Abstract
1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg2+ were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm3), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm3).
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Abbreviations
- PAL:
-
L-phenylalanine ammonia-lyase
- Tris:
-
tris-(hydroxymethyl)aminomethane
- EDTA:
-
ethylenediamine tetraacetic acid
- ATPase:
-
ATP phosphohydrolase
References
Alibert, G., Ranjeva, R., Boudet, A.: Recherches sur les enzymes catalysant la biosynthese des acides des phenoliques chez Quercus pendunculata. II. Localisation intracellulaire de la phenylalanine ammoniaque-lyase, de la cinnamate 4-hydroxylase et de la benzoate synthase. Biochim.Biophys. Acta 279, 282–289 (1972a)
Alibert, G., Ranjeva, R., Boudet, A.: Recherches sur les enzymes catalysant la biosynthese des acides des phenoliques chez Quercus pendunculata. III. Formation sequentielle, a partir de la phenylalanine, des acides cinnamique, p-coumarique et cafeique, par des organites cellulaires isoles. Physiol. Plant. 27, 240–243 (1972b)
Amrhein, N., Zenk, M.H.: Untersuchungen zur Rolle der Phenylalanin-Ammonium-Lyase (PAL) bei der Regulation der Flavonoidsynthese im Buchweizen(Fagopyrum esculentum Moench). Z. Pflanzenphysiol. 64, 145–168 (1971)
Arnon, D.J.: Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15 (1949)
Baginski, E.S., Foa, P.P., Zak, B.: Glucose-6-phosphatase. In: Methoden der enzymatischen Analyse, pp. 839–843. Bergmeyer, H.-U. ed., Weinheim: Verlag Chemie 1970
Benveniste, I., Durst, F.: Physiologie Vegetale, Mise en evidence dans les tissus de tubercule de Topinambour (Helianthus tuberosus L., variete blanc commun) d'une enzyme a cytochrome P-450, l'acide trans-cinnamique 4-hydroxylase (=CAH). C.R. Acad. Sci. (Paris) 278D,1487–1490 (1974)
Bergmeyer, H.-U., Gawehn, K., Graßl, M.: Enzyme. In: Methoden der enzymatischen Analyse, pp. 388–483. Bergmeyer, H.-U., ed. Weinheim: Verlag Chemie 1970
Camm, E.L., Towers, G.H.N.: Phenylalanine ammonia-lyase, a review. Phytochemistry 12, 961–973 (1973a)
Camm, E.L., Towers, G.H.N.: Effect of ageing on enzymes of phenylpropanoid metabolism in Solanum tuberosum discs. Phytochemistry 12, 1575–1580 (1973b)
Charriere-Ladreix, Y.: Sur la presence de deux isoenzymes de la phenylalanine-ammoniac-lyase chez Aesculus hippocastanum. Phytochemistry 14, 1727–1731 (1975)
Czichi, U., Kindl, H.: A model of closely assembled consecutive enzymes on membranes: Formation of hydroxycinnamic acids from L-phenylalanine on thylakoids of Dunaliella marina. Hoppe Seylers Z. physiol. Chem. 356, 475–485 (1975a)
Czichi, U., Kindl, H.: Formation of p-coumaric acid and o-coumaric acid from L-phenylalanine by microsomal membrane fractions from potato: evidence of membrane-bound enzyme complexes. Planta (Berl.) 125, 115–125 (1975b)
Donaldson, R.P., Tolbert, N.E., Schnarrenberger, C.: A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue. Arch. Biochem. Biophys. 152, 199–215 (1972)
Duke, S.O., Naylor, A.W.: Light effects on phenylalanine ammonia-lyase substrate levels and turnover rates in maize seedlings. Plant Sci. Lett. 6, 361–367 (1976)
