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Aspects of secondary metabolism in basidiomycetes: I. biological and biochemical studies on Psilocybe… Wang, Wei-Wei 1978

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ASPECTS OP SECONDARY METABOLISM IN BASIDIQMYCETES BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF LENTINUS AND LENTINELLUS B.Sc, National Taiwan University, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF BOTANY) We accept 'this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1977 f7\ Wei-Wei Wang, 1977 by by.' . WEI-WEI/WANG In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements f o r an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of t h i s t h e s i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or p u b l i c a t i o n of th i s thesis fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of Botany The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date December 6. 1977 i i ABSTRACT I. Psilocybe cubensis was cultured s u c c e s s f u l l y i n two media. Medium A was devised by Catalfomo and Tyler and Medium B was a modification of a medium which has been used f o r ergot a l k a l o i d production by Claviceps  purpurea. Only when the fungus was kept on Sabouraud agar p l a t e s . d i d i t subsequently produce p s i l o c y b i n when transferred to l i q u i d media. A quantitative time-course study of p s i l o c y b i n production i n the two media was c a r r i e d out. Maximal production appeared on the f i f t h day. The a c t i v i t i e s of an acid phosphatase, acting on p s i l o c y b i n , were measured from mycelia grown i n the two media. Enzyme a c t i v i t y from the A culture was very high and a blue color caused by oxidation of p s i l o c i n formed i n f i v e minutes. The e f f e c t of adding L-tryptophan on a l k a l o i d production as well as 14 the f a t e of tryptophan-C was also investigated. Tryptophan stimulated s i g n i f i c a n t l y p s i l o c y b i n production i n the very beginning i n the B medium. The degradation of tryptophan was d i f f e r e n t i n the two media. I t was converted to kynurenine and a n t h r a n i l i c acid i n A medium and to tryptamine i n tryptophan added B medium (B' medium). Radioactive D,L-tryptophan side chain labeled, gave labeled p s i l o c i n and p s i l o c y b i n . Potassium deficiency decreased p s i l o c y b i n production while a potassium supplement had no e f f e c t . The fungus did not produce polyacetylenic compounds i n the medium but ergosterol was detected as a major acetate derived metabolite when the fungus was kept on MYP agar plates and transferred subsequently to l i q u i d media. P s i l o c i n has very s l i g h t a n t i -b i o t i c a c t i v i t y against Candida albicans whereas p s i l o c y b i n has none. i i i I I . Eight species of Lentinus and Lentinellus . were investigated f o r the occurrence of a phenol-O-methyltransferase. Only Lentinus lepideus and Lentinus pbnderbsus showed enzyme a c t i v i t y i n both l i g h t and dark conditions. The s p e c i f i c i t y of the enzyme f o r a number of substrates was also examined. Of s i x compounds tested, methyl p-coumarate, methyl caffeate and methyl ferulate.served-as substrates. The products of enzymic a c t i v i t y were i d e n t i f i e d - b y radioautography. iv TABLE OF CONTENTS PAGE ABSTRACT i i TABLE OF CONTENTS;. iv LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT ix I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS INTRODUCTION 1 REVIEW OF LITERATURE 3 MATERIALS AND METHODS A. Chemicals 13 B. Medium and culture 15 C. Analytical methods 17 D. Radioautography 20 E. Psilocybin phosphatase activity 21 F. Potassium nutrition and psilocybin production 23 G. Test of UV-mediated antibiotic and phototoxic activities of medium, psilocin and psilocybin 24 EXPERIMENTAL AND RESULTS A. Chromatographic separation and identification of tryptophan metabolites 25 B. pH, Growth and morphological difference of cultures 26 C. Psilocybin production 27 D. Formation of psilocybin and psilocin from tryptophan 28 V E. Phosphatase a c t i v i t y 39 F. P s i l o c y b i n production and potassium n u t r i t i o n 39 G. E h r l i c h - p o s i t i v e compounds i n the e x t r a c t s 42 H. Tryptophan con c e n t r a t i o n i n the medium 42 I. A n t i b i o t i c a c t i v i t y of p s i l o c i n , p s i l o c y b i n and medium f i l t r a t e 49 J . I d e n t i f i c a t i o n of d(-)mannitol 49 DISCUSSION 54 LITERATURE CITED 63 I I . A SURVEY OF PHENOL-0-METHYLTRANSFERASE IN SPECIES OF LENTINUS AND LENTINELLUS INTRODUCTION 69 MAT ERI AL S A ANDI1ME THOD S A. Chemicals 73 B. Medium and c u l t u r e 74 C. E x t r a c t i o n of enzyme 75 D. Enzyme assay 75 E. I d e n t i f i c a t i o n of r e a c t i o n products 76 F. P r o t e i n c o n c e n t r a t i o n 76 RESULTS AND DISCUSSION 77 LITERATURE CITED 81 v i LIST OF TABLES page I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS 1. Some Tryptamine Derivatives Found in Fungi 3 2. Fungi Containing Psilocybin 6 3. Composition of A-medium 15 4. Composition of B-medium 16 5. Fluorescence, Color Reaction and R^  Values of Some Tryptophan Metabolites 25 6. Distribution of Radioactivity in Tryptophan, Psilocybin and Psilocin of Mycelium Extract, 14 Administered D,L-Tryptophan, side chain-3-C 28 7. The Growth, pH of Medium and Psilocybin Production of B-medium, Potassium Deficient and Supplement Media 39 II. A SURVEY OF PHENOL-0-METHYLTRANSFERASE IN SPECIES OF LENTINUS AND LENTINELLUS 1. Substrates Specificity of Methylating Enzymes from Lentinus ponderosus and Lentinus lepideus 78 v i i LIST OF FIGURES page I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS 1. Locations of some standard tryptophan metabolites on a two-dimensional cellulose thin layer plate 29 2. pH of A and A' media 30 3. pH of B and B' media 31 4. Growth rate of fungal cells in A and A' media 32 5. Growth rate of fungal cells in B and B' media 33 6. UV spectrum of authentic psilocybin -.34 6!. UV spectrum of psilocybin isolated from fungal cells 35 7. Psilocybin production from A and A' media 36 8. Psilocybin production from B and B' media 37 9. Radioautograph of chromatographed tryptophan metabolites of a mycelium extract of Psilocybe cubensis, administered D,L-tryptophan 38 10. Blue color formation when psilocybin incubated with enzyme extract 40 11. Activities of acid phosphatases of the mycelia from two different media 41 12. Chromatograms of mycelium extract and medium extract of different growth period 43 13. Tryptophan concentration of A' and B' media 48 14. Antibiotic activity of psilocin 50 15. IR spectrum of white crystals 51 16. NMR spectrum of acetate derivative of white crystals 53 17. UV spectrum of ergosterol 55 v i i i A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF LENTINUS AND LENTINELLUS 1. Radioautographs of the chromatographed methylated clnnamate products i x ACKNOWLEDGEMENT I wish to express my appreciation to Dr. G. H. N. Towers for h i s suggestion of t h i s project and h i s advice, c r i t i c i s m and encouragement. I l i k e to thank s i n c e r e l y to Dr. C. K. Wat for her h e l p f u l a s s i s t -ance and guidance; to the members of committee i n reading the manuscript and making c r i t i c a l comments; and.to the f a c u l t y , s t a f f and students of the Department of Botany f o r being h e l p f u l i n many ways. I also acknowledge with gratitude to Mr. Terry Q. F. Ching f o r h i s invaluable assistance i n the preparation of the manuscript; to Miss Yvonne Tang f o r proofreading the f i n a l manuscript; and to Mr. H. P. Hsu for h i s contribution i n many ways. X I, I. BIOLOGICAL AND BIOCHEMICAL STUDIES ON PSILOCYBE CUBENSIS 1 INTRODUCTION Intoxication, following the ingestion of certain kinds of mushrooms has. been known as early as the tenth century in the Sung Dynasty of China (69) and an 11th Century tale from Japan is about the eating of "dancing mushrooms" and "laughing mushrooms" (4, 69). Of the psychotropic materials utilized by ancient Nahuatl people of the New World, teonanacatl ("God's mushrooms" or "God's flesh") especially stands out in Mexican history (3). Since the pre-Columbian era, the Indians of Mexico have made the eating of certain fungi a part of their religious rites (15). The f i r s t record of the use of hallucinogenic mushrooms dates from 1502 at the Aztec Coronation festivals of Montezuma II (3, 70). This was recently rediscovered by R. G. Wasson and U. P. Wasson in 1957 (70). The active hallucinogenic principle was isolated from Psilocybe and therefore named psilocybin (4-phosphoryl-N,N-dimethyltryptamine). Psilocin is the dephosphorylated derivative. They are the f i r s t natural indole derivatives found to possess an OH group in position 4 and psilocybin i s the f i r s t one known to contain a phosphate group (71). The chemical synthesis of these two indole alkaloids has been well 14 studied. The biosynthesis has only been studied by feeding C -labeled precursors and from these results a pathway has been proposed. Enzymological studies are meager. One of the objectives of the present study was to find better growth conditions for the production of psilocybin so as to lead to further studies on enzymology of biosynthesis and psychopharmaco-logy. Psilocybin has been demonstrated to be derived from tryptophan. An objective of this study concerned the fate of added tryptophan and of labeled tryptophan. 