Partial Purification and Properties of S-Adenosylmethionine. (R), (S

Home , Apparicine, Reticuline

Planta Journal of Medicinal Plant Research medica Organ der Editor-in-Chief A. Baerheim-Svendsen, E. Hecker, Heidelberg J. M. Rowson, Mablethorpe Gesellschaft für E. Reinhard, Tübingen Leiden R. Hegnauer, Leiden M. v. Schantz, Helsinki Arzneipflanzen• Pharmazeutisches Institut H. Böhm, Halle W. Herz, Tallahassee K. F. Sewing, Hannover forschung Auf der Morgenstelle 8 F. Bohlmann, Berlin K. Hostettmann, Lausanne E. J. Shellard, London D-7400 Tübingen A. Cave, Chatenay-Malabry H. Inouye, Kyoto S. Shibata, Tokyo P. Delaveau, Paris M. A. Iyengar, Manipal Ch. Tamm, Basel Editorial Board Ding Guang-sheng, F. Kaiser, Mannheim W. S. Woo, Seoul H. P. T. Ammon, Tübingen Shanghai F. H. Kemper, Münster Xiao Pei-gen, Beijing W. Barz, Münster C. -J. Estler, Erlangen W. R. Kukovetz, Graz E. Reinhard, Tübingen N. Farnsworth, Chicago J. Lemli, Leuven •O. Sticher, Zürich H. Floss, Columbus Liang Xiao-tian, Beijing H. Wagner, München H. Friedrich, Münster M. Lounasmaa, Helsinki M. H. Zenk, München D. Fritz, Weihenstephan M. Luckner, Halle A. G. Gonzalez, La Laguna J. Lutomski, Poznan Advisory Board O. R. Gottlieb, Sao Paulo H. Menßen, Köln N. Anand, Lucknow E. Graf, Köln E. Noack, Düsseldorf R. Anton, Strasbourg H. Haas, Mannheim J. D. Phillipson, London

Contents

Volume 49,1983

ISSN 0032-0943 (f) Hippokrates Hippokrates Verlag Stuttgart II

Contents 49,1983

Atta-ur-Rahman, Bashir, M.\ Isolation of New Alkaloids Cannabinoids in Phelipaea ramosa, a Parasite of Canna• from Catharanthus roseus 124 bis sativa) 250 Atta-ur-Rahman, Nisa, M., Farhi, S.: Isolation of Moenjod- aramine from Buxus papilosa 126 Galun, £., Aviv, D., Dantes, A., Freeman, A. \ Biotransfor• mation by Plant Cells Immobilized in Cross-Linked Poly- Balsevich, J., Kurz, W. G. W.: The Role of 9- and/or 10- acrylamide-Hydrazide. Monoterpene Reduction by En- oxygenated Derivatives of Geraniol, Geranial, Nerol, trapped Mentha Cells 9 and Neral in the Biosynthesis of Loganin and Ajmalicine 79 Ghosal, Sh., Singh, A.K., Biswas, K.: New6-Aryl-2-pyrones Bassleer, R., Marnette, J.-M., Wiliquet, Ph., De Pauw-Gillet, from Gentiana pedicellata 240 M.-CL, Caprasse, M., Angenot, L. \ Etüde complemen- taire de la cytotoxicite de la melinonine F, alcaloide derive Hafez, A., Adolf, W., Hecker, E.\ Active Principles of the de la ß-carboline (Complementary Study of Cytotoxic Thymelaeaceae. III. Skin Irritant and Cocarcinogenic Activity of Melinonine F) 158 Factors from Pimelea simplex 3 Becker, H., Chavadej, S., Weberling, F.: Valepotriates in Valeriana thalictroides 64 Ieven, M., van den Berghe, D. A., Vlietinck, A. J.\ Plant Becker, H., Herold, S.: RP-8 als Hilfsphase zur Akkumula• Antiviral Agents. IV. Influence of Lycorine on Growth tion von Valepotriaten aus Zellsuspensionskulturen von Pattern of Three Animal Viruses 109

Valeriana wallichii (RP-8 Auxiliary Phase for the Accu- Ishiguro, K., Yamaki, M.y Takagi, S.: Studies on Iridoid- mulation of Valepotriates from Cell-Suspension-Culture related Compounds; III: Gentiopicral, the Aglucone of of Valeriana wallichii) 191 Gentiopicroside 208 Bounthanh, C, Richert, L., Beck, J. P., Haag-Berrurier, M., Anton, R.: The Action of Valepotriates on the Synthesis Jakovlev, V., Isaac, O., Flaskamp, E.: Pharmakologische of DNA and Proteins of Cultured Hepatoma Cells. ... 138 Untersuchungen von Kamillen-Inhaltsstoffen, VI. Unter• Briangon-Scheid, F., Lobstein-Guth, A., Anton, R.: HPLC suchungen zur antiphlogistischen Wirkung von Chama- Separation and Quantitative Determination of Biflavones zulen und Matricin (Pharmacological Investigations with in Leaves from Ginkgo biloba 204 Compounds of Chamomile, VI. Investigations on the Antiphlogistic Effects of Chamazulene and Matricine) 67

Caputo, O., Delprino, L., Viola, F., Caramiello, R.f Bal- Joshi, K. C, Singh, P., Taneja, S.: A Sesquiterpenoid Naph- liano, G.: Biosynthesis of Sterols and Triterpenoids in thol from Kydia calycina 127 Tissue Cultures of Cucurbita maxima 176 Jossang, A., Lebceuf, M., Cabalion, P., Cave, A.: Alcaloides Chagnon, M., Ndibwami, A., Dube, S., Bumaya, A.: Ac- des Annonacees. XLV: Alcaloides de Polyalthia niti- tivite Anti-Inflammatoire d'Extraits de Crassocephalum dissima (Alkaloids from Annonaceae. XLV: Alkaloids multicorymbosum (Anti-inflammatory Action of an Ex- of Polyalthia nitidissima) 20 tract from Crassocephalum multicorymbosum) 255 Chattopadhyay, S., Chattopadhyay, U., Mathur, P. P., Saini, Kimura, Y., Ohminami, H., Okuda, H., Baba, K., Kozawa, K. S., Ghosal, S.: Effects of Hippadine, an Amarylli- M., Arichi, S.: Effects of Stilbene Components of Roots daceae Alkaloid, on Testicular Function in Rats 252 of Polygonum ssp. on Liver Injury in Peroxidized Oil-fed Chen Weiming, Yan Yaping, LiangXiaotian: Alkaloids from Rats 51 Roots of Alstonia yunnanensis 62 Kisiel, W.: 8-Epidesacylcynaropicrin from Crepis capillaris 246 Kiso, Y., Suzuki, Y., Watanabe, N., Oshima, Y., Hikino, H.: Dehaussy, H., Tits, M., Angenot, L.\ La guattegaumerine, Antihepatotoxic Principles of Curcuma longa Rhizomes 185 nouvel alcaloide bisbenzylisoquinoleinique de Guatteria Kiso, Y., Tohkin, M., Hikino, H.: Assay Method for Anti• gaumeri (Guattegaumerine, New Bisbenzylisoquinoline hepatotoxic Activity Using Carbon Tetrachloride In- Alkaloid from Guatteria gaumeri) 25 duced Cytotoxicity in Primary Cultured Hepatocytes . . 222 Dominguez, X. A., Franco, R., Cano, G., Ma. Consue lo Kram, G., Franz, G.\ Untersuchungen über die Schleim- Garcia, F., Xorge A. Dominguez, S. Jr., Leonardo de la polysaccharide aus Lindenblüten (Analysis of Linden