Ebel, J., Schaller-Hekeler, B., Knobloch, K.-H., Wellmann, E., Grisebach, H., Hahlbrock, K.: Coordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soy bean cell suspension cultures. Biochim. Biophys. Acta 362, 417–424 (1974)
Frühling, J., Pecheux, F., Penasse, W.: Localisation ultrastructurale et distribution biochimique subcellulaire de l'Inosine-Diphosphatase dans le cortex surrenalien du rat. Histochemistry 42, 145–162 (1974)
Gestetner, B., Conn, E.E. The 2-hydroxylation of transcinnamic acid by chloroplasts from Melilotus alba Desr. Arch. Biochem. Biophys. 163, 617–624 (1974)
Gregor, H.D.: Studies on phenylalanine ammonia-lyase from castor bean endosperm. I. Subcellular localization and induction of the enzyme. Z. Pflanzenphysiol. 77, 454–463 (1976)
Hagel, P., Kindl, H.: p-Hydroxybenzoate synthase: a complex associated with mitochondrial membranes of roots of Cucumis sativus. FEBS Lett. 59, 120–124 (1975)
Hertel, R., Thomson, K., Russo, V.E.A.: In vitro auxin binding to particulate cell fractions from corn coleoptiles. Planta (Berl.) 107, 325–340 (1972)
Hill, A.C., Rhodes, M.J.C.: The properties of cinnamic acid 4-hydroxylase of aged swede root disks. Phytochemistry 14, 2387–2391 (1975)
Jangaard, N.O.: The effect of herbicides, plant growth regulators and other compounds on phenylalanine ammonia-lyase activity. Phytochemistry 13, 1769–1775 (1974)
Kindl, H.: The regulation of the L-tyrosine ammonia-lyase activity by phenolic compounds. Hoppe Seylers. Z. physiol. Chem. 351, 792–798 (1970)
Kindl, H.: Zur Frage der ortho-Hydroxylierung aromatischer Carbonsäuren in höheren Pflanzen. Hoppe-Seylers Z. Physiol. Chem. 352, 78–84 (1971)
Kindl, H.: Metabolism of aromatic acids in plant organelles. Hoppe-Seylers Z. Physiol. Chem. 353, 133 (1972)
Leigh, R.A., Williamson, F.A., Wyn-Jones, G.: Presence of two different membrane bound, KCl-stimulated adenosine triphosphatase activities in maize roots. Plant Physiol. 55, 678–685 (1975)
Lembi, C.A., Morree, D.J., Thomson, U.St., Hertel, R.: N-1-Naphtylphthalamic-acid-binding activity of a plasma membrane-rich fraction from maize coleoptiles. Planta (Berl.) 99, 37–45 (1971)
Löffelhardt, W., Ludwig, B., Kindl, H.: Thylakoid-gebundene L-Phenylalanin-Ammoniak-Lyase. Hoppe-Seylers Z. Physiol. Chem. 354, 1006–1017 (1973)
Löffelhardt, W., Kindl, H.: The conversion of L-phenylalanine into benzoic acid on the thylakoid membrane of higher plants. Hoppe-Seylers Z. Physiol. Chem. 356, 487–493 (1975)
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951)
Marsh, B.: The estimation of inorganic phosphate in the presence of ATP. Biochim. Biophys. Acta 32, 357–361 (1959)
McFarlane, I.J., Lees, E.M., Conn, E.E.: The in vitro biosynthesis of Dhurrin the cyanogenic glycoside of Sorghum bicolor. J. biol. Chem. 250, 4708–4713 (1975)
Monties, B.: Sur la presence de phenylalanineammonialyase dans les chloroplastes des cellules foliaires. C.R. Acad. Sci. (Paris) 278, 1465–1467 (1974)
Potts, J.R.M., Weklych, R., Conn, E.E.: The 4-hydroxylation of cinnamic acid by Sorghum microsomes and the requirement for cytochrome P-450. J. biol. Chem. 249, 5019–5026 (1974)
Pridham, J.B., Woodhead, S.: Multimolecular forms of phenylalanine ammonia lyase in Alternaria. Biochem. Soc. Trans. 1070–1072 (1974)