2 I t was i n t e r e s t i n g to f i n d out i f there i s any b i o l o g i c a l s i g n i f i c a n c e i n the production of these two indole compounds. A b r i e f examination of cultures f o r other obvious secondary metabolites, such as polyacetylenes, was also c a r r i e d out. 3 REVIEW OF LITERATURE A. The occurrence of tryptamine derivatives in higher fungi Certain tryptamine derivatives (I) to which psychotomimetic, properties have been ascribed occur in a number of the Basidiomycetes (1, 2, 3, 4). Serotonin (II), N-methylserotonin (III), bufotenine (IV) bufotenine N-oxide (V), N,N-dimethyltryptamine (VI) and 5-methoxy-N,N-dimethyltryptamine (VII) have been found in either carpophores or mycelia of species of Amanita (Amanitaceae), Panaeolus, Coprinus (Coprinaceae), Boletus (I('B6>letaeeaeO) and Sarcodpn (Thelephoraceae) (Table 1) (5, 6, 7, 8, 9, 10). Table 1. Some Tryptamine Derivatives Found in Fungi Species Compound Amanita citrinaa. II, Amanita mappa IV Amanita muscaria IV Amanita pantherina IV Amanita porphyria II, Amanita tomentella !V Ranaeolus foenisecii II Panaeolus semiovatus II Panaeolus sphinctrinus II Panaeolus subbalteatus II Coprinus atramentarius I Coprinus comatus I Coprinus micaceous I Boletus erythropus I Sarcodon imbricatum I Psilocybin (VIII) and psilocin (IX) have been shown to be responsible for the hallucinogenic effects of mushrooms of the genus Psilocybe (Strophariaceae). Two analogs of psilocybin, baeocystin (X) and 4 •N-CH, N-Methylserotonin III Bufotenine (5-Hydroxy-N,N-dimethyltryptamine) IV 0H-•N-CH, Bufotenine N-oxide V N,N-Mmethyltryptamine VI 5 0 II _ HO-P-0 5-Methoxy-N,N-dimethyltryptamine Psilocybin (4-Phosphoryl-N-N-dimethyltryptamine) VII VIII -N-CH-I 3 Psilocin (4-Hydroxy-N,N-dimethyltryptamine) IX Baeocystin (4-Phosphoryl|rN^monomethyltryptamine) X Nor-baeocystin H (4-Phosphory1tryptamine) Ergoline ring XI XII 6 nor-baeocystin (XI) have been found in Psilocybe baeocystis (11, 12, 13) and recently baeocystin was detected in Psilocybe semilanceata (14). Baeocystin and nor-baeocystin are found to be toxins (74). B. Chemical synthesis of psilocybin and i t s physical properties Hofmann (15) reported the chemical synthesis of psilocybin i n 10 stages from O-nitrocresoll'.- This was a commercial synthesis and is outlined in Scheme I. Psilocybin and psilocin have been isolated from Psilocybe and identified c r i t i c a l l y (15, 16, 17). Psilocybin forms colorless, monoclinic crystals, soluble in methanol but practically insoluble in the usual organic solvents. The melting point i s 185-195 °C and UV absorption maxima in methanol are 220, 267, 290 nm (loge 4.6, 3.8, 3.6). C. Distribution of psilocybin Psilocybin has been identified in mycelia, spores, carpophores or s'cierbt:ia<rocf 34 species of the genera Conocybe (Bolbitiaceae) .Panaeolus and Psilocybe (Table 2). Table 2. Fungi Containing Psilocybin Species Reference Conocybe cyanopus 9, 12 Conocybe smithii 9, 12 Panaeolus africanus 4 Panaeolus ater 4 Panaeolus cambodginiensis 4, 18 Panaeolus campanulatus 4 Panaeolus castaneifolius 4 Panaeolus cyanescens 4 Panaeolus fimicola 4 Panaeolus foenisccii 4, 20 7 Panaeolus r e t i r u g i s 4 Panaeolus sphinctrinus 4, 9, 12 Panaeolus subbalteatus 4, 9, 18 Panaeolus t r o p i c a l i s 4 Psilocybe aztecorum 9, 12 Psilocybe baeocystis 9, 12 Psilocybe b o n e t i i 18 Psilocybe caerulescens 12 Psilocybe eaerulipes 4, 12 Psilocybe c o l l y b i o i d e s 3 Psilocybe cubensis 1, 9, 12 Psilocybe cyanesis 9, 12 Psilocybe f i m e t a r i a 9, 12 Psilocybe mexicana 9, 12, 20 Psilocybe mixanesis 9, 12, 21 Psilocybe p e l l i c u l o s a 9 , 12 Psilocybe quebecensis 22 Psilocybe semilanceata 9, 12 Psilocybe semperviva 9, 12, 21 Psilocybe s t r i c i t i p e s 25 Psilocybe s t u n t z i i 18 Psilocybe subaeruginosa 3, 24 Psilocybe wassonii 9, 12 Psilocybe zapotecorum 9, 21 Other species of Psilocybe have been reported to be hallucinogenic and/or Coxi" , bul whether ;'• v" • - v : - . ..• ^  . j c y ^ i u .-- ; • -/ and/or t o x i c , but whether or not they contain p s i l o c y b i n i s not known eg,. Psilocybe r> o: i s s i r \ , Psilocv.Le agge- id.-r.-4 , IVixocvbe p.-_-•ea g> 0Psi1 ocybggacu 11ssima-, Psilcycybe\ agg.er.i.cola,^Psilocybe-,bolivari, PsixocyBe b6 o^sbligehii^ aPsilb'cybe c a l l o s a , Psilocybe candidipes, Psilocybe cordispora, Psilocybe f a g i c o l a , Psilocybe f a s c i a t a , Psilocybe  i s a u r i , Psilocybe kumaenorum, Psilocybe macrocystis, Psilocybe  s i l v a t i c a , Psilocybe subcaerulipes, Psilocybe yungensis (3)i and Psilocybe argentipes (26). 8 OCH_C,H_ I 2 6 5 0=P-0CHoC,H Cl Scheme I Nutritional studies Heim and Cailleux succeeded in growing several mushrooms belonging to the family of Strophariaceae and even obtained fruiting bodies, on natural substrates (15). Submerged cultures of Psilocybe was f i r s t attempted by Catalfomo and Tyler (1). They developed conditions for the production of mycelial rpellets yielding psilocybin in shaken culture of Psilocybe cubensis. However, Psilocybe cyanescens and Psilocybe  pelliculosa failed to produce psilocybin under these conditions. Of various species of the family Strophariaceae studied by Leung (12), only Psilocybe baeocystis was successfully grown on a modified nutrient solution of Catalfomo and Tyler. The fungus was found to produce psilocybin and baeocystin under these conditions. The nutrient solution used for producing ergot alkaloids of Claviceps species has been tried with Psilocybe but without success. The influence of phosphate on the distribution pattern of nitrogen between soluble and insoluble cellular components was studied on Psilocybe cubensis, Psilocybe cyanescens and Panaeolus campanulatus (27). Kitamoto et a l . (28) showed that Psilocybe  panaepllf-ormisjformed fruiting bodies within two weeks on a liquid medium consisting of glucose, amino acids, mineral salts and various growth factors. Ammonium and nitrate nitrogen were utilized for mycelial growth but not for fruiting. Casamino acids were a good nitrogen source for fruiting. Biogen;esiso:5fp psMocy.bin The structural similarity between psilocybin and tryptophan suggests that i t i s derived from that amino acid. Brack et a l . (23) demonstrated that psilocybin isolated from surface-cultures mycelium of Psilocybe  semperviva was derived from labeled tryptophan added to the medium. 10 14 Agurell and Nilsson (2, 29, 30) using C -labeled substrates demonstrated a biosynthetic sequence from tryptophan to psilocybin via decarboxylation, N-methylations, hydroxylation and phosphorylation (Scheme I I ) . Their results indicated however that this i s not the only pathway to psilocybin and that a grid of pathways probably exists in the fungus. H H Scheme II F. Blueing phenomenon in psilocybin-containing mushrooms Psilocybin-containing mushrooms are well known for the blueing which results from damage, Although the blueing reaction has not been studied in hallucinogenic mushrooms per se, a number of other studies 11 shed light on this phenomenon (4). Blaschko and Levine (31, 32) found that an enzyme from the g i l l plates of the mussel (Mytilus edulis) or ceruloplasmin, a copper containing oxidase, from pig plasma, converted psilocin to a blue product. Horita and Weber (33) found that psilocybin was readily dephosphorylated by various mammalian alkaline phosphatases and that the liberated psilocin was oxidized to blue metabolites. Using a preparation of cytochrome oxidase, they (34) found psilocin was oxidized to a dark blue product and the synthetic hallucinogen 4-hydroxy-N,N-diethyltryptamine to a bluish-green metabolite. 4-Hydroxy-tryptamine gave a blackish-brown pigment. Levine (35) found that the oxidative formation of a blue color could also be eli c i t e d without enzyme in the presence of f e r r i c ion. EDTA blocked this reaction. With psilocybin, neither formation of blue color nor uptake of 0^ occurred during incu-bation with ceruloplasmin or fe r r i c ion. para-Diphenol oxidase (laccase), a copper containing enzyme from the fungus Polyporusv versicolor, i s capable of oxidizing psilocin to a blue product (36). The blue product i s hydrophilic and has a;;sp'ectrum withtabsorption at 610 nm and 395 nm. The blue product was readily reduced to a colorless product by ascorbic acid and is thus probably quinonoid in nature. It appears that a free OH group i s essential for oxidation because psilocybin is not oxidized under identical conditions. Bocks;(37) also found a laccase type of enzyme in homogenates of the mycelium of Psilocybe cubensis which is capable of catalyzing the oxidation of 2,6-dimethoxyphenol and of psilocin. The mechanism of the catalytic action of laccase on 2,6-dimethoxyphenol could be written as follows (38). 79); " 12 Adventitious browning occurs in the flesh of certain ripe fruits such as apples and pears following injury and this rapid browning results from the oxidation of endogenous phenols by enzymes having laccase and/or phenolase activity. The blueing reaction in Psilocybe is most lik e l y a similar type of reaction mediated by these types of oxidizing enzymes (4). G. Other chemical constituents of Psilocybe Very l i t t l e chemical work has been carried out on members of this genus. Brack e_t a l . (73) reported the production of certain ergot alkaloids in Psilocybe semperviva. Polyacetylenic compounds, such as the triynol, CH3-CsC-C2C-C=C-CH20H (2,4,6-octatriynol) and triynoic acid CH3-C=C-C=C-C=C-COOH (2,4,6-octatriynoic acid) were found in the culture fluids of Psilocybe merdaria (39). The polyacetylene, HC=C-C=C-C=C-C=C-COOH (4,6,8-nonatriyn-2-enoic acid) was reported from Psilocybe sarcocephala (40). These fungal acetylenes often show anti-biotic activity. 13 MATERIALS AND METHODS Chemicals: The source of the chemicals used was as follows: Chemical Source MgS04. 7H20 Fisher Scientific Co. (NH 4) 6MS 70 2 4. 4H20 Fisher ZnSO,. 7H.0 4 2 B. D. H. Laboratory MnCl 2. 4H20 B. D. H. FeSO.. 7H.0 4 2 Allied Chemical Ltd. CuS04. 5H20 Fisher N a N 02 , Mallinckrodt Inc. KH2P04 Fisher NaHoP0. 2 4 Fisher KCI Fisher NaOH Mallinckrodt NH. OH 4 Allied H2 S°4 Allied HC1 Allied NaCI Amachem NaCN Mallinckrodt (NH 4) 2C0 3 J. T. Baker Chemical Co Acetic acid, glacial Allied Isopropanol Amachem Methanol Fisher n-Butanol Fisher Thiamine hydrochloride J. T. Baker D(-)Glucose Mallinckrodt D(-)Mannitol ICN Sucrose Fisher 14 Yeast extract Sabouraud dextrose agar Malt extract Soytone Glycine L-Tryptophan p-Dimethylaminobenzaldehyde Tryptamine hydrochloride N-Methyltryptamine N, N-5-Dimethyltryptamine Psilocin Psilocybin 5-Hydroxytryptophan 5 -Hydroxy t ryp t amine (oxalate in ) (serotonin) Kynurenine Kynurenic acid Anthranilic acid 3-Hydroxyanthranilic acid 3-Methy1indole(skatole) r3=Ethanol indole(tryptophol) i i u a Urea Allantoic acid Allantoina N-Acetyl-D, L-tryptophan 14 D, L-Tryptophan(side chain-3-C ) specific activity 3.76 mCi/m mole (2.71 mg 50 uCi) Aquasol Dowex 50 WX-8 Difco Laboratory Difco Difco Difco Fisher Sigma Matheson Coleman and Bell Co. Sigma Sigma Gifts from the Dept. of National'-•Health and Welfare, Health Protection Branch, Ottawa Sigma Sigma Sigma Sigma Calbiochem Mann Research Laboratory Sigma Sigma Fisher ICN Eastman OrganicCChemicals Sigma ICN New England Nuclear J. T. Baker 15 Phenolphthalein . diphosphate (PDP) Sigma l-Nitroso-2-naphthol BDH Ethylene dichloride Matheson Coleman and Bell Manufact-uring Chemists Ammonium succinate was prepared by adding 40 ml NH^ OH to 11.8 gm succinic acid.. To this solution, 95% ethanol was added unt i l white crystals precipitated. The crystals were collected and air dried. Medium and culture Medium: Two kinds of liquid media were employed in this work. A-medium was devised by Catalfomo and Tyler (1) (Table 3). B-medium was a modified version of Stoll's medium for ergot alkaloid production from Claviceps purpurea (41) (Table 4). Table 3. Composition"offA^medium-s Ammonium succinate 1.0 gm Glycine 9.0 gm D(-)Glucose 10.0 gm Yeast extract 0.5 gm KH2P04 0.1 gm Thiamine hydrochloride 3.0 mg MgS04. 