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Datura stramonium-tropic acid biosynthesis Johnson , Anker Lenard 1969

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DATURA STRAMONIUM-TROPIC ACID BIOSYNTHESIS by ANKER LENARD JOHNSON B.S.P., University of British Columbia, 1$6? A THESIS SUBMITTED IN PARTIAL FULFIB1ENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Division of Pharmacognosy of the Faculty of Pharmaceutical Sciences « We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1969 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h C olumbia, I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and Study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of P h a r m a c e u t i c a l S c i e n c e s The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada D a t e 28 August, 