Pena9M.: Isolation of a New Furano-l,4-Naphthaquinone, Flower Mucilage) 149 Diodantunezone from Lanthana achyranthifolia .... 63 Krüger, D., Junior, P., Wichtl, M.\ Neue Cardenolidglyko- side aus Digitalis lanata (New Cardiac Glycosides from

Endo, K., Oshima, Y.t Kikuchi, H., Koshihara, Y., Hikino, Digitalis lanata) 74 H.: Hypotensive Principles of Uncaria Hooks 188 Engelshowe, R.: Dimere Proanthocyanidine als Gerbstoff• Langhammer, L., Schulze, G., Gujer, R., Magnolato, D., vorstufen in Juniperus communis (Tannin Producing Horisberger, M.\ Isolation and Structure of a Rarely Dimeric Proanthocyanidins in Juniperus communis). . . 170 Occurring Cyanidanol Glycoside from Cortex Betulae . 181 Esterl, A., Gab, S., Bieniek, D.\ Zur Kenntnis der Inhalts• Lemli, J., Cuveele, J., Verhaeren, E.\ Chemical Identification stoffe von Isertia hypoleuca (On The Constituents of of Alexandrian and Tinnevelly Senna. Studies in the Isertia hypoleuca) 244 Field of Drugs Containing Anthracene Derivatives XXXIV 36 Fournier, G., Paris, M.: Mise en Evidence de Cannabinoi'des Long-Ze Lin, Wagner, H., Seligmann, ö.\ Thalifaberine, Chez Phelipaea ramosa, Orobanchacees, Parasitant le Thalifabine and Huangshanine, Three New Dimeric Chanvre, Cannabis sativa, Cannabinacees (Detection of Aporphine-Benzylisoquinoline Alkaloids 55 Contents III

Man-Po Wong, Teh-Chang Chiang, Hson-Mou Chang: Chem• Rueffer, M., Nagakura, N., Zenk, M. H.: Partial Purification ical Studies on Dangshen, the Root of Codonopsis pilo- and Properties of S-Adenosylmethionine: (R), (S)-Nor- sula 60 laudanosoline-6-O-Methyltransferase from Argemone Misawa, M., Hayashi, M., Takayama, S.: Production of platyceras Cell Cultures 131 Antineoplastic Agents by Plant Tissue Cultures. I. In- duction of Callus Tissues and Detection of the Agents in Sariyar, G.: Alkaloids from Papaver triniifolium of Turkish Cultured Cells 115 Origin 43 Seip, E. H., Ott, H. H., Hecker, £.: Skin Irritant and Tumor Nahrstedt, A., Wray, V., Grotjahn, L., Fikenscher, L. H., Promoting Diterpene Esters of the Tigliane Type from Hegnauer, R.: New Acylated Cyanogenic Diglycosides the Chinese Tallow Tree (Sapium sebiferum) 199 from Fruits of Anthemis cairica 143 Sepulveda-Boza, S., Friedrichs, £., Puff, H., Breitmaier, £.:

Nomura, T, Fukai, T.} Shimada, T, Ih-Sheng Chen: Com- Ein iso-Homoprotoberberin-Alkaloid aus den Wurzeln ponents of Root Bark of Morus australis. I. Structure of von Berberis actinacantha (An iso-Homoprotoberberin- a New 2-Arylbenzofuran Derivative, Mulberrofuran D. 90 Alkaloid from the Roots of Berberis actinacantha). ... 32 Shoyama, Y., Hatano, K., Nishioka, /.: Clonal Multiplica- Obata-Sasamoto, H., Komamine, A.: Effect of Culture Con- tion of Pinellia ternata by Tissue Culture 14 ditions on DOPA Accumulation in a Callus Culture of Stoianova-lvanova, B., Budzikiewicz, H., Koumanova, B., Stizolobium hassjoo 120 Tsoutsoulova, A., Mladenova, K., Brauner, A.: Essential Ohiri, F. C, Verpoorte, R., Baerheim Svendsen, Ar. !H NMR Oil of Chrysanthemum indicum 236 Chemical Shift Values for Aromatic Protons in 2,3,9,10- and 2,3,10,11-tetrasubstituted Tetrahydroprotoberberine Teh-Chang Chiang, Hson-Mou Chang, Mak, Th. C. W.: New Alkaloids 162 Oleanene-type Triterpenes from Abrus precatorius and Ohiri, F. C, Verpoorte, R., Baerheim Svendsen, A:Tertiary X-ray Crystal Structure of Abrusgenic Acid-Methanol Phenolic Alkaloids from Chasmanthera dependens ... 17 1:1 Solvate 165 Ojewole, J. A. O., Adesina, S. K.: Mechanism of the Hypo- tensive Effect of Scopoletin Isolated from the Fruit of Tetrapleura tetraptera 46 van derSluis, W. G., van der Nat, J. M., Spek, A. L., Ike- shiro, Y., Labadie, R. P.: Gentiogenal, aConversion Pro- Ojewole, J. A. O., Adesina, S. K. \ Cardiovascular and Neuro- duct of Gentiopicrin (Gentiopicroside) 211 muscular Actions of Scopoletin from Fruit of Tetrapleura tetraptera 99 Okogun, J. /., Adeboye, J. O., Okorie, D. A.\ Novel Struc- Wagner, H., Schwarting, G., Varljen, J., Bauer, R., Ham- tures of two Chromone Alkaloids from Root-Bark of dard, M. £., El-Faer, M. Z., Beal, J.: Die chemische Zu• Schumanniophyton magnificum 95 sammensetzung der Convolvulaceen Harze IV. Die Gly- kosidsäuren von Ipomoea quamoclit, I. lacunosa, I. pan- Palacios, P., Gutkind, G., Rondina, R. V. D., de Torres, R., durata und Convolvulus al-sirensis (Chemical Consti• Coussio, J. D.: GenusBaccharis. II. Antimicrobial Activ• tuents of the Convolvulaceae-Resins IV. The Glycosidic ity of B. crispa and B. notosergila 128 Acids of Ipomoea quamoclit, I. lacunosa, I. pandurata Pandey, V. B., Singh, J. P., Mattocks, A. R., Bailey, £.: A and Convolvulus al-sirensis) 154 Note on "Isolation and Pharmacological Action of Helio- Watanabe, K., Watanabe, H., Goto, Y., Yamaguchi, M., trine, the Major Alkaloid of Heliotropium indicum Seeds" 254 Yamamoto, N., Hagino, K.: Pharmacological Properties Pathak, V. P., Saini, T. R., Khanna, R. N.: A New Furano- of Magnolol and Hönokiol Extracted from Magnolia offi- flavone from Seeds of Pongamia glabra 61 cinalis: Central Depressant Effects 103 Perera, P., Sandberg, F., van Beek, T. A., Verpoorte, R.\ Willuhn, G., Röttger, P.-M., Matthiessen, U.: Helenalin- Tertiary Indole Alkaloids of Tabernaemontana dicho- und 11,13-Dihydrohelenalinester aus Blüten von Arnica toma Seeds 28 montana (Helenalin- and 11,13-Dihydrohelenalinester Perera, P., van Beek, T. A., Verpoorte, /?.: Dichomine, a from Flowers of Arnica montana) 226 Novel Type of Iboga Alkaloid 232 Witte, L., Berlin, J., Wray, V., Schubert, W., Kohl, W., Höfle, G., Hammer, J.: Mono- and Diterpenes from Cell Ravid, U., Putievsky, E.: Constituents of Essential Oils from Cultures of Thuja occidentalis 216 Majorana syriaca, Coridothymus capitatus and Satureja thymbra 248 Yang Minghe, Chen Yanyong: Steroidal Sapogenins in Dios- Röder, E., Wiedenfeld, H., Honig, A.: Pyrrolizidinalkaloide corea collettii 38 aus Senecio aureus 57 Yu De-Quan, Das, B. C.: Structure of Hydroxymuscopyri- Rueffer, M., Nagakura, N., Zenk, M. H.\ A Highly Specific O- dine A and Hydroxymuscopyridine B, Two New Constit• Methyltransferase for Nororientaline Synthesis Isolated uents ofMusk 183 from Argemoneplatyceras Cell Cultures 196 Yu De-quan, Y., Das, B. C: Alkaloids of Aconitum barbatum 85 IV