7H20 0.5 gm (NH 4) 6Mo 70 2 4. 4H20 0.05 mg ZnSO.. 7H„0 4 2 0.3 mg MnCl 2. 4H20 0.35 mg FeSO.. 7H£0 4 2 2.5 mg CuSO,. 5H-0 4 2 0.5 mg D i s t i l l e d water 1 l i t e r The pH was adjusted^fco::-555vwith"HGl. 16 Table 4. Composition;.of B-medium--D(-)Mannitol 50.0 gm Sucrose 50.0 gm Succinic:..acid" 5.4 gm Yeast extract 1.0 gm KH2P04 1.0 gm FeS0 4. 7H20 O.Olgm MgS04. 7H20 0.3 gm ZnSO^. 7H20 4.0 mg Di s t i l l e d water X>1 l i t e r The pH was adjusted to 5.4 with NH^ OH. One hundred ml of each medium in a 500 ml Erlenmeyer flask with a cotton plug was sterilized at 15 lb, 121 °C for 15 minutes. L-Tryptophan containing media, A'-medium and B'-medium, were prepared by adding aseptically 10 ml L-tryptophan solution (2.5 mg/ml A-medium or B-medium) to a flask containing 90 ml sterilized A-medium or B-medium. Culture: Psilocybe cubensis was obtained from Dr. S. H. Pollock, Dept. of Pharmacology, University of Texas, USA. It was reported to produce both psilocybin and psilocin. The fungus was maintained on slants of malt extract-yeast extract-soytone (MYP, 7 gm—0.5 gm—1 gm) agar. After growth, the cultures were covered with sterile mineral o i l and stored in closed screw-cap tubes at 4 C. Mycelium from a slant was transferred to a Sabouraud agar plate (neopeptone-dextrose-agar, 10 gm--~40 gm —15 gm) and incubated at room temperature for approximately two weeks. After growth, this plate culture served as a "working plate". Mycelial tissues from the working plate were transferred to a sterilized Waring Blendor and homogenized 17 for 30 seconds with sterile d i s t i l l e d water (50 ml for half a plate). Ten ml of the resulting suspension was pipetted under ste r i l e conditions into a 500 ml Erlenmeyer flask, containing 100 ml A-medium: or B-medium. The culture was incubated for 5 days at 23+1 °C on a reciprocal shaker (80 rpm) and served as "liquid inoculum". The mycelial pellets of this liquid inoculum were aseptically transferred from flask to a sterile Waring Blendor and homogenized for 30 seconds. Five ml aliquots were added to 500 ml Erlenmeyer flasks containing 100 ml of A-medium or B-medium. To investigate the effect of tryptophan, the liquid inoculum was homogenized as above and a five ml portion added to A'-medium or B'-medium. A l l cultures were incubated at 23+1°C on a reciprocal shaker (80)rpm) and illuminated for 8 hours a day. Cultureswwerehharvested on alternate days five to fifteen days after inoculation. C. Analytical methods 1. Preparation of samples for chromatography: Six replicate cultures were sampled each time. Each culture was f i l t e r e d through four-layered cheesecloth and the mycelial pellets washed thoroughly with d i s t i l l e d water and freeze dried (VIRTIS) for two days. The weight of the freeze-dried pellets was recorded. Using a Waring Blendor, freeze-dried pellets were homogenized with d i s t i l l e d methanol and then decanted into a 500 ml Erlenmeyer flask. The blender was rinsed with methanol three times and the washings combined with the homogenate and the flask covered with a paraffin membrane. The flask was agitated on the rotary 18 shaker (80 rpm) overnight. The suspension was f i l t e r e d with suction through a Buchner funnel and the residue washed with methanol three times, the washings being added to the f i l t r a t e . The methanolic extract was transferred to a round bottom flask and evaporated to dryness in vacuo at 40 °C and the residue redissolved i n 1-2 ml methanol. A 1 x 5 cm column of ion exchanger (Dowex 50 WX-8(H^ ')) was washed in sequence with 4 ml 1 N NaOH, 8 ml d i s t i l l e d water, 4 ml 1 N HC1, 8 ml d i s t i l l e d water and 5 ml 80% methanol. The concentrated methanolic extract was added to the top of the column and washed in with 5 ml 80% methanol. The effluent was discarded since i t did not show psilocybin-type ultraviolet absorption. The column was then eluted with 20 ml of 5% concentrated NH.OH in 80% methanol. The 4 solvent was evaporated and the residue dissolved in 0.6 ml d i s t i l l e d methanol. Extracts prepared in.this manner were stored in a r e f r i g -erator un t i l used in chromatographic and UV spectral analyses. The f i l t e r e d medium of A and-B media, after determination of the pH was extracted with ethyl ether. The ethereal layer was tested for antibiotic activity after concentration to 5 ml (see below) and the aqueous phase was evaporated to dryness in a rotatory evaporator at 40 °C under reduced pressure. The residue was extracted with 2-5 ml portion of d i s t i l l e d methanol and transferred to a small v i a l . After standing for a while, the solution was refiltered through cotton and evaporated to dryness. Using 1 ml d i s t i l l e d methanol, the residue was redissolved and passed on to the cation exchanger as above and the f i n a l concentrated extract (0.6 ml) was stored until ready to use. 19 A 10 ml aliquot of the f i l t e r e d medium (A'-medium or B'-medium), after determination of the pH, was used for determination of trypto-phan concentration. The remainder of.the medium was evaporated and treated as above for medium A or B, i.e. subjected to ion exchange, fractionation.etc. Location and identification of.Ehrlich-positive compounds on chroma-tograms Two directional thin layer chromatography (2-D TLC) was employed. 2 A 30 ul aliquot of extract was spotted on a 10 x 10 cm cellulose plate (Eastman Chromatogram 13254 cellulose) and the plate developed in two directions using n-Butanol-Acetic acid-Water (BAW, 4:1:1) followed by Isopropanol-Ammonium hydroxide-Water (IAW, 8:1:1). After drying, the plate was examined for fluorescence withwaishort".wave- ; length UV lamp before spraying with Ehrlich reagent (1 gm p-dimethyl-aminobenzaldehyde dissolved in a mixture of 75 ml d i s t i l l e d methanol and 25 ml cone. HCI). Isolation and purification of psilocybin Descending paper chromatography was employed. An aliquot of a suitable extract (200 ul) was streaked on Whatman #1 chromatography paper and an aliquot of 50 ul was spotted with authentic psilocybin solution.^. In addition, authentic psilocybin was spotted alone. The paper was developed in the BAW system overnight. The developed chromatogram was air dried and examined for fluorescent compounds with UV light. The band corresponding to reference psilocybin was eluted with 80% methanol and the eluate concentrated under reduced pressure. It was streaked on paper again and developed with solvent IAW overnight. The band migrating at the same rate as reference 20 psilocybin was eluted as above and the eluate evaporated to dryness and the residue redissolved in 1 ml methanol. The ultraviolet absorp-tion spectrum of the solution was obtained (UNICAM sp 800) and the optical density at 267 nm was recorded (UNICAM sp 500 PYE Series 2). The concentration of psilocybin could then be calculated from a standard curve of psilocybin in which optical density was plotted against concentration. This was linear over the range from 0 to 50 ug/ml. 4. Determination of tryptophan concentration in A' and B' media Tryptophan- was assayed in the aqueous medium f i l t r a t e by the Spiess and Chambers (42) method. Eight ml of 23.8 N l^SO^ and 1 ml of 2 N H^ SO^  containing 30 mg of p-dimethylaminobenzaldehyde were o mixed and.cooled to 25 C. To this solution was added 1 ml of medium f i l t r a t e . The mixture after shaking and cooling to 25 C was kept in the dark for one hour after which 0.1 ml of 0.04% NaM^ solution was added. The mixture was shaken and the color allowed to developififor 30 minutes at room temperature in the dark. The optical density at 600 nm was read and converted to the concentration of tryptophan from standard curves. D. Radioautography 14 The fate of C -labeled tryptophan in the fungal cells was investi-gagated by radioautography of the chromatographically separated compounds in the fungal extracts. 14 D,L-Tryptophan, side chain-3-C , 5uCi/250 mg/100 ml B-medium was added aseptically to a 3-day-old culture. Three flasks were used. After 48 hours, the pellets were collected and the medium f i l t r a t e 21 processed as above. The extracts were spotted on paper together with authentic tryptophan metabolites and developed i n two directions, BAW followed by IAW. After drying and examination under UV light, the chromatogram was exposed to Kodak X-ray film (Blue brand) for two weeks. The film was developed in Kodak X-ray developer for 4-5 minutes, washed with dilute acetic acid and transferred to a fixative solution for about 10 minutes. After washing i n running tap water for 20-30 minutes i t was dried and examined. The dark spots on the radioautograph represented the radioactive substances on the chromatogram. Radioactive spots were eluted with 80% methanol and the eluates evaporated to dryness under reduced pressure at 40 °C. The residue was redissolved in 0.7 ml methanol and pipetted into a s c i n t i l l a t i o n v i a l . A 9.3 ml aliquot of s c i n t i l l a t o r (Aquasol) was added and the mixture counted in a Searle Isocap/300. E. Psilocybin phosphatase activity Method 1: Enzyme extract: A l l extraction steps were carried out in a cold room. Eight-day-old cultures from A ordB medium were collected and the pellets thoroughly washed with d i s t i l l e d water after determination of the fresh weights, the pellets were homogenized in a chilled Waring Blendor for about 30 seconds with glycine-NaOH buffer (pH 9.0, contain-ing 2 mM mercaptoethanol) and polyclar AT (0.1% of fresh weight) was added. The slurry was f i l t e r e d through 4 layers of cheesecloth and the suspension centrifuged at 7,000 g (SORVALL RC 2-B) for 15 minutes. The supernatant was used as crude enzyme extract. Enzyme assay: Psilocybin aqueous solution (1.76 mM) was used as 22 substrate. An aliquot of 0 . 