically intact mitochondria from Euglena gracilis. Z. Agr. Biol. Chem. 7, 1439–1440Google Scholar
  57. Vaisberg, A.J., Schiff, J.A. (1976) Events surrounding the early development of Euglena, chloroplasts. 7. Inhibition of carotenoid biosynthesis by the herbicide SAN 9789 (4-chloro-5-(methylamino)-2-α,α,α-trifluoro-m-tolyl)-3 (2H-pyridazinone)) and its developmental consequences. Plant Physiol. 57, 260–269Google Scholar
  58. Weinstein, J.D., Beale, S.I., (1983) Separate physiological roles and subcellular compartments for two tetrapyrrole biosynthetic pathways in Euglena gracilis. J. Biol. Chem. 6799–6807Google Scholar
  59. Weinstein, J.D., Castelfranco, P.A. (1977) Protoporphyrin IX biosynthesis from glutamate in isolated greening chloroplasts. Arch. Biochem. Biophys. 178, 671–673Google Scholar
  60. Weinstein, J.D., Castelfranco, P.A. (1978) Mg-protoporphyrin IX and 5-aminolevulinic acid synthesis from glutamate in isolated greening chloroplasts. Arch. Biochem. Biophys. 186, 376–382Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • B. Gomez-Silva
    • 1
  • M. P. Timko
    • 1
  • J. A. Schiff
    • 1
  1. 1.Institute for Photobiology of Cells and OrganellesBrandeis UniversityWalthamUSA

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