Subject Index 49,1983

A Cocarcinogens 3 Gentiogenal 211 Convolvulaceae resins 154 Gentiopicral 208 Abietane 220 Coreximine 14 Gentiopicrin 211 Abrusgenic acid 165 Coronaridine 28 Gentiopicroside 208, 211 Abruslactone 165 Coumaric acid 186 Geranial 79 Acevaltrate 64,191 Coumarins 46, 99 Geraniol 79 Adaptogenes 240 Cucurbitacin 176 Ginkgetin 204 Alkaloids 17, 20, 25, 28, 32, 43, 55, 57, 62, Curcumin 185 Glycosidic acids 154 79, 85, 95,109,124,126,131,158, 162, Curcuminoids 185 Glycyrrhetinic acid 224 188,196, 232, 244, 252, 254 Cyanogenic glycosides 143 Glycyrrhizin 224 Amyrin 177 Cycloartenol 177 Govanine 17 Antineoplastic agents 115 Cymenol 216 Guajazulene 67 Antiviral agents 109 Cynarin 224 Guattegaumerine 25 Apigenin 128 Cynaropicrin 246 Aporphine alkaloids 20 Cytotoxic action 158 Aporphine derivatives 43 H Apparicine 232 Arabinose 149 D Helenalin 226 Arylbenzofuran derivatives 90 Helenalinester 226 Daphnane derivatives 3 Avenasterol 177 Helenanolides 226 Dauricine 20 Heliotrine 254 Daurisoline 20 Hepatoma cells 138 Dehydrohelenalinester 226 B Hibiscone 127 Deoxyphorbol 199 Hibiscoquinone 127 Dichomine 232 Benzylisoquinoline alkaloids 131 Hinikione 220 Didrovaltrate 64, 138, 191 Berberine alkaloids 32 Hinokiol 220 Diginatigenin derivatives 74 Biflavones 204 Hippadine 252 Digoxigenin derivatives 74 Bilobetin 204 Homovaltrate 64, 191 Dihydrocadambine 188 Honokiol 103 Biosynthesis of alkaloids 131, 196 Dihydroquinamine 244 Huangshanine 55 Biosynthesis of ajmalicine 79 Diodantunezone 63 Hydroxymuscopyridine 183 Biosynthesis of loganin 79 Diosgenin 38 Hydroxymusizin glycoside 36 Biosynthesis of sterols 176 Diterpene esters 3, 199 Biosynthesis of triterpenoids 176 Diterpenes 216 Biotransformation 9 Diterpenoid alkaloids 85 Bisabolol 67 Dopa 120 Bisbenzylisoquinoline alkaloids 20, 25, 55 Iboga alkaloids 232 Bisnorargemonine 14 Ibogamine 28 Borneol 236 Iboxygaine 232 Bornyl acetate 236 Immobilized cells 9 Enzymes 196 Indicine 254 Epilumicin 143 Indole alkaloid glucoside 188 C Epoxyphorbol 199 Indole alkaloids 28, 62, 79, 124. 158, 188, Erythrocentaurin 212 232 Cadambine 188 Essential oils 9, 67, 216, 236, 248 Iridoids208,211 Caffeic acid 186 Isochinoline alkaloids 196 Camphor 216 Isodihydrocadambine 188 Cannabinoids 250 Isoginkgetin 204 Cardenolides 74 Isoquinoline alkaloids 32 Carvacrol 216 Fenchone 220 Isothujone 220 Catechin 181 Ferruginol 220 Isovaltrate 64, 191 Catechinxylopyranoside 181 Ferulic acid 186 Cell cultures 9, 14 79, 115, 120, 131, 176, Flavonoids 61, 63, 90, 128, 204 191,196,216 Fluorocarpamine 124 K Chamazulene 67 Fridelin 60 Cholestanol 177 Furoflavones 61 Kuwanon 90 Chromone alkaloids 95 Chrysanthenone 236 Chrysosplenetin 63 G L Cinchonamine 244 Cinnamic acid 186 Galactose 149 Lasiocarpine 254 Clonal multiplication 14 Genkwanin 128 Laudanosoline 196 Subject Index V

Levomenol 67 Pedicellin 240 Steroidal sapogenins 38 Liliolide 240 Penduletin 63 Stigmastenol 177 Lindolhamine 20 Perakine 62 Stilbene51 Linoleic acid 220 Perivine 232 Sugiol 220 Liriodenine 20 Phorbol 199 Swertiamarin 240 Lumicin 143 Phytosterols 60, 176 Lycorine 109 Piceid51 Picroside 224 Pimelea factors 3 M Pleiocarpamine 124 Tabersonine 28 Podophyllotoxin 224 Tacaman alkaloids 232 Magnoiol 103 Polysaccharides 149 Tannins 181 Mandelonitril glycosides 143 Proanthocyanidins 170 Taraxerol 60 Matricine 67 Protoberberin derivatives 32 Terpineol 216 Melionine 158 Protoberberine alkaloids 20 Tertiary phenolic alkaloids 14 Mentenediol 220 Protocatechuic acid 186 Tetrahydroalstonine 62 Menthone 9 Pseudoguaianolides 226,246 Tetrahydroisochinoline alkaloids 196 Methionine 224 Puberaconitidine 85 Tetrahydroprotoberberine alkaloids 162 Methylabrusgenat 165 Puberaconitine 85 Thalifaberine 55 Methylnorlaudanosoline 196 Puberanidine 85 Thalifabine 55 Methyltransferase 131, 196 Puberanine 85 Thujaplicin derivatives 216 Moenjodaramine 126 Pulegone 9 Thujone 220 Monoterpenes 216 Pyrrolizidine alkaloids 57 Tigliane derivatives 3 Morphinane derivatives 43 Tinnevellin glycoside 36 Morusin 90 Tripdiolide 109 Mucilage 149 Triterpenoids 60, 165 Myrtanol 216 Tropolone 220 Tumor promotors 199 Resins 154 Resveratrol 51 N Reticuline 20, 131 U Rhamnose 149 Naphthalene glycosides 36 Rhoeadine derivatives 43 Ursolic acid 240 Naphthaquinones 63 Ushinsunine 20 Neolignane derivatives 103 Neomenthol 9 S Neral 79 V Nerol 79 Sabinene 220 Norlaudanosoline 131 Valepotriates 64, 138, 191 Sapogenins 38 Normacusine 62 Valtrate 64, 138,191 Saponins 38, 60 Nororientaline 196 Vellosimine 62 Sarpagine 62 Norreticuline 131 Vindolinine derivatives 124 Schumannificine 95 Vinorine 62 Sciadopitysin 204 Voacangine 28 Scopoletin 46, 99 Voacristine 232 O Secoiridoids 208,211 Voaphylline 28, 232 Sesquiterpene lactones 226, 246 Vobasine 232 Opium alkaloids 131 Sesquiterpenes 127 Orientaline 196 Silybin 224 Simplexin 3 Y Sinapic acid 186 P Sitosterol 220 Skin irritants 199 Yamogenin 38 Pallidine 14 Spinasterol 177 Palmatic acid 220 Stemmadenine 28 Z Papaver alkaloids 43 Stepholidine 20 Pedicellatin 240 Steroidal alkaloids 126 Zierinxyloside 143 VI