5 ml crude enzyme extract and 0 . 4 ml p s i l o c y b i n s o l u t i o n were incubated at 3 0 °C f o r 3 0 - 9 0 minutes and 0 . 5 ml of the reagent (2';gm p-dimethylaminobenzaldehyde dissolved i n the mixture of 65 ml cone. U^SO^ and 35 ml H^ O containing 0 . 1 ml 3% FeCl^) was ,]) added to the test.tube a f t e r the appropriate time ( 2 9 ) . The mixture was s t i r r e d and i r r a d i a t e d f o r 15 minutes under an U V lamp. The o p t i c a l density of the s o l u t i o n which turned purple on i r r a d i a t i o n , was deter-mined at 5 7 0 nm. A blue product (max. absorption at 6 3 0 nm) was always produced during the incubation, however, and t h i s color i n t e r f e r r e d with the purple color developed by p s i l o c y b i n . The concentration of p s i l o c y b i n could not be measured and t h i s method was therefore abandoned. Method 2 : Enzyme extract: A l l the extraction procedures were s i m i l a r to those described in.Method.1 except that a succinate-NaOH buffer (pH 5 . 0 , containing 2 mM mercaptoethanol and 0 . 0 0 1 M NaCN) replaced the glycine-NaOH buffer. Enzyme assay: Three substrates were employed i n t h i s assay. Phenolphthalein diphosphate (PDP, 0 . 1 M, 0 . 1 ml) was added to 1 ml of enzyme extract and incubated at 3 4 *C. A f t e r an appropriate incubation period, 3 ml of 2 M (NH^^CO^ was added and the absorbance of the red color which developed was measured at 5 4 0 nm. P s i l o c y b i n ( 1 . 7 6 mM, 0 . 4 ml) was added to 1 ml of enzyme extract and the reaction mixture was incubated at 34°C. A f t e r an appropriate incubation period, 5 ml of n-butanol was added and the p s i l o c i n l i b e r a t e d was extracted and i t s concentration determined. P s i l o c i n aqueous s o l u t i o n ( 2 . 4 6 mM, 0 . 4 ml, prepared freshly) was added to 1 ml of enzyme extract and the 23 mixture was incubated at 34 °C. After an appropriate incubation.period, 5 ml of n-butanol was added and the residual psilocin was extracted and i t s concentration determined. Colorimetric analysis for psilocin: To the reaction mixture (enzyme extract, substrate and 5 ml of n-butanol), 0.5 ml borate buffer (KCl-H 3B0 3, pH 9.0) and 1 gm NaCl were added (33, 43, 44). The test tube was shaken and then.the supernatant was pipetted into a centrifuge tube. The organic phase was washed by shaking with 2 ml borate buffer and the butanol phase was transferred to another centrifuge tube containing 5 ml heptane and 0.4 ml 0.1 N HCI. The mixture was shaken. The super-natant was discarded. To the acidic phase, 1 ml of l-nitroso-2-naphthol reagent (0.1% l-nitroso-2-naphthol in 95% ethyl alcohol) and 1 ml of nitrous acid reagent (0.2 ml of 2.5% NaN02 in 5 ml of 2 N l^SO^, pre-pared freshly) were added. The mixture was stirred and incubated in a water bath at 55 C for 5 minutes. Ten ml of ethylene dichloride was added and the tube was centrifuged at a low speed for 5 minutes and the absorbance at 430 nm of the supernatant (brownish orange color) was recorded. The psilocin concentration was calculated from a standard curve. This was linear over the range from 0 to 0.5 mg/ml. Protein concentration was determined by the method of Lowry ejt a l . (45). Enzyme activity was expressed as mg psilocin released/mg protein. F. Potassium nutrition and psilocybin production Two treatments were studied, potassium deficiency and high levels of potassium. Replacement cultures were used. K +-deficiency: Instead of 1 gm of KJ^PO^ as used in B-medium, 1 gm NaH0PO/ and 20 mg KHoP0/i were used. After growing for 5 days, the pellets 24 were harvested and the concentration of. psilocybin determined. K+-?supplement: In addition to 1 gm KR^PO^ as used in B-medium, 2 gm of KCI was added. After growing for 5 days, the pellets were collected and the concentration of psilocybin determined. A culture growing in B-medium served as control. G. Test of UV-mediated antibiotic and phototoxic activities of medium, psilocybin and psilocin A Candida albicans assay was employed (46) . Candida albicans was streaked on Sabouraud's agar plate with sterile cotton swabs in one direction and cross-wise in other direction. Several small f i l t e r paper discs were sterili z e d . About 200ul of an ethereal extract of medium was dropped on to the paper disc and allowed to dry. Discs were placed uniformly around the agar plate so that zones of inhibition would not overlap. A control consisting of solvent alone and a sample of 8-methoxy-psoralen, a known phototoxic compound was also included in the test. Test plates were incubated at room temperature for at least 12 hours under an UV lamp. Addtiplicate plate was incubated in the dark at room temperature. Extracts which caused a greater area of clearing after light incubation than after dark were termed phototoxic. Those extracts which caused the k i l l i n g of Candida in both light and dark were termed antibiotic. Authentic crystals of psilocybin and psilocin were tested as above. 25 EXPERIMENTAL AND RESULTS Chromatographic separation and identification of tryptophan metabolites The color reactions, Rjl values and fluorescence colors in UVor, of f 254 some tryptophan metabolites are li s t e d in Table 5. Figure 1 i s a map showing the location of these compounds on a two-dimensional chromatogram using the BAW and IAW solvent systems. Table 5. Fluorescence and Color Reactions of Some Tryptophan Metabolites Color reactions Fluorescence color in UV Compounds 254 Ehrlich Ninhydrin 1. L-Tryptophan 2. 5-OH-Tryptophan 3. Tryptamine 4. 5-OH-Tryptamine (Serotonin) 5. N-Methyltryptamine 6. N,N-Dimethyltryptamine 7. Psilocybin 8. Psilocin 9. Kynurenine 10. Kynurenic acid 11. 3-Methylindole 12. Indole-3-ethanol 13. N-Acetyltryptophan 14. Allantoic acid 15. Allantoin 16. Anthranilic acid 17. 3-0H-Anthranilic acid 18. Urea 19. Indole-3-acetic acid dark blue black dark' blue dark blue dark blue yellowish blue dark blue dark blue azure yellowish blue dark blue dark blue dark blue light blue light blue grey brownish green brownish purple brownish purple purple purple violet purple purple violet violet purple brownish purple pink blue orange purple violet violet purple -bright yellow yellow bright . yellow yellowish -orange bright yellow -brown 26 Table 5 (continued). Rf Values of Some Tryptophan Metabolites Cellulose plate Whatman #1 paper # Compounds BAW LAW BAW IAW 1. L-Tryptophan 0.44 0.24 0.39 0.26 2. 5-OH-Tryptophan 0.18 0.15 0.12 0.16 3. Tryptamine 0.68 0.75 0.61 0.73 4. 5-OH-Tryptamine 0.39 0.57 0.34 0.49 5. N-Methyltryptamine 0.78 0.86 0.65 0.81 6. N,N-Dimethyltryptamine 0.78 0.95 0.68 0.86 7. Psilocybin 0.38 0.02 0.31 0.06 8. Psilocin 0.72 0.90 0.62 0.82 9. Kynurenine 0.37 0.26 0.26 0.25 10. Kynurenic acid 0.53 0.55 0.55 0.40 11. 3-Methylindole 0.96 0.98 0.92 0.88 12. Indole-3-ethanol 0.93 0.92 0.87 0.85 13? «NzrAeetyit ryp t ophan- 0.90 0.60 0.87 0.60 14. Allantoic acid o.nio 0.02 0.16 0.15 15. Allantoin 0.26 0.07 0.18 0.13 16. Anthranilic acid 0.91 0.46 0.85 0.39 17. 3-OH-Anthranilic acid 0.80 0.33 0.76 0.27 18. Urea 0.4'6 0.44 0.45 0.50 19. Indole-3-acetic acid 0.86 0.51 0.86 0.39 B. pH, Growth and morphological difference of cultures Cultures of the organism in A, A', B and B' media were harvested on alternate days, five to fifteen days after inoculation. The pH of the medium and the dry weight of the mycelium were determined at the termination of this experiment. The results are summarized in Figures 2, 3, 4 and 5. These data revealed that the pH of B and B' media remained acid in the region of 5.35-5.55 through the whole growth period. The pH of the A and A' media decreased gradually from the f i f t h day to the ninth day. A f t e r the nint h day, the pH of the A'-medium increased gradually to 7.35 on the f i f t e e n t h day. The pH of the A-medium, a f t e r the ninth day, increased r a p i d l y u n t i l a plateau was reached near pH 7.4. The dry weight of mycelia from A and A' media increased l i n e a r l y from the f i f t h day to the nineth day and continued to increase to a maximum on the eleventh day. There was then a rapid decrease i n the dry weight and a f i n a l l e v e l l i n g o f f . The growth rate was poor i n e i t h e r B or B' medium i n comparison with A and A' media. The dry weight of mycelia from B-medium gradually increased u n t i l the eleventh day and then leve l e d o f f . The dry weight of mycelia from B-medium increased slowly i n the beginning and then r a p i d l y increased u n t i l a plateau was reached. The mycelial p e l l e t s growing i n A or A' medium were round, smooth and bigger compared with those i n B or B' medium. P e l l e t s i n B or B 1 medium were fuzzy. The p e l l e t s of e a r l i e r stages from A-medium were b l u i s h green i n color and gradually turned dark grey. P e l l e t s from A'-medium were also b l u i s h green color at an e a r l i e r stage, but they turned brown to dark brown a f t e r eleven days. The color of A'-medium f i l t r a t e at a l a t e r stage was dark brown while A-medium f i l t r a t e remained yellowish. The p e l l e t s of e a r l i e r stages from B and B' media were s i l v e r y grey. At a l a t e r stage, p e l l e t s from B-medium were s t i l l s i l v e r y grey while those from B'-medium were dark brown. The B-medium f i l t r a t e gave a dark brown color at a l a t e r stage and B-medium f i l t r a t e remained yellowish. P s i l o c y b i n production P s i l o c y b i n was i d e n t i f i e d by chromatography, color reactions and i t s UV spectrum. The spectra of authentic p s i l o c y b i n and that i s o l a t e d from 28 fungal c e l l s are shown i n Figures 6 and 6'. P s i l o c y b i n was produced on both A and B media. Media to which tryptophan had been added gave better y i e l d s i n the e a r l i e r periods but decreased l a t e r . P s i l o c y b i n was never detected i n the medium. The data are shown i n Figures 7 and 8. I t can be seen that the fungus produced p s i l o c y b i n very early and that maximum production was on the f i f t h day. D. Formation of p s i l o c y b i n and p s i l o c i n from tryptophan 14 D,L-Tryptophan, side chain-3-C , was administered to 3-day-old cultures f or 48 hours. A f t e r treatment, extracts of the p e l l e t s and the medium were chromatographed two-dimensionally and the chromatograms