Ray, P.M., Shininger, T.L., Ray, M.M.: Isolation of β-glucan synthetase particles from plant cells and identification with Golgi membranes. Proc. nat. Acad. Sci. (Wash.) 64, 605–612 (1969)
Rich, P.R., Bendall, D.S.: Cytochrome components of plant microsomes. Europ. J. Biochem. 55, 333–341 (1975)
Ruis, H., Kindl, H.: Distribution of ammonia-lyases in organelles of castor bean endosperm. Hoppe Seylers Z. physiol. Chem. 351, 1425–1427 (1970)
Ruis, H., Kindl, H.: Formation of α, β-unsaturated carboxylic acids from amino acids in plant peroxisomes. Phytochemistry 10, 2627–2631 (1971)
Rungie, J.M., Wiskich, J.T.: Salt-stimulated adenosine triphosphatase from smooth microsomes of turnip. Plant Physiol. 51, 1064–1068 (1973)
Russell, D.W.: The metabolism of aromatic compounds in higher plants. X. Properties of the cinnamic acid 4-hydroxylase of pea seedlings and some aspects of its metabolic and developmental control. J. biol. Chem. 246, 3870–3878 (1971)
Russell, D.W., Conn, E.E.: The cinnamic acid 4-hydroxylase of pea seedlings. Arch. Biochem. Biophys. 122, 256–258 (1967)
Schnarrenberger, C., Oeser, A., Tolbert, N.E.: Development of microbodies in sunflower cotyledons and castor bean endosperm during germination. Plant Physiol. 48, 566–574 (1971)
Stafford, H.A.: The metabolism of aromatic compounds. Ann. Rev. Plant Physiol. 25, 459–486 (1974a)
Stafford, H.A. Possible multienzyme complexes regulating the formation of C6-C3 phenolic compounds and lignins in higher plants. In: Recent Advances in Phytochemistry. Vol. 8, pp. 53–79. Runeckles, V.C., Conn, E.E., eds. New York: Academic Press 1974b
Thomson, K.-S., Hertel, R., Müller, S.: 1-N-Naphthylphthalamic acid and 2, 3, 5-triiodobenzoic acid. In vitro binding to particulate cell fractions and action on auxin transport in corn coleoptiles. Planta (Berl.) 109, 337–352 (1973)
Van der Woude, W.J., Lembei, C.A., Morre, D.J., Kindinger, J.I., Ordin, L.: β-Glucan synthetase of plasma membrane and Golgi apparatus from onion stem. Plant Physiol. 54, 333–340 (1974)
Weissenböck, G.: Aktivitätsverlauf der Phenylalanin-, Tyrosin-Ammonium-Lyase (PAL, TAL) und Chalkon-Flavanon-Isomerase im Vergleich zur C-Glycosylfavon-Akkumulation im wachsenden Hafersproß (Avena sativa L.) bei Belichtung und Dunkelheit. Z. Pflanzenphysiol. 74, 226–254 (1975)
Wellmann, E., Baron, D.: Durch Phytochrom kontrollierte Enzyme der Flavonoidsynthese in Zellsuspensionskulturen von Petersilie (Petroselinum hortense Hoffm). Planta (Berl.) 119, 161–164 (1974)
Wellmann, E., Schopfer, P.: Phytochrome-mediated de novo synthesis of phenylalanine ammonia-lyase in cell, suspension cultures of Parsley. Plant Physiol. 55, 822–827 (1975)
Wellmann, E., Hrazdina, G., Grisebach, H.: Induction of anthocyanin formation and enzymes related to its biosynthesis by UV-light in cell cultures of Haplopapus gracilis. Phytochemistry 15, 913–915 (1976)
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Czichi, U., Kindl, H. Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution. Planta 134, 133–143 (1977). https://doi.org/10.1007/BF00384962
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DOI: https://doi.org/10.1007/BF00384962