Index of Names of Organisms 49,1983

Please note that the following plant families are listed as indicated below: Alsinaceae sub Caryophyllaceae Cacalia floridana 57 Fagara zanthoxyloides 116 Apiaceae sub Umbelliferae Caesalpinia gilliesii 116 Fumaria officinalis 133 Arecaceae sub Palmae Caesalpiniaceae 36 Asteraceae sub Compositae Campanulaceae 60 Brassicaceae sub Cruciferae Cannabis sativa 250 Clusiaceae sub Guttiferae Cassia angustifolia 36 Fabaceae sub Leguminosae (including Mi- Cassia senna 36 Gentiana pedicellata 240

mosoideae = Mimosaceae y Caesalpini- Catharanthus roseus 79,124 Gentianaceae 208, 211, 240 oideae = Caesalpiniaceae and Papilion- Celastraceae 115 Ginkgo biloba 204 oideae = Papilionaceae = Fabaceae Cephalotaxaceae 115 Ginkgoaceae 204 sensu stricto) Cephalotaxus harringtonia 115 Glaucium flavum 133 Hypericaceae sub Guttiferae Chamomilla recutita 67 Guatteria gaumeri 25 Lamiaceae sub Labiatae Chasmanthera dependens 17, 162 Oenotheraceae sub Onagraceae Chelidonium majus 133 Poaceae sub Gramineae Chlorella ellipsoidea 178 H Chrysanthemum indicum 236 Cissampelos mucronata 133 Heliotropium indicum 116,254 Clivia miniata 109 Heliotropium supinum 254 Codonopsis pilosula 60 Hibiscus species 127 Abrus cantoniensis 165 Colchicum speciosum 116 Holacantha emoryi 116 Abrus precatorius 165 Compositae 57,67,128,143,226,236,246, Hura crepitans 3 Aconitum barbatum 85 255 Adlumia fungosa 133 Convolvulaceae 154 Albertisia papuana 22 Convolvulus al-sirensis 154 I Alstonia yunnanensis 62 Convolvulus microphyllus 154 Althaea officinalis 152 Coridothymus capitatus 248 Ipomoea lacunosa 154 Amaryllidaceae 109, 252 Corydalis pallida 133 Ipomoea operculata 154 Annonaceae 20, 25 Corydalis sempervirens 133 Ipomoea pandurata 154 Anthemis altissima 143 Crassocephalum multicorymbosum 255 Ipomoea quamoclit 154 Anthemis cairica 143 Crepis capillaris 246 Ipomoea turpethum 154 Anthocephalus cadamba 189 Crepis virens 246 Isertia hypoleuca 244 Apocynaceae 28, 62,79,124, 232 Crinum asiaticum 252 Araceae 14 Crinum augustum 252 Argemone intermedia 133 Crinum latifolium 252 Argemone platyceras 131, 196 Crinum pratens 252 Arnica montana 226 Cucumis sativus 176 Juniperus communis 170 Artemisia caruthii 67 Cucurbita maxima 176 Cucurbita pepo 177 Cucurbitaceae 176 B Cupressaceae 170, 216 Curcuma longa 185 Kydia calycina 127 Baccharis crispa 128 Baccharis megapotamica 116 Baccharis notosergila 128 Baliospermum montanum 200 D Berberidaceae 32,133 Labiatae 9,248 Berberis actinacantha 32 Dictiostelium discoideum 177 Lantana achyranthifolia 63 Berberis henryana 133 Digitalis lanata 74 Leguminosae 36,46, 61, 99, 120,165 Berberis stolonifera 133 Digitalis schischkinii 74 Lindera oldhamii 22, 26 Berberis wilsonae 133 Dioscorea collettii 38 Loganiaceae 158 Betula species 181 Dioscoreaceae 38 Betulaceae 181 Blackstonia perfoliata 211 M Bombax malabaricum 127 Boraginaceae 254 Magnolia obovata 103 Brucea antidysenterica 116 Magnolia officinalis 103 Bryonia dioica 176 Erythrina lithosperma 22 Magnoliaceae 103 Buxaceae 126 Eschscholtzia tenuifolia 133 Majorana syriaca 248 Buxus papulosa 126 Euphorbiaceae 199 Malvaceae 127 Index of Names of Organisms Vll

Maytenus bucchananii 116 Penicillium expansum 211 Strychnos melinoniana 158 Menispermaceae 17, 133, 162 Phelipaea ramosa 250 Strychnos usambarensis 158 Menispermum dauricum 22 Pimelea linifolia 7 Mentha species 9 Pimelea prostrata 3 Mimosaceae 46, 99 Pimelea simplex 3 T Moraceae 90 Pinellia ternata 14 Morus alba 90 Podophyllum hexandrum 223 Tabernaemontana dichotoma 28, 232 Morus australis 90 Polyalthia beccarii 20 Tabernaemontana eglandulosa 232 Moschus moschiferus .183 Polyalthia emarginata 20 Taxus brevifolia 116 Mycobacterium species 10 Polyalthia nitidissima 20 Tetrapleura tetraptera 46, 99 Polyalthia oligosperma 20 Thalictrum dasycarpum 116 Polyalthia oliveri 20 Thalictrum faberi 55 N Polyalthia suaveolens 20 Thalictrum sparsiflorum 133 Polygonaceae 51 Thalictrum tuberosum 133 Nicotiana tabacum 250 Polygonum cuspidatum 51 Thuja occidentalis 216 Polygonum multiflorum 51 Thymelaeaceae 3 Polypodium vulgare 181 Thymus capitatus 248 O Pongamia glabra 61 Tilia species 149 Putterlickia verrucosa 115 Tiliaceae 149 Ochrosia moorei 116 Tripterygium wilfordii 115 Origanum compactum 249 Origanum floribundum 249 R Origanum hirtum 249 U Origanum majorana 249 Ranunculaceae 55, 85,133 Origanum maru 248 Rhus coriaria 248 Ulmus americana 181 Origanum smyrnaeum 249 Rubiaceae 95,188,244 Uncaria sinensis 188 Origanum syriacum 248 Uncaria species 188 Orobanchaceae 250 S

P Sapium sebiferum 199 V Satureja capitata 248 Panax ginseng 60 Satureja peltieri 249 Valeriana edulis ssp. procera 64 Panax pseudo-ginseng 60 Satureja thymbra 248 Valeriana kilimandscharica 64 Papaver armeniacum 43 Schistosoma japonicum 199 Valeriana thalictroides 64 Papaver cylindricum 43 Schumanniophyton magnificum 95 Valeriana wallichii 138,191 Papaver fugax 43 Schumanniophyton problematicum 97 Valerianaceae 64, 138, 191 Papaver Orientale 196 Scolytus multistriatus 181 Verbenaceae 63 Papaver somniferum 131,196 Scolytus ratzeburgi 181 Papaver tauricola 43 Scrophulariaceae 74 Papaver triniifolium 43 Senecio aureus 57 Z Papaveraceae 43,131, 196 Stizolobium hassjoo 120 Papilionaceae 120 Streptomyces clavuligerus 10 Zingiberaceae 185 VIII

Pharmacology Index 49,1983

Biological Systems Pharmacological Effects / Plant/Constituent Page Organs / Diseases Effects on