radioautographed. In extracts of the medium, no radioactive metabolites were i d e n t i f i e d d on chromatograms, the bulk of the r a d i o a c t i v i t y being i n unchanged tryptophan. In extracts of p e l l e t s , p s i l o c y b i n and p s i l o c i n were labeled although most of the r a d i o a c t i v i t y was s t i l l i n unchanged tryptophan. P s i l o c i n was only f a i n t l y d i s c e r n i b l e on radioautograph. The d i s t r i b u t i o n of r a d i o a c t i v i t y i n tryptophan, p s i l o c i n and p s i l o c y b i n i s shown i n Table 6. A photograph of the radioautograph of the mycelium extract i s present i n Figure 9. Table 6. D i s t r i b u t i o n of R a d i o a c t i v i t y i n Tryptophan, P s i l o c i n and P s i l o c y b i n of Mycelium Extract, Administered D,L-Tryptophan, side chain-3—C ,14 Percentage D i s t r i b u t i o n of R a d i o a c t i v i t y Tryptophan 0.65 P s i l o c i n 0.004 P s i l o c y b i n 0.062 29 0.9 0.7 10..S 4-0.3 + 0.1 + + 16 + 19 + 13 + 17 + •5 + 12 + 6 + 3 + 8 + 10 + 7 1 + 9 + 18 + 4 15 + 2 14 e °-l 0.3 0.5 0.7 0.9 Figure 1. Location of some standard tryptophan metabolites on a two-dimensional cellulose thin-layer plate. Numbering of spots corresponds to that in Table 5 3 0 l_7y 1 1 1 — : 1 1 ,_ 0 5 7 9 1 1 1 3 1 5 days Figure 2. pH of A and A' media 31 Figure 3. pH of B and B' media 32 Figure 4. Growth, rate of fungal c e l l s i n A and A' media 33 Figure 5. Growth"rate of fungal c e l l s i n B and B' media wavelength millimicrons Figure 6. UV spectrum of authentic p s i l o c y b i n 200 225 250 275 300 325 3* w a v e l e n j ^ Figure 6'. UV spectrum of p s i l o c y b i n i s o l a t e d from fungal c e l l 36 Figure >7-. Psilocybin production from A and A' media 37 A B-Medium • L-// 1 1 I \ \ h 0 5 7 9. . 11 13 .15 days Figure 8'. P s i l o c y b i n production from B and B' media 38 Trp -Pb -Pc -Tryptophan P s i l o c y b i n P s i l o c i n 39 Phosphatase activity Incubation of psilocybin or psilocin with a crude enzyme extract resulted in the formation of a.blue color usually after 5-15 minutes incubation and gave a linear rated over 180-minute-period (Figure 10). The addition of NaCN was effective in delaying the blue color formation, but did not influence the phosphatase activity'. The dephosphorylation of PDP by a crude enzyme extract proceeded at a rapid rate as shown the liberation of phenolphthalein (30-120 seconds for enzyme extract A and 5-25 minutes for enzyme extract B). The dephos-phorylation of psilocybin was rapid for both enzyme extracts. The blueing phenomena occurred in 3-5 minutes incubation in enzyme extract A and after 15 minutes in enzyme B. The liberation of psilocin reached a maximum just before the reaction mixture turned blue (Figure 11). When the psilocin was used as substrate, i t s concentration remained unchanged before i t turned blue. Partial purification of enzyme was attempted. The fraction which precipitated with ammonium sulfate^ at a concentration of 40-70% showed most activity. Psilocybin production.and potassium nutrition The pH, growth and psilocybin content of 5-day-old cultures of K+-deficient, K+-supplement and control conditions were compared. The data are indicated in Table 7. Table 7. The Growth, pH of Medium and Psilocybin Production of B-medium, Potassium Deficient and Supplement Media B-medium K +-deficient K+-supplement pH £.533 E 5.3 5.45 Growth 70.0 78.1 70.0 mg/100.ml medium Psilocybin 3.51 1.92 3.51 mg/g dry wt 0. D. at 630 nm - Figure ,10. Blue color formation when.psilocybin incubated with "enzyme extract 41 O A-Medium A B-Medium i i i i 0 5 10 15 20 . minutes Figure H-. A c t i v i t i e s of a c i d phosphatases of the mycelia from two d i f f e r e n t media 42 G. Ehrlich-positive compounds in extracts Diagrams of chromatograms visualized with Ehrlich.reagent are shown in Figure 12. Spot Y, near the origin gave a yellow color after spraying with Ehrlich reagent and a purple color with Ninhydrin reagent. On chroma-tograms of extracts of mycelium of the thirteenth day and the fifteenth day of growth on B'-medium, there was a spot Z close to psilocin which gave a purple color with either Ehrlich or Ninhydrin reagent. Attempts were made to identify these two compounds. After streaking and eluting in two solvent systems, the R^  values of spot Y were 0>.ll and 0.19 in BAW and IAW respectively. It was identified as glycine on the basis of i t s color reactions and co-chromatography with this amino acid. The R^  values of spot Z were 0.61 and 0.73 in BAW and IAW respectively. A methanolic solution of this compound gave an UV spectrum of an indolyl derivative with maximum at 290, 281, 270 and 220 nm. The R^  values and color reactions with Ehrlich and Ninhydrin reagents were the same as those of tryptamine and i t was identified as such. H. Tryptophan concentration in the medium The tryptophan concentrations of the f i l t r a t e s of medium A' and B' were determined. The data are indicated in Figure 13. Tryptophan was uti l i z e d by fungal cells more in A'-medium than in B-medium. From the beginning to. the f i f t h day, there was a great decrease in tryptophan in both media. After five days, tryptophan levels i n B'-medium did not changed too much, while in A-medium, a slow decrease was followed by a sudden drop and a levelling off. Figure 12. Chromatograms of mycelium extract and medium extract of d i f f e r e n t growth period 44 0 mycelium extract p s i l o c y b i n p s i l o c i n tryptophan kynurenine a n t h r a n i l i c a c i d unknown yellow spot unknown spot, no color reaction with E h r l i c h reagent, showing l i g h t blue fluorescence 45 z unknown purple spot 46 48 L-// 1 1 1 1 1 f-0 5 7 9 11 13 15 days Figure 13.. Tryptophan concentration of A' and B' media 49 I. Antibiotic activity of psilocin, psilocybin and medium f i l t r a t e Neither A nor B medium f i l t r a t e showed antibiotic activity. The characteristic polyacetylenic UV spectra in which normally two sets of absorption bands are observed were not obtained. Authentic psilocybin crystals did not show any antibiotic activity towards Candida albicans while psilocin showed only very slight activity. With psilocin, there was a clear zone in light and in dark. The control, 8-methoxypsoralen gave a clear zone in UV light but not in the dark. The agar plate indicating the activity i s diagrammed in Figure 14. J. Identification of D(-)mannitol Mycelial extracts from B-medium usually gave a deposit of white crystals after evaporation of the methanol. The crystals melted at 164.5 °C and the IR spectrum (UNICAM 200G) proved to be that of a sugar alcohol (Figure 15). The acetate derivative was prepared with anhydrous pyridine and acetic anhydride (47) and the NMR spectrum was taken (EM-390 90 MHz NMR Spectrometer)(Figure 16). The IR and NMR spectra were found to be identical to those of D(-)mannitol. 50 Figure 14, Antibiotic activity of psilocin (shaded area covered with Candida albicans) Figure 15. IR spectrum of white crystals Figure 16. NMR spectrum of acetate derivative of white crystals 54 DISCUSSION It was noticed that only when the fungus was maintained on Sabouraud agar plates^ (neopeptone-dextrose, 10 gm-r-40 gm). would, i t produce psilocybin on subsequent, transfer, to . liquid, medium. ... If ..it was kept on malt extract-yeast-soytone agar plate (MYP, 7 gm—0.5 gm—1 gm) , i t did not produce psilocybin . even after a 15-day-period of incubation and the c e l l extracts gave an UV spectrum of ergosterol (Figure 17) instead of that.of an indole. When fungus was kept on MYP agar plates, A-medium without yeast extract and A-medium without glycine were tried. However, there was no indole metabolites visual-ized from chromatograms and the UV absorption spectra. However, this does not preclude the possibility that the fungus when grown on MYP agar w i l l eventually produce psilocybin i f a longer incubation is allowed. Catalfom6 and.Tyler (1) as well as Leung (12) maintained their cultures on potato-dextrose agar plates (PDA, 200. gm—15 gm) and succeeded in obtaining psilocybin. The sugar contents of either Sabouraud or PDA is larger that that of MYP. Amici e_t a l . (48) found that the capacity to produce ergot alkaloids is correlated with the uti l i z a t i o n of large amounts of sucrose and c i t r i c acid. Amici suggested that the simultaneous utilizationcolf sucrose and c i t r i c acid i s favorable to the accumulation of "primary precursors" as, for instance, acetyl CoA and phosphoenolpyruvic acid. Catalfomo and Tyler found that higher levels of glucose resulted in higher yields of psilocybin in Psilocybe cubensis. Abe was also successful in promoting alkaloid synthesis in Claviceps by using a medium containing high concentration of sucrose and mannitol (49, 50). The nitrogen source is also important in revealing alkaloid-producing strains (49). The differences in nitrogen sources in MYP and Sabouraud may also account for the alkaloid production. After trying cellulose and s i l i c acid TLC.and paper chromatography for 56 quantitative analyses, paper chromatography was f i n a l l y selected because of reproducibility, although the recoveries were low (47+5%). Psilocybin i s sensitive to shorter wavelength UV light and authentic psilocybin on paper turned pale yellow (without changing i t s UV spectrum) i f i t was exposed to UV light for more than 1 minute. S i l i c acid plates, although they gave better resolution, led to the decomposition of the indole compounds when they were developed in the solvent systems used here. In conclusion, methods of purifying psilocybin are too long to allow rapid screening of different media. Recently, a sensitive GLC-Mass spectral analysis of psilocybin and psilocin was reported by Repke et a l . (51) and this appears to be the method of choice. Unfortunately the instrumentation was not available for this study. '.'VV • . " - l i f f t o u d c & d Psilocybin was produced early in the development of the culture in both A and B media. This might be expected because i t s formation does not i involve condensation with other carbon compounds such as amino acids or isoprene units which are involved in ergot alkaloid biosynthesis (52). The formation of the latter type of compound requires concerted condensations and perhaps the formation or the action of such enzymes i s suppressed by primary metabolites (52) so that the biosynthesis of these compounds occurs only i n the idiophase of growth. Fluctuating rates of psilocybin production could be seen in both A and B media. Rahacek et a l . (53) suggested in Claviceps that alkaloid production commences while tryptophan synthetase activity is increasing. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan levels in balance. This could partially explain the fluctuation of psilocybin production in A and B media. Another reason for these fluc-tuations could be the heterogenous growth of the mycelium. Comparing the . 57 y i e l d of p s i l o c y b i n on two d i f f e r e n t media, fungus from B-medium gave a better y i e l d but a poorer growth rate. Abe and co-workers stress that the fact that slow growth i s required f o r a l k a l o i d production (54). Demain (55) suggested that rapid growth during trophophase involves a balanced assimi-l a t i o n of a l l e s s e n t i a l nutrients with a minimum of accumulation of meta-b o l i t e s . By v i s u a l comparison of color i n t e n s i t i e s and s i z e of spots on chromato-grams, the p e l l e t s from A-medium were found to contain more p s i l o c i n than those from B-medium. The p e l l e t s from the B-medium were l e s s b l u i s h green than those from the A-medium. The a c t i v i t i e s of acid phosphatases from these two cultures were quite d i f f e r e n t . Enzyme extract from A cultures dephosphorylated p s i l o c y b i n very r a p i d l y and gave a blue color a f t e r f i v e minutes incubation. The enzyme preparation from B cultures gave a blue color only a f t e r f i f t e e n minutes. The cause of the blue color has not been explained s a t i s f a c t o r i l y . I t i s suggested to be an enzymic oxidation (32, 34, 35, 37, 38, 44, 56) by some while, others i n d i c a t e that oxygen i s not required (57). The blue color can be e l i c i t e d without enzyme, i n the presence of f e r r i c ion (35) and i t can be blocked by EDTA. I t has been shown for several b a c t e r i a l enzymes that the rates of enzyme synthesis are subject to regulation e i t h e r by s p e c i f i c "inducer" substances or by s p e c i f i c "repressor" substances. The amount of a l k a l i n e phosphatase i n E_. c o l i was markedly reduced when there was an excess of phosphate a v a i l a b l e (75, 76) and phosphate was demonstrated to beraprepressof of a l k a l i n e phosphatase formation (75, 76, 77). The amount of acid phosphatase formed i n _E. c o l i was independent of phosphate concentration (75). That phosphate ion i n h i b i t s various acid and a l k a l i n e phosphatases has been reported (72, 75, 77). Neal et a l . (27) who worked on the r e l a t i o n s h i p s 58 between phosphate n u t r i t i o n and accumulated p s i l o c y b i n found excess phosphate i n the mycelia grown i n phosphate-rich medium. Comparing the phosphate . content of two media used here, B-medium had more than A-medium. The dif f e r e n c e i n phosphatase a c t i v i t y could be due to higher endogenous phosphate concentration i n B culture i n h i b i t i n g the enzyme a c t i v i t y . In the present studies, 8-day-old cultures s t i l l had a creamy white appearance. Af t e r eight days, the p e l l e t s i n A-medium gradually turned blue. The blue color would not be produced a f t e r eight days, however, i f shaking of the culture was stopped. Acid phosphatases have been reported to occur i n fungicsuch as A s p e r g i l l u s (58). Wakao et a l . (58) have shown that two types of acid phosphatases i n A s p e r g i l l u s oryzae located either i n the c e l l w a i l or the cytoplasmic membrane. The p e l l e t s used here for enzyme prepara-tions were white before extraction. A f t e r homogenization the insoluble residue was quite greenish. It would seem therefore that some of the acid phosphatases are located on the c e l l w a l l . The occurrence of acid phosphatases could possibly explain the decrease of p s i l o c y b i n production a f t e r nine days. P s i l o c i n aqueous s o l u t i o n should be prepared f r e s h l y or d'ferturnsn. brown e a s i l y . L-Tryptophan does not appear to have a clear-cut r o l e i n regulating p s i l o c y b i n biosynthesis i n a l l species which produce i t . Several workers have shown (1, 12) that there i s no consistent r e l a t i o n s h i p between tryptophan addition and p s i l o c y b i n production. In t h i s work, L-tryptophan stimulated p s i l o c y b i n production by almost a f a c t o r of two i n the very beginning i n B'-jmedium. On the other hand, in- Aiamedium, the stimulation was not so s i g n i -f i c a n t . In the study of Claviceps s t r a i n 47 A (78), the r e s u l t s indicated that additions of D,L-phehylalanine Tfavorablyfinfluenced3ergot a l k a l o i d accumulation. 59 Sucrose was also noted to stimulate the accumulation of alkaloids in strain 47 A. Additions of sucrose and D,L-phenylalanine were synergistic in their influence i f the phosphate concentration was high (0.1%), but added amino acid had l i t t l e influence in the presence of a low phosphate concentration (0.01%). Brady and Tyler (78) suggested that at least two limiting factors are involved in clavine alkaloid biosynthesis. One of the limiting factors is apparently an aromatic amino acid or a related metabolic substance; the other appears to be a component associated closely with carbohydrate meta-bolism. Floss and Mothes (59) showed that for the maximum effect on ergot alkaloid production of adaaftfg tryptophan, i t must be done at the beginning of the fermentation long before alkaloid synthesis begins. They concluded that this "training" phenomenon involved some kind of inducer action and suggested de-repression or activation of the enzyme system as the most li k e l y mechanism (59, 60). Whether or not tryptophan acts as an'inducef ofetheVehzymei necessary for psilocybin production has not been investigated. B-medium seems to be a promising medium to grow Psilocybe cubensis from the point of view of low acid phosphatase activity and tryptophan enhancement in early stage. It was observed (see Figure 12) that the residual tryptophan concentration in the medium fluctuated with the age of the culture. Teuscher reported (61) that there exists a proportionality between uptake and degradation of trypto- . phan. Besides the participation in alkaloid biosynthesis, many other path-ways of tryptophan metabolism are operative in fungi and plants as shown in Scheme IV (62). In A and A' media, the fungus metabolized tryptophan mainly through kynurenine and anthranilic acid; while in B'-medium instead of kynurenine, tryptamine was formed. Kaplan et a l . (63) concluded that the degradation of tryptophan by the kynurenine pathway did not play a s i g n i f i -60 Aromatic NAD pathway Quinaldine pathway pathway jSiAD Scheme IV. Outline of Tryptophan Metabolism in Plants and Microorganisms. 61 cant r o l e i n the metabolism of tryptophan, under condition of high a l k a l o i d production i n Claviceps s t r a i n SD 58. There i s some.evidence (49) that the fate of tryptophan added to culture i s d i f f e r e n t i n ergot a l k a l o i d producing and non-producing s t r a i n s . The formation of a n t h r a n i l i c a c i d was observed i n a culture which f a i l e d to produce a l k a l o i d . The appearance of kynurenine and a n t h r a n i l i c a c i d i n the l a t e r stage of growth might also explain the decrease i n psilocybin.production. A f t e r feeding labeled D,L-tryptophan to Psilocybe cubensis grown on B-medium, three radioactive spots were i d e n t i f i e d by radioautograpfty as tryptophan, p s i l o c y b i n and p s i l o c i n although the l a s t one was only f a i n t l y d i s c e r n i b l e . The percentage incorporated i n t o p s i l o c y b i n was 0.062 here and 0.43 i n the report of A g u r e l l (exposed f o r 5-6 days) (29) and 7.97 i n the work of Brack et a l . (exposed for 60 days) (23). The diamine, putrescine (NH 2CH 2CH 2CH 2CH 2NH 2) i s well-established as a constituent of microorganisms, animals and plants (64). The l e v e l of putre-scine i s greatly increased i n potassium-deficient plants (65, 66). Coleman et a l . (66) suggested that b a s i c i t y may conceivably help to explain why t h i s p a r t i c u l a r substance accumulated i n potassium st a r v a t i o n f o r i f a l k a l i metal deficiency s h i f t s the i n t e r n a l balance between inorganic anions and cations i n the d i r e c t i o n of. increased a c i d i t y , basic metabolites maiy become important as compensatory factors and t h e i r metabolic s t a b i l i t y increased. The feeding of hydrochloric a c i d also caused s i g n i f i c a n t increases i n amines such as arginine and putrescine content (67) . In t h i s work, potassium d e f i c i e n t condition decreased the p s i l o c y b i n y i e l d without changing growth or.medium pH. Potassium supplement had no ef f e c t at a l l . Vining and Nair, however, showed (50) that increasing the concentration of potassium caused an increased i n a l k a l o i d production i n 62 Claviceps. 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Some aspects of n i t r o g e n metabolism i n b a r l e y and other p l a n t s i n r e l a t i o n to potassium d e f i c i e n c y . Annals of Botany 20: 393-409. 67. Smith, T. A. and S i n c l a i r , C. (1967). The e f f e c t of a c i d feeding on amine formation i n b a r l e y . Annals of Botany 3_1: 103-111. 68. S t e i n b e r g , R. A. (1951). M i n e r a l n u t r i t i o n of p l a n t s . Ed. by E. Truog, Univ. of Wisconsin Press, pp. 359-386. 69. Yu, C. J . (1959). The C o n t i n e n t a l Magazine _19(8): 1-4. Taiwan, ROC. 70. H o f f e r , A. and Osmond, H. (1967). The h a l l u c i n o g e n s . Academic Press, pp. 480-500. 71. Hoffmann, A., Heim,-R. , Brack. A., Kobel, H., Frey, A., Ott, H., .. P e t r z i l k a , Th. and T r o x l e r , F. (1959). P s i l o c y b i n und P s i l o c i n , zwei psychotrope W i r k s t o f f e aus mexikanischen Rauschpilzen. Helv. Chim. Acta 42: 1557-1572. C i t e d from Chemical A b s t r a c t s 54: 5830g (1960). 68 72. Colowick, S. P. and Kaplan, N. 0. (1955). "Methods in Enzymology". Vol. II, pp. 523-616. Academic Press, N. Y. 73. Brack, A., Brunner, R. und Kobel, H. (1962). Mikrobiologische Hydroxyl-ierungen an Mutterkornalkaloiden von clavine Typus mit dem rcexikanischen Rauschpilz Psilocybe semperviva Heim et Cailleux. Helv. Chim. Acta 45: 276-281. Cited from Chemical Abstracts 57: 6443e (1962). 