Central nervous System anticonvulsive Magnolia officinalis, Magnoliaceae 103 muscle relaxant Magnolia officinalis 103 sedative Magnolia officinalis 103 Peripheral nervous System muscle relaxant Tetrapleura tetraptera, Mimosaceae 99 Autonomous nervous System spasmolytic Tabernaemontana dichotoma, Apocynaceae 28 Cardiovascular System hypotensive Tetrapleura tetraptera, Mimosaceae 46 hypotensive Tetrapleura tetraptera, Mimosaceae 99 Hormonal System antifertility Hippadine, Amaryllidaceae 252 Infectious deseases antibacterial Baccharis crispa, Asteraceae 128 antiviral Cliviaminiata, Amaryllidaceae 109 Tumors tumor promotion Pimelea simplex, Thymelaeaceae 3 Inflammation antiinflammatoric Chamomillareculita, Asteraceae 67 antiinflammatoric Crassocephalum multicorymbosum 255 Journal of Planta 1983, Vol. 49, pp. 131-137, ©Hippokrates Verlag GmbH Medicinal "^5" . —Plant Research lllGIlICa

Partial Purification and Properties of S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-0-Methyltransferase from Argemone platyceras Cell Cultures

M. Rueffer*, N. Nagakura** and M. H. Zenk*

* Lehrstuhl Pharmazeutische Biologie der Universität München, D-8000 München 2, Federal Republic of Germany ** Kobe Women's College of Pharmacy, Kobe 658, Japan

Received: August 7,1983; accepted: August 31,1983

Key Word Index: formation from dopamine and 3,4-dihydroxypheny- lacetaldehyde. Argemone platyceras; Papaveraceae; Cell Suspen• Further down the pathway, reticuline is proven to sion Cultures; Alkaloid Biosynthesis; S-Adenosyl• be a branch point intermediate in the biosynthesis of methionine: (R), (S)-Norlaudanosoline-6-OMethyl- a vast array of structure types of benzylisoquinoline transferase. alkaloids, as for instance: morphinanes, protoberbe- rines, proaporphines, cularines, dibenzopyrrocoli- nes etc. (e.g. 3]. This means that NLS must be trans- Abstract formed to reticuline by three methylation reactions, two O-methylations at positions 6 and 4' and one N- A new enzyme, S-adenosylmethionine: (R), (S)- methylation (at atom 2). Since nor-reticuline has norlaudanosoline-6-O-methyltransferase, was isola• been amply demonstrated by feeding experiments to ted from the soluble protein extract of A. platyceras be a precursor of reticuline in vivo [4, 5], one has to cell cultures and purified approximately 80-fold. This assume that O-methylation of NLS precedes N-me- enzyme catalyses the formation of 6-O-methylnor- thylation for instance in opium alkaloids. O-Methy- laudanosoline, and, to a minor extent, 7-O-methyl- lation of NLS is therefore an important reaction in norlaudanosoline from SAM and (S), as well as (R), the early steps of the biosynthesis of reticuline, the norlaudanosoline. The apparent molcular weight of universal branch point intermediate. O-Methyltrans- the enzyme is 47000 Dalton. The pH-optimum of the ferases have been reported to mediate the transfer of enzyme is 7.5, the temperature Optimum, 35° C. Ap• methyl groups from SAM to phenylpropanoid and parent KM values for (S) and (R)-norlaudanosoline flavonoid Compounds mainly at the meta position of were 0.2 mM, and for SAM, 0.05 mM. The transfera- the aromatic System, though para-O-methylation is se shows high Substrate specificity for tetrahydro- not uncommon [6 and literature cited therein]. Speci- benzylisoquinoline alkaloids. Simple orthophenols, fic-O-methyltransferases derived from plants and ac- like phenylpropane derivatives, coumarins or fla- ting on tetrahydrobenzylisoquinolines have so far not vonoids, are not accepted as Substrates. The enzyme been reported. Yet incubation of poppy latex with is widely distributed in benzylisoquinoline-contain- (R,S)-norlaudanosoline and 14C-SAM has led to the ing plant cell cultures and is present in differentiated formation of labelled opium alkaloids, thus demon- plants like Papaver somniferum. strating indirectly the presence of methyltransferase enzymes [7]. There are, however, several reports on NLS-O-methylation, by animal enzymes [e.g. 8]. Introduction In our attempt to elucidate the enzymatic steps involved in isoquinoline biosynthesis in plants, we (S)-NLS is now firmly established as the first inter- have investigated the O-methylation of NLS. In this mediate in benzylisoquinoline alkaloid biosynthesis report, we present the partial purification and pro- [1]. A recently discovered enzyme [2] catalyses its perties of a new enzyme named norlaudanosoline-6- O-methyltransferase from A. platyceras cell Suspen• sion cultures. This enzyme catalyses the predominant Abbreviations: NLS = Norlaudanosoline; SAM = S-Adeno- syl-L-methionine; SAH = S-Adenosylhomocysteine, NLS-OMT transfer of the S-methyl group of SAM to the pheno- = S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-0-Me- lic OH-group at position 6 of NLS, and to a lesser ex• thyltransferase. tent, to the 7-O-position. 132 Rueffer, Nagakura, Zenk

R!0

R20

R30

R«0 3.

R1 R2 R3 R« R5

Norlaudanosoline H H H H H

6- •0- methylnorlaudanosoline CH3 H H H H

7- •0- methylnorlaudanosoline H CH3 H H H

5- •0- methylnorlaudanosoline H H CH3 H H

U- -0- methylnorlaudanosoline H H H CH3 H

Laudanosoline H H H H CH3

Norreticuline CH3 H H CH3 H

Nororientaline CH3 H CH3 H H

Laudanidine CH3 CH3 H CH3 CH3

1H = a = (S); 1H = p = (R)

Materials and Methods no enzyme or no Substrate (NLS) yielded a blank value of about 8 % radioactivity, a value which was subtracted from all incubation Plant Material mixtures. A. platyceras cell culture was initiated and maintained since For product identification, the products were separated by 1975 on Linsmeyer and Skoog (LS) medium [9]. Batch cultures in HPLC using a Nucleosil-SA-column (25 mm x 3.2 mm i.d.) and 1 litre Erlenmeyer flasks containing 250 ml medium were agitated 0.5 ammonium phosphate : methanol (80:20) as a solvent System. on a gyratofy shaker (100 rpm) in diffuse light (750 lux) at 24° C Retention times of the potential products were: 5'-0-methyl-NLS, and were subcultured at weekly intervals using about 10 % inocul- 6.45 min.; 7-O-methyl-NLS, 7.35 min.; 4'-0-methyl-NLS, 8.31 um. The cells were harvested after 8 days, frozen in liquid nitrogen min.; 6-O-methyl-NLS, 9.18 min. Preparative isolations were and stored at -20° C. Cell fresh weight was determined after filte- done by using the above incubation mixture x 100. The reaction ring the cells through a fritted glass funnel using suction. Aliquots product was extracted by ethylacetate and subjected to TLC (Si- were used for dry-weight determination. All other cell cultures gel; solvent System: Chloroform : methanol: acetic acid : water = were from our culture collections and were grown under identical 18:6:3:0.3). The zone containing radioactivity was scraped off, pu- conditions as given above. rified for a second time in the solvent System: Chloroform : n-pro-

panol : methanol : water = 45:15:60:40 (CHCl3-phase), and its Chemicals mass spectrum was measured in a Finnigan MAT 44S instrument. The following Compounds were obtained from the indicated sources: S-adenosyl-L-methionine hydrogen sulphate, Combithek Enzyme purification 2 (calibration proteins), S-adenosylhomocysteine, all from Boehrin- Step 1:100gfrozen tissue wasallowed tothawin0.1 MKP04 - ger, Mannheim; S-adenosyl-L-methyl-3H-methionine was prepa- buffer, pH 7.5, containing 20 mM ß-mercaptoethanol, stirred for red enzymatically from methionine -S-methyl-3H, (Radiochemical 20 minutes, pressed through cheese cloth, and centrifuged at Centre Amersham) using Standard methods. All benzylisoquinoli• 48000 xg for 10 min. Ammoniumsulphate precipitation was done ne alkaloids were synthesized according to Standard procedures. from 0-70 % Saturation, centrifuged again for 10 min. at 48000 xg. 2 ACA 34 was purchased from LKB, DEAE-cellulose-microgranu- The pellet was taken up in 0.1 M KP04 *-buffer, pH 7.5, containing lar form from Whatman, and hydroxyapatite from Bio-Rad. All 20 mM ß-mercaptoethanol. other materials were of reagent grade. Liquid scintillation count- Step 2: The crude extract from step 1 (15 ml) was put on an Ul- ing was performed in a toluene mixture (Rotiszint 22, Roth). trogel ACA 34 column (1 = 90 cm, 0 = 2.5 cm), equilibrated with 2 10 mM KP04 -buffer, pH 7.5, 20 mM ß-mercaptoethanol. Frac- O-Methyltransferase Assay and Product Identification tions of 4 ml were collected at a flow rate of 8 ml/h. The fractions Düring enzyme purification, OMT activity was assayed against (45-61) containing the enzyme activity were pooled and subjected R/S-norlaudanosoline, except that with crude enzyme prepara- to the next step. tions, laudanosoline was substituted for its nor-derivative. The as• Step 3: The fractions containing the enzyme (59 ml), were sub• say mixture consisted of KPO42* -buffer, pH 7.5 (130 mM), ascor- jected to ionexchange chromatography on DEAE-cellulose (1 = bate (130 mM) R/S-norlaudanosoline (0.3 mM), 3H-SAM (0.1 10 cm, 0 = 1.5 cm). The column was equilibrated with 10 mM 2 mM, 10000 cpm), and varying amounts of enzyme in a total KP04 *-buffer, pH 7.5, containing 20 mM ß-mercaptoethanol, and volume of 150 ul. a gradient was applied from 0-300 mM KCl (8 hrs). 4 ml Fractions The mixture was incubated for 45 minutes at 35° C and the reac- were collected at a flow rate of 40 ml/h. The enzyme was found in tion was terminated by addition of 200 ul Na2C03-buffer (1 M, pH fractions 56-64. The fractions containing the enzyme were pooled 9.5). The methylated products were extracted by adding 400 ul and applied to the next step. isoamylalcohol and shaking for 45 min. The turbid mixture was Step 4: The protein Solution was added to a hydroxyapatite co• 2 cleared by centrifugation in an Eppendorf centrifuge for 5 min. 200 lumn (1 = 10 cm, 0 = 1 cm), equilibrated with 10 mM KP04 "-buf- ul of the organic phase were transferred to scintillation vials and fer, pH 7.5, 20 mM ß-mercaptoethanol, and a gradient of 10-200 2 counted for radioactivity. Recovery of the methylated products mM KP04 , pH 7.5, was applied (8 hrs). 2 ml Fractions were col• was 95 % under these conditions. Blank mixtures containing either lected at a flow rate of 30 ml/h. Enzyme activity was found in frac- Enzymes from Argemone Cell Cultures 133 tions 43-54 with NLS as a Substrate. Soluble protein was determi- Table I ned as described previously [10] or in more highly purified samp- Survey of distribution of (R, S)-laudanosoline-methyltransferase les, with an optical method [11]. activity in species of different isoquinoline alkaloid containing Molecular Weight Determination families (laudanosoline served as Substrate) The molecular weight determination of the purified O-methyltransferase was carried out by gel filtration on a calibrated G-100- Plant material Family Enzyme activity superfine column. Although only the Stokes radii of the proteins pkat/l pkat/mg can be determined by this method, it is often used for the determin• medium protein ation of the molecular weight assuming globular shape for the prot• eins. The column, 168 ml (1 = 95 cm, 0 = 1.5 cm), equilibrated

2 Cell cultures: with 10 mM KP04 -buffer, pH 7.5, 20 mM ß-mercaptoethanol, Papaveraceae 6350 13.8 was eluted at a flow rate of 15 ml/h in 100 fractions of 2 ml. The co• Argemone platyceras lumn was calibrated with the proteins of the Combithek. Ferritin Corydalis sempervirens „ 4620 12.2 (MW: 450000) was used for the determination of the void volume Glauciumflavum 2855 27.7 of the column. The Standards were monitored by the absorbance at Fumaria officinalis 1950 11.1 280 nm. The results are given as Stokes radii. Corydalis pallida „ 1480 8.2 Argemone intermedia 1360 33.7 Adlumia fungosa „ 1070 12.5 Results Papaver somniferum 1020 9.3 Chelidonium majus „ 915 6.8 The presence of O-methyltransferases in crude en• Eschscholtzia tenuifolia 590 4.2 zyme extracts of plant cell cultures of different taxo- Berberis henryana Berberidaceae 2050 38.7 nomic origin was investigated using (R, S)-laudano- Berberis wilsonae „ 1650 13.9 3 soline and C H3-SAM as Substrates. Laudanosoline Berberis stolonifera 1260 18.9 was chosen as an initial methyl group acceptor to Thalictrum tuberosum Ranunculaceae 2060 26.6 avoid interference with N-methyltransferases potenti- Thalictrum sparsiflorum 570 2.4 ally present in the assay. Using these Substrates in the » Standard assay, O-methyltransferases were detected Cissampelos mucronata Menispermaceae 940 3.7 in all cell cultures containing isoquinoline alkaloids Differentiated plant: pkat/gdwt pkat/mg thus far tested (Table I). Cell cultures of four diffe• Papaver somniferum 218 1.6 rent plant families tested for the presence of O-methyltransferase enzymes showed positive results. By the culture filtrate. The enzyme was isolated and far the highest absolute amount of O-methyltransfe- partly purified by ammonium sulphate precipitation, rase per unit culture fluid was found in Argemone ACA 34, DEAE-cellulose, and hydroxyapatite chro- platyceras (Papaveraceae), a plant species known to matography as described under "Materials and Me• contain isoquinoline alkaloids of the pavine, proto- thods". This procedure yielded a purification of ap- berberine, and aporphine types [12]. It was there- proximately 80-fold with a recovery of 13 %. The da• fore decided to use cells of this species to purify and ta for a typical purification procedure are summari- characterize the O-methyltransferase. (R, S)-norlau- zed in Table II. The protein Solution at the stage of danosoline was used as a Substrate in the purification highest purification did not contain any other enzy• of the enzyme since it was possible to demonstrate mes of the isoquinoline biosynthetic pathway thus far that using this plant tissue, there was no interference tested. A typical elution profile of a hydroxyapatite with N-methyltransferases under the conditons cho• column is shown in Fig. 1. sen for the assay. All of the NLS-methylating emzyme activity was Properties ofthe O-Methyltransferase detected in the 100000 xg supernatant of the homo- The 80-fold purified enzyme was used to deter- genate of the Argemone cells. It could not be found in mine the catalytic properties. The activity of the

Table II Purification procedure for O-methyltransferase from Argemone platyceras (R, S-norlaudanosoline as Substrate)

Purification step Total activity Total protein Specific activity Recovery Purification (pkat) (mg) pkat/mg (%) -fold

Crude extract 3626 259.6 14 100 1 Ammoniumsulf ate-precipitation (0-70%) 3409 178.3 19 94 1.4 Gel filtration (ACA = 34) 2290 15.2 150 63.2 10 DEAE-cellulose chromatography 1124 2.5 459 31.0 32 Chromatography on hydroxyapatite 477 0.4 1173 13.2 83 134 Rueffer, Nagakura, Zenk

•160

Fraction

Fig. 1. Elution profile ofNLS-OMT from a hydroxyapatite column.