74. Bandoni, R. J. and Szczawinski, A. F. (1975). "Guide to Common Mushrooms of British Columbia". Published by the British Columbia Provincial Museum, Victoria, Canada. 75. Torriani, A. (1960). Influence of inorganic phosphate in the formation of phosphatase by_E. c o l i . Biochim. Biophys. Acta 3_8: 460-469. 76. Garen, A. and Levinthal, C. (1960). A fine-structure genetic and -chemical study of the enzyme alkaline phosphatase of E.ccoli. Biochim. Biophys. Acta 38: 470-483. 77. Weinberg, E. D. (1973). Secondary metabolism: Control by temperature and inorganic phosphate. Developments in Industrial Microbiology 15: 70-81. 78. Brady, L. R. and Tyler, V. E. Jr. (1960). Alkaloid accumulation in two clavine-producing strains of Claviceps. Lloydia 23: 8-20. 79. Benfield, G., Bocks, S. M., Bromley, K. and Brown, B. R. (1964). Studies of fungal and plant laccases. Phytochem. 3_: 79-88. 68b II. A SURVEY OF PHENOL-O-METHYLTRANSFERASE IN SPECIES OF LENTINUS AND LENTINELLUS 69 INTRODUCTION The Basidiomycete, Lentinus lepideus, is a frequent cause of brown rots in timber. It i s able to attack many woods on account of it s tolerance to comparatively high concentrations of creosote (1). In culture on wood or oh glucose i t frequently produces a strong, sweet, aromatic odor due to the synthesis of methyl cinnamate (I), methyl p-methoxycinnamate (II) and methyl anisate (III). Crystalline deposits of methyl p-methoxycinnamate are often obtained butiif the culture flasks in which the crystals have accumulated are continuously shaken, the amount of the deposit diminishes rapidly and after a few days, almost completely disappears. After 1 or 2 days of shaking, methyl p-coumarate (IV) was present in the medium (2). In surface as well as shaken cultures, the presence of methyl isoferulate (V) has also been established %(3). (I) (II) (III) (IV) (V) When Lentinus lepideus was grown in media containing radioactive glucose, radioactivity was significantly incorporated into (II). It indicated that this compound may be synthesized via shikimic acid (4) and that i t i s not a product of the degradation of lignin (5). Among seven species of Lentinus and Lentinellus, a closely related genus, only Lentinus lepideus and Lentinus ponderosus were found to produce, either in light or dark conditions, methyl esters of phenolic acids (6). Lentinus 70 ponderosus yielded compounds I, II, III and V. Neither L_. lepideus nor _L. ponderosus produced detectable amounts of free p-OH-cinnamic acid (VI), caffeic acid (VII) or isoferulic acid (VIII). OH OH 0CH3 (VI) (VII) (VIII) An enzyme, L-phenylalanine ammonia lyase (PAL) (E.C. 4.3.1.5) which converts L-phenylalanine directly to cinnamic acid with the liberation of ammonia (7) and which often has a similar action on L-tyrosine (TAL) has been found in Basidiomycetes including Lentinus lepideus. In some fungi, or in higher plants, light triggers the appearance of PAL (26). Cinnamic acid i s esterified enzymically to give methyl cinnamate, the methyl donor being S-adenosylmethionine. Methyl cinnamate may undergo one or two hydroxy-lations and the phenols in turn undergo O-methylation. Biological methylations in nature are sufficiently important that special enzymes for carrying out these reactions have been developed during the course of evolution (8). Keller et a l . (9) gave a direct quantitative demonstration that the methyl group was transferred as an intact unit. S-AdenosyMethionine (SAM) has been established as the direct methyl donor and there are more than thirty reactions in which SAM has been demonstrated to serve as a methyl donor (8). The methyl group may be transferred to the nitrogen of primary, secondary or tertiary amines or of heterocyclic comr pounds, the sulfur of thioesters, the oxygen of phenols, carboxylate _ 71 structures and carbon atoms of a number of compounds. The methyl transferases, enzymes catalyzing transmethylation reactions have been found in a wide range of biological forms. In general, each methyl-transferase catalyzes the transfer of a methyl group to only a specific acceptor. For example, Fales et a l . (8, 10) showed that the ratio of para to meta methylation of the diphenol norbelladine was 22:1, when the reaction was catalyzed by a plant enzyme, whereas the ratio was 0.28:1, when the reaction was catalyzed by rat liver catechol O-methyltransferase. A number of enzyme preparations from higher plants have been described which catalyze the transfer of the methyl group of ,SAM to the meta position of v i c i n a l polyphenolic compounds (11, 12, 13, 14, 15, 16, 17). Enzymatic para-O-methylation by catechol O-methyltransferase from the rat was reported (18). The partially purified preparation of norbelladine O-methyltransferase from Nerine bowdenii i s the f i r s t reported cell-free system from a higher plant that catalyzes para-O-methylation predominantly (19). Using the c e l l -free enzyme system of Foeniculum vulgare, 4-OH-cinnamate is methylated to 4-methoxycinnamic acid (20). The partially purified enzyme from peyote (Lophophora williamsii) Catalyzes O-methylation in the meta position but also in the para position depending upon the substrate (12). Partially purified catechol O-methyltransferase from pampas grass (Cortaderia selloana) catalyzes the methylation at both meta and para positions,bu6',vcwheh acting . on caffeic acid, the preparations catalyze methylation of the meta hydroxyl (14). A phenolic O-methyltransferase from Lentinus lepideus (21) was shown to catalyze para-O-methylation but only with methyl esters of the hydroxy-cinnamic acids as substrates. The same enzyme preparations catalyze the formation of the methyl ester of cinnamic acid from the free acid. 72 There is l i t t l e evidence for the participation of cofactors or other activators in transmethylation reactions. An occasional methyltransferase has been found to be stabilized by mercaptoethanol (13, 15, 21), but the activity of a partially purified methyltransferase from Vinca rosea was reduced by mercaptoethanol and enhanced by dithiothreitol (22). Requirement I | for Mg or other metal ions is not clear-cut (.'(21, 23). The objective of this study was to survey the existence of the phenol-O-methyltransferase in eight species of Lentinus and Lentinellus and also to study the effect of light on the levels of enzyme activity. 73 MATERIALS AND METHODS A. Chemicals: The source of the chemicals used was as follows: Chemical Source Cinnamic acid Aldrich Chemical Company, Inc p-Hydroxybenzoic acid Eastman Organic Chemicals Methyl p-OH-cinnamate Methyl 3,4-dimethoxycinnamate Methyl caffeate Methyl ferulate Methyl isoferulate Sephadex G-25 Polyclar AT ) (polyvinylpolypyrrolidone) (NH 4) 2S0 4 NaHoP0.. H„0 2 4 2 Na2HP04 A l l these methyl esters were obtained ^from Dr. C. K. Wat, Dept. of Botany, U. B. C., Vancouver Mercaptoethanol Soytone Omnifluor Dithioerythritol (DTE) Dithiothreitol (DTT) Glutathione (reduced form) S-Adenosyl-L-methionine,methyl-CJ Specific activity 51.8 mCi/m mole (10 uCi, 0.11 ml) ICN Pharmaceuticals Inc. Pharmacia Fine Chemicals Sigma Chemical Company BDH, Chemicals LTD Fisher Fisher Calbiochem Difco New England Nuclear Sigma Sigma Sigma ,14 74 Medium and culture Medium: Seven gm of malt extract, 0.5 gm of yeast extract and 1 gm of soytone were dissolved in 1 l i t e r of d i s t i l l e d water. Two hundred ml of the medium in a 500 ml Erlenmeyer flask was sterilized at 15 lb, o 121 C for 15 minutes and used for culturing the fungi. Culture: Lentinus edodes (UBC 767) and L_. lepideus (UBC 718) were obtained from Dr. R. J. Bandoni, Dept. of Botany, Univ. of British Columbia, Lentinellus vulpinus (177 A) from Dr. R. S. Smith, Western Forest Products Laboratory, Vancouver, B. C., Lentinus tigrinus (RLG-9953-Sp), L_. sulcatus (OKM-8302-Sp) and L_. ponderosus (OKM-3120-S) from Dr. J . G. Palmer, USDA, Madison, Wisconsin, Lentinellus cochleatus (DAOM 22534) and Lentinus kauffmanii (DAOM 11660, 17180) from Dr. J . H. Ginns, Agriculture Canada, Ottawa. The fungus was maintained on MYP (malt-yeast-soytone, 7 gm—0.5 gm-1 gm) agar slants. After growth, the cultures were covered with sterile mineral o i l and stock cultures were stored in closed screw-cap tubes at 4°C. Mycelia from a slant were transferred to a MYP agar plate and incubated at room temperature for approximately two weeks. After growth, this plate culture served as a "working plate". Mycelial tissuesffromiaaworkihggplalHeewereetransferred to a sterilized Waring Blendor and homogenized for 30 seconds with sterile d i s t i l l e d water (100 ml water for one plate). Ten ml of the resulting suspension was pipetted under sterile conditions into a 500 ml Erlenmeyer flask containing 200 ml medium. A l l cultures were kept at 25°C with 8 hours 75 of fluorescent light per day. C. Extraction of enzyme A l l steps were carried out in a cold room. Three replicate cultures were sampled each time. The medium of a 3-4-week-old culture was decanted and the mycelial mat was washed three times with d i s t i l l e d water. The wet weight was determined. The mycelial mat was frozen in liquid ^ and ground. Polyclar AT was added (5% of wet weight) and the mixture was stirred for 20 minutes in phosphate buffer (0.1 M, pH 7.0 containing 2 mM mercaptoethanol). The suspension was fi l t e r e d through two layers of cheesecloth and the f i l t r a t e centrifuged at 7,000 g (SORVALL RC 2-B) for 15 minutes. The supernatant was further fractionated by addition of solid (NH^^SO^. The 20 to 60% ammonium sulfate precipitate was dissolved in 2 ml buffer and desalted by passage through Sephadex G-25 column. The protein was eluted from the column and a 5 ml aliquot was used in the enzyme assay and for the determination of protein. D. Enzyme assay The assay mixture consisted of 0.1 ml of substrate (0.1 u mole), 0.1 ml SAM^C 1^ (0.1 uCi in 0.0525 u mole) and 0.5 ml of the above enzyme (21). The reaction mixture was incubated at 30°C for 30 minutes and terminated by the addition of 0.5 ml 5% HG1. The reaction mixture was extracted with 10 ml anhydrous diethyl ether. A 2.5 ml aliquot of ethereal extract was pipetted into a s c i n t i l l a t i o n v i a l and placed in a fume hood. The ether was evaporated and then 10 ml of s c i n t i l l a t o r (6 gm Omnifluor dissolved in a mixture of 625 ml toluene and 375 ml ethanol) was added. A l l readings (Searle Isocap/300) were corrected 76 using efficiency curves calibrated from standard quenching solution by the channel ratio method. The substrate specificity was calculated from the activity obtained and the specific activity of SAM. Identification of reaction products The remainder of the above ethereal extract (7.5 ml) was pipetted to a v i a l and the ether was evaporated in the fume hood. The residue was redissolved in 0.2 ml ether and the solution was spotted on a 10 x 2 10 cm s i l i c acid plate (Eastman Si gel chromatogram with fluorescent indicator) with reference compounds. The plate was developed two-dimensionally in benzene-acetone (9:1) followed by benzene-acetic acid-isooctane (90:10:15). After drying, the plate was examined under shorter wavelength UV lamp for the fluorescent spots. A radioautograph was prepared by exposing the chromatogram to a Kodak X-ray film for one week. The film was developed in Kodak X-ray developer for 4-5 minutes, washed with dilute acetic acid solution and transferred to a fixative solution for about 10 minutes. After washing.in running tap water for 20-30 minutes i t was dried and examined. The dark spots on the radio-autograph represented the radioactive substances on the chromatogram. Protein concentration Protein concentration was determined by the method of Lowry et a l . (24). The concentration of protein was calculated from a standard curve of Bovine Serum Albumin (BSA). 