pkdt enzyme was measured at a ränge of pH 5-9 with different buffers as shown in Fig. 2. The O-methyltransferase from Argemone shows a clear pH-optimum at pH 7.5. The enzyme exhibits a temperature optimum at 35° C. The molecular weight of the O-methyltransferase determined by gel filtration on Sephadex G 100 was 47000 daltons. The enzyme was inhibited by preincubation for 15 min. at 35° C with the following metal ions added as sulpha- tes to a final concentration of 5 mM, and the reaction started by the addition of the labelled Substrate: Cu++ (100% Inhibition), Mn++ (15%), Zn++ (100%), Mg++ (0%), Co++ (81%), Ni++ (100%), Sn++ (55 %), Hg++ (100 %), Fe++ (90 %). No Inhibi• tion was found after preincubation with 25 mM ED- TA. Of the organic enzyme inhibitors tested only p- chloromercuribenzoate (25 mM, 70 %) and iodoben- zoic acid (10 mM, 73 %) resulted in substantial Inhi• bition of the enzyme. This indicates that there is an SH-group requirement for füll enzyme activity. A competitive inhibition was observed using S-adenosyl-homocysteine in the presence of 3H-SAM and NLS, as Substrates. A Ki of 10 \im was determined for SAH. The enzyme shows a half life of 8 hrs at 30° C. At 4° C after a 4 week storage period, 50 % of the in• itial activity of the enzyme was lost. All enzyme acti• vity was lost when the enzyme was frozen in 30 % gly- Fig, 2. pH profile of the catalytic activity of purified NLS-OMT from cerol Solution. A, platyceras cell cultures. Buffers used: A - A Na-maleate/ As shown in Table III, the purified enzyme is abso- NaOH; • - • K2HP04/KH2P04; • - • Tris/HCI; • - • Na-bora- te. lutely specific for the tetrahydrobenzylisoquinoline Enzymes from Argemone Cell Cultures 135

Table III Substrate specificity of partlally purified NLS-OMT from A. platyceras cell cultures

Substrate Enzyme activity Reaction product pkat/mg enzyme %*

(S)-Norlaudanosoline 313 100 6- O-methyl norlaudanosoline: 7- O-methylnorlaudanosoline = 8:2 (R)-Norlaudanosoline 288 92 sameas above (R, S) 4'-0-methylnorlaudanosoline 107 34 Norprotosinomenine (R, S) 5'-0-methylnorlaudanosoline 255 81 Nororientaline (R, S)-Laudanosoline 247 79 6-O-methyllaudanosoline (R, S)-Norlaudanosoline-1 -carboxylic acid 15 5 nd (R, S)-Norreticuline 3 1 nd (R, S)-Nororientaline 0 0 (R, S)-Laudanidine 0 0 - (S)-Scoulerine 3 1 -Tetrahydrocolumbamine 2,3-Dihydroxy-9,10-dimethoxy protoberberine 22 7 Jatrorrhizine (R, S)-2,3-Dihydroxy-9,10-dimethoxy tetrahydroprotoberberine 15 5 Tetrahydrojatrorrhizine Adrenaline 0 0 Aesculetine 0 0 - Caffeicacid 0 0 - Catechol 0 0 - Dopamine 0 0 - Quercetin 0 0 - relative to (S)-NLS; nd = not determined nucleus. None of the phenylpropanoids, phenolics, tion mixture containing (R, S)-laudanosoline as Sub• biogenic amines, coumarins or flavonoids were me• strate, mass spectroscopy of the purified major un- thylated by action of this enzyme. The best Substrates known product showed a clear fragment (3,4-dihy- were the ones with four free phenolic-hydroxy-func- dro-6-methoxy-7-hydroxy-N-methyl-isochinolinium tions, followed by mono-methyl-derivatives. Di-O- cation) with m/z 192 (100%) containing one N and methylated alkaloids are very poor Substrates. KM one O-methyl-group which further fragmented to ml values for this enzyme were determined using the fol- z 177 (192-CH3, 30 %), and m/z 162 (177-CH3,4 %).

lowing Substrates: KM (S)-norlaudanosoline0.2mM; The spectrum clearly indicated methylation at the A- (R)-noi iaudanosoline 0.2 mM; (R,S)-4'-0-methyl- ring and was identical in every respect with the mass norlaudanosoline 1.1 mM; (R, S)-laudanosoline 0.3 spectrum of authentic 6-O-methyllaudanosoline. mM. SAM 0.05 mM (with (S)-NLS as Substrate). Thus the major product of the NLS-OMT reaction The 80-fold purified OMT is absolutely free of any was proven to be 6-O-methylnorlaudanosoline. N-methylating activity as demonstrated by its lack of The time course of the enzyme formation in Sus• activity on norreticuline, nororintaline or tetrahy- pension cultures of Argemone cells is shown in Fig. 3. dropapaverine. The enzyme is present in the inoculum only in low The best enzyme Substrate of the Compounds amounts. The activity peaks at day 8 of cultivation tested so far proved to be (S)-norlaudanosoline. The exactly at the point when the culture is leaving the lo- product of the reaction catalysed by the transferase garithmic growth phase. A 30-fold increase in activity using (S)-NLS and 14C-SAM as Substrates was subse- can be seen as compared with only 10-fold increase in quently investigated. HPLC-chromatography under dry cell matter. Düring stationary phase there is a the conditions given, showed that only two radioacti- drastic decrease of total activity of the enzyme. ve products were formed, one Compound with a retention time of 7.2 min. (20 yield) and the major Com• pound with a retention time of 9.2 min. (80 % yield). Discussion The retention times and co-chromatography with all four possible authentic monomethylated norlauda- Isoquinoline alkaloids comprise the largest group nosolines indicated the minor Compound to be 7-0- of alkaloids in plant kingdom. Relatively little is methyl-norlaudanosoline and the major Compound known about their biosynthesis at the cell-free level. to be 6-O-methyl-norlaudanosoline. Preparative iso- In an attempt to elucidate the enzymatic steps invol- lation of the major Compound from a 15 ml incuba• ved in benzylisoquinoline synthesis, it is our primary 136 Rueffer, Nagakura, Zenk

aim to isolate and characterize those enzymes which labelled product at pH 9.5 with isoamylalcohol lea- are involved in reticuline biosynthesis, the central ving residual 3H-SAM in the aqueous Solution. and branch-point intermediate in benzylisoquinoline By using this assay the methylating enzyme could metabolism in plants [3]. In previous studies, we be purified about 80-fold and the major (80 %) pro• were able to demonstrate that the initial reaction in duct of reaction could unequivocally be identified as formation of the benzylisoquinoline skeleton is the 6-O-methylnorlaudanosoline by HPLC and mass stereospecific condensation of dopamine and 3,4-di- spectroscopy and comparison with the other four au• hydroxyphenylacetaldehyde to yield (S)-norlauda- thentic mono-methyl-NLS species. The minor reac• nosoline [1,2]. The aldehyde, rather than the 3,4-di- tion product (20%) was identified as 7-O-methyl- hydroxyphenylpyruvate, is the Substrate for this con• NLS. It is noteworthy that using the plant enzyme densation reaction. The previously postulated inter• there was not much difference in the methylation rate mediate norlaudanosoline-1-carboxylic acid [13-15] using the stereoisomers (S)- or (R)-NLS. Further- is most probably an artefact. Between the now reco- more there was no difference in methylation pattern gnized first metabolite in the pathway norlaudanoso• observed using the pure optical isomers as Substrates. line and reticuline, three methylation steps are in• In both cases 80 % of the product formed was 6-0- volved, two O-methylations at carbon atom 6 and 4' methyl-NLS, formed regardless of whether (R)- or and one N-methylation at atom 2. Since nor-reticuli- (S)-NLS was used as Substrate. This demonstration is ne has been amply demonstrated by in vivo experiments to be the immediate precursor of reticuline [e.g. 4, 5], the question arises, which of the phenolic Table IV groups of the norlaudanosoline molecule is methyla• Comparison of Norlaudanosoline-O-methyltransferases of plant ted first on its way to nor-reticuline, the one in 6 or and animal origin the one in 4' position. A survey of plant cell cultures Characteristics Enzyme source using (R,S)-laudanosoline and 3-H-SAM as Substra• tes (in order to prevent N-methylation) demonstra• A. platyceras Rat [17] Mouse* ted that all isoquinoline containing species contained _ good methylating activity. No methyltransferase ac• (S)-NLS:KM 0.2 mM 0.8 mM 3 - tivity was observed using (R,S)-NLS and H-SAM as (R)-NLS:KM 0.2 mM 2.2 mM - - Substrate with for instance Catharanthus roseus (R,S)-NLS:KM 1.3mM

(Apocynaceae), a species which is known notio con• SAM:KM 0.05 mM 6.2 mM 1.0 mM tain any benzylisoquinoline alkaloids. The highest pH-optimum 7.5 7.7-8.0 7.5 amount of methyltransferase per volume of medium Position of methylation 6 and 7 6 and 7 6 and 7 was observed in A. platyceras cell cultures, and it was Ratio for (S)-NLS 80:20 79:14 53:47 decided to use this species for the isolation and cha- Ratio for (R)-NLS 80:20 26:68 47:53 Caffeic acid methylated No N.d. Yes racterization of the NLS-O-methyltransferase enzy• me. The test used for the analysis of the methylated * This investigation was carried out in our laboratory using a NLS formed involved differential extraction of the mouse liver preparation according to [17] Enzymes from Argemone Cell Cultures 137

N-H H3C0

HCK^^ HO' 80% 20% (R, S)- Norlaudanosoli ne 6-O-Methyl - 7-0 - Methyl - (R.S)-Norlaudanosoline (R.S)-Norlaudanosoline

Fig. 4. Reaction sequence catalysed by S-Adenosyl-L-methionine: (R), (S)-Norlaudanosoline-6-0-methyltransferase.

at variance with observations using a purified O-me• the enzyme Systems introducing the second O-methyltransferase from rat liver [8, 16, 17], where it thyl-group are probably rather unspecific. could be clearly shown that methylation of (S)-NLS The search for methylating enzymes in the path- yielded predominantly (79%) 6-O-methyl-NLS way leading to reticuline must now concentrate on an while the (R)-NLS isomer gave rise to 7-O-methyl- O-methyltransferase methylating the 4'-position in NLS (68 %). Thus in the animal System, the position 6-O-methyl-NLS, as well as on a norreticuline-N-me- of O-methylation (either at atom 6 or 7) is largely di- thyltransferase. rected by the particular isomeric form of the Substra• te. A comparison of NLS-O-methyltransferases of Acknowledgements plant and animal origin are given in Table IV. There are distinct differences between the animal This investigation was supported by SFB 145 of Deutsche For• and plant enzyme. The plant enzyme shows a consid- schungsgemeinschaft, Bonn, as well as by Fonds der Chemischen Industrie. The technical assistance of GABRIELE WEBER is gratefully erably higher affinity for the Substrates than the ani• acknowledged. We thank Dr. E. FANNING for kind linguistic help.

mal enzymes, as demonstrated by the low KM value for NLS and SAM. The animal Systems which do not References show, in contrast to the plant enzyme, a high Substra• te specificity clearly demonstrate that the stereoiso- (1) Rueffer, M., H. El-Shagi, N. (10) Bensadoun, A. and D. Wein• Nagakura and M. H. Zenk: FEBS- stein: Anal. Biochem. 70, 241 meric form of the alkaloid Substrate dictates the site Letters 729,5 (1981). (1976). of enzymatic O-methylation. The plant enzyme pre- (2) Schumacher, H.-M., M. Ruef• (11) Warburg, O. and W. Chri• 310, fers the 6 positions for methylation regardless of the fer, N. Nagakura and M. H. Zenk: stians: Biochem. Z. 384 Planta Med. 48, 212 (1983). (1941). steric configuration of the NLS Substrate. (3) Cordeil, G. A.: Introduction to (12) Santavy, F. and R. H. F. It is also noteworthy that the previously assumed Alkaloids, New York 1981, John Manske (ed.): The alkaloids, Vol. Wily & Sons. XVII, 385, New York, San Francis• intermediate, (R,S)-norlaudanosoline-l-carboxylic (4) Battersby, A. R., D. M. Foul- co, London, 1979, Academic Press. acid [13-15] shows only about 5 % of the activity of kes, M. Hirst, G. V. Parry and J. (13) Wilson, M. L. and C. J. Cos- (S)-NLS. This fact would indicate that even if the car- Staunton: J. Chem. Soc. (C) 1968, cia: J. Am. Chem. Soc. 97, 431 210. (1975). boxylic acid were a natural intermediate in benzyliso• (5) Brochmann-Hanssen, E., C- (14) Battersby, A. R., R. C. F. Jo• quinoline biosynthesis, decarboxylation would have H. Chen, C. R. Chen, H.-C. nes and R. Kazlauskas: Tetrahe• to precede methylation of this molecule. Chiang, A. Y. Leung and K. dron Lett. 1975,1873. McMurtrey: J. Chem. Soc. Perkin (15) Scott, A. I., S.-L. Lee and T. The Observation presented above that the first me• I, 1975, 1531. Hirata: Heterocycles 11, 159 thylation reaction of NLS occurs at the 6-O-position (6) Tsang, Y. F. and R. K. Ibra• (1978). him: Phytochemistry 18, 1131 (16) Collins, A. C, J. L. Cashaw (see Fig. 4) is in perfect agreement with a finding by (1979). and V. E. Davis: Biochem. Phar- BROCHMANN-HANSSEN et al. [5]. Their results, ob- (7) Antoun, M. D. and M. F. Ro• macol. 22,2337 (1973). tained from in vivo experiments, indicate that during berts: Planta Med. 28, 6 (1975). (17) Meyerson, L. R., J. L. Cas• (8) Davis, V. E., J. L. Cashaw, K. haw, K. D. McMurtrey and V. E. the early steps in papaverine formation 6-O-methyla- D. McMurtrey, S. Ruchirawat and Davis: Biochem. Pharmacol. 28, tion must precede 7-O-methylation. This is also con- Y. Nimit, in: Beta-Carbolines and 1745 (1979). 36, sistent with the sequence of O-methylation of phene- Tetrahydroisoquinolines. Bloom, (18) Paul, A. G.: Lloydia 36 F. et al. eds.: p. 99, New York 1982, (1973). thylamine and tetrahydroisoquinolines in the biosyn• Alan R. Liss Inc. (19) Tewari,S.,D.S. Bhakuniand thesis of peyote alkaloids [18]. The Observation that (9) Linsmaier, E. and F. Skoog: R. S. Kapil: J. Chem. Soc. Chem. 18, (R,S)-[l-^H]-4'-0-methylnorlaudanosoline (0.18 %) Physiol. Plant 100(1965). Commun. 1975, 554. is slightly better incorporated into reticuline than (R,S)-[l-3H]-6-0-methylnorlaudanosoline (0.12 %) Address: Prof. Dr. M. H. Zenk, Pharmazeutische Biologie, in feeding experiments to Litsea glutinosa [19] may Karlstraße 29, be within experimental error, but demonstrates that D-8000 München 2, Feder al Republic ofGermany