77 RESULTS AND DISCUSSION Among eight species (Lentinus ponderosus, L_. edodes, L_. kauffmanii (DAOM 11660, 17180), L_. tigrinus, L_. sulcatus, L_. lepideus, Lentinellus  vulpinus and Lentinellus cochleatus), in addition to Lentinus lepideus, only Lentinus ponderosus was found to contain the phenol-O-methyltransferase. The data are indicated in Table 1 (columns 5 and 7). Of six substrates tested, methyl p-OH-cinnamate had the highest activity. The para-specificity of this fungal enzyme was clearly demonstrated in i t s abil i t y to methylate methyl caffeate and methyl ferulate but not methyl isoferulate. Cinnamic acid was reported to be the only free acid to serve as substrate for the enzyme from Lentinus lepideus (21). In this investiga-tion, however, i t did not show activity, and the radioautograph did not give any corresponding spot either. Radioautograms of the methylated reaction products distinctly identified the labeled main products as methyl p-methoxycinnamate *f(#2) , methyl isoferu-late (#3) and methyl 3,4-dimethoxycinnamate (#4). This i s shown in Figure 1. Lentinus lepideus was reported to produce methyl p-methoxycinnamate either in light (21) or in dark (25). Lentinus ponderosus was also reported to produce methyl p-methoxycinnamate in light and dark (6). In the study of Polyporuss (26) PAL and other enzymes associated with styrylpyrone biosyn-thesis are influenced by light. In this case, the effect of light and dark on phenol-O-methyltransferase activity in these two species was also studied. The data shown in Table 1 indicates there is no difference between light and dark grown cultures of Lentinus ponderosus and _E_. lepideus. Light, therefore, does not seem to play a role in the regulation of cinnamic metabolism in these two species, although the increase in PAL activity occurs when certain Basidiomycetes are cultured in light (7). Table 1. Substrate Specificity of Methylating Enzymes from Lentinus poridero.siusaaridlLenfcinus lepideus Rf Values Enzyme Specific Activity* Benzene Benzene L. s.poriderosus L. lepideus # Substrate Product Acetone Acetic acid Isooctane light dark light dark 1. Cinnamic acid - - - 0 0 0 0 2. Methyl pj^OH-cinnamate Methyl p-methoxycinnamate 0.66 0.62 37.63 36. 60 37.80 36.80 3. Methyl caffeate Methyl isoferulate 0.32 0.40 66:19 10. 80 6.50 7.04 4. Methyl ferulate Methyl 3, 4-dimethoxy- 0.54 0.59 6.89 6. 93 5.88 6.96 cinnamate 5. Methyl isoferulate - - - 0 0 0 0 6. p-Hydroxybenzoic acid — — _ 0 0 0 0 Specific activity expressed as the number of n mole product/mg protein/hr. Figure 1. Radioautographs of the chromatographed methylated cinnamate products #2 methyl p-methoxycinnamate #3 methyl i s o f e r u l a t e #4 methyl 3,4-dimethoxycinnamate 80 The addition of 0.05 mM DTE (dithioerythritol), DTT (dithiothreitol) and glutathione did not increase the enzyme activity. From this study and from previous reports, i t seems that only species which produce methyl p-methoxycinnamate or methyl isoferulate have the phenol-O-methyltransf erase. Although both Lentinus ponderosus and L_. lepideus produce methyl anisate, none of the benzoic acids or their esters were active substrates for these enzyme preparations (6, 21). Thus i t would seem that in Lentinus the methylating enzyme for benzoic acid derivative must be a different one to that for cinnamic acid (21) or that methyl -anisate i s formed from methyl p-OH-cinnamate. Lentinus ponderosus ,aL. lepideus and L_. kauffmanii belong to a class of fungi, members of which cause brown rots in wood, and which bring about the preferential decomposition of cellulose. Although the wood i s destroyed when attacked by this type of mold, i t is interesting that the metabolism of some members of the group differs from that of others despite the fact that the overall process, the decay of the wood is the same-. 81 LITERATURE CITED 1. Blrkinshaw, J . H. and Findlay, W. P. K. (1940). Biochemistry of the wood-rotting fungi I. Metabolic products of Lentinus lepideus Fr. Biochem. J . 34: 82-88. 2. Shimazono, H. and Ford, F. F. (1958). I d e n t i f i c a t i o n of methyl p-coumarate as a metabolic product of Lentinus lepideus. Arch. Biochem. Biophys. 78: 263-264. 3. Shimazonop5HH- (1959). Investigations on l i g n i n s and l i g i n i f i c a t i o n . XXI. I d e n t i f i c a t i o n of phenolic esters i n the culture medium of Lentinus lepideus and the O-methylation of methyl p-coumarate to methyl p-methoxycinnamate i n vivo. Arch. Biochem. Biophys. 83: 206-215. 4. Shimazono.pEHn,.., Schubert, W. J . and Ford, F. F. (1958). Investigations on l i g n i h s s and l i g n i f i c a t i o n . XX. The biosynthesis of methyl p-methoxycinnamate from e s p e c i a l l y labeled D-glucose by LentihusV-lepideus. J . Am.; Chem. Soc. 80: 1992-1994. 5. Danielsson, H. and Bloch, K. (1957). On the o r i g i n of C„„ i n ergosterol. J . Am. Chem. Soc. 79: 500-501. 6. Wat, C. K. and Towers, G. H. N. (1977). Production of methylated phenolic acids by species of Lentinus (Basidiomycetes). Phytochem 16: 290-291. 7. Power, D. M., Towers, G. H. N. and Neish, A. C. (1965). Biosynthesis of phenolic acids by c e r t a i n wood-destroying Basidiomycetes. Can. J . of Biochem. 43: 1397-1407. 8. Mudd, S. H. (1973). "Biochemical mechanisms i n methyl group t r a n s f e r . "Metabolic Conjugation and Metabolic Hydrolysis". Vol. I I I . Ed. by W. H. Fishman, Academic Press. 9. K e l l e r , E. B., Rachele, J . R. and du Vigneaud, V. (1949). A ^ t u d y of transmethylation with methionine containing deuterium and C i n the methyl group. J . B i o l . Chem. 177: 733-738. 10. Fales, H. M., Mann, J . and Mudd, S. H. (1963). In v i t r o a l k a l o i d b i o -synthesis i n the Amaryllidaceae norbelladine O-methylpheraserai-J . Am. Chem. Soc. _85: 2025-2026. 11. F i n k l e , B. J . and Nelson, R. F. (1963). Enzyme reactions with phenolic compounds: a meta-O-methyltransferase i n plants. Biochim. Biophys. Acta 78: 747-749. 12. Basmadjian, G. P. and Paul, A. G. (1971). The i s o l a t i o n of an 0-methyl-transferase from peyote and i t s r o l e i n the biosynthesis of mescaline. L l o y d i a 34: 91-93. 82 13. Ebel, J . , Hahlbrock, K. and Grisebach, H. (1972). P u r i f i c a t i o n and properties of an O-dihydric phenol meta-O-methyltransferase from c e l l suspension cultures of parsley and i t s r e l a t i o n to flavonoid biosyn-t h e s i s . Biochim. Biophys. Acta 269: 313-326. 14. Fi n k l e , B. J . and K e l l y , S. H. (1974). Catechol O-methyltransferase i n pampas grass: d i f f e r e n t i a t i o n of m- and p- methylating a c t i v i t i e s . Phytochem. 13: 1719-1725. 15. Kuroda, H., Shimada, M. and Higuchi, T. (1975). P u r i f i c a t i o n and prop-e r t i e s of O-methyltransferase involved i n the biosynthesis of Gymno-sperm l i g n i n . Phytochem. J ^ : 1759-1763. 16. Yamada, Y. and Kuboi, T. (1976). Significance of c a f f e i c acid-O-methyl-transferase i n l i g n i f i c a t i o n of cultured tobacco c e l l s . Phytochem. 15: 395-396. 17. Kuboi, T. and Yamada, Y. (1976). C a f f e i c acid-O-methyltransferase i n a suspension.of c e l l aggregates of tobacco. Phytochem. 15: 397-400. 18. Senoh, S., Daly, J . , Axelrod, J . and-Witkop, B. (1959). Enzymatic p-O-methylation by catechol O-methyltransferase. J . Am. Chem. Soc. 81:6240. 19. Mann, J . D., Fales, H. M. and Mudd, S. H. (1963). Alkal o i d s and plant metabolism. VI. O-methylation i n v i t r o of norbelladine, a precursor of Amaryllidaceae a l k a l o i d s . J . B i o l . Chem. 238: 3820-3823. 20. Kaneko, K. (1962). Biogenetic studies of natural products. VIII. Biosynthesis of anethole by Foeniculum vulgare. Chemical and Pharma-i e u t T c e e u t i c a l B u l l e t i n 10: 1085-1087. 21. Wat, C. K. and Towers, G. H. N. (1975). Phenolic O-methyltransferase from Lentinus lepideus (Basidiomycete's) •. Phytochem. 14_: 663-666. 22. Madyastha, K. M., Guarnaccia, R., Baxter, C. and Coscia, C. J . (1973). S-adenosyl-L-methionine: loganic acid methyltransferase. A carboxyl--ajLkylating enzyme from Vinca rosea. J . B i o l . Chem. 248: 2497-2501. 23. Axelrod, J . and Tomchiek,RR. (1958). Enzymatic O-methylation of e p i -nephrine and other catechols. J . B i o l . Chem. 233: 702-705. 24. Lowry, 0. H., Rosebrough, N. J . , Farr, A, L. and Randall, R. J . (1951). Protein measurement with the f o l i n phenol reagent. J . B i o l . Chem. 193: 265-275. 25. Nord, F. F. and V i t u c c i , J . C. (1947). On the mechanism of enzyme action XXX. The formation of methyl p-methoxycinnamate by the action of Lentinus lepideus on glucose and xylose. Archivesaof^Biochemistry 14: 243-247. 26. Towers, G. H. N., Vance, C. P. and Nambudiri, A. M. D. (1974). Photo-regulation of phenylpropanoid and styrylpyrone biosynthesis i n Polyporus  hispideus. Recent Advances i n Phytochemistry 8_: 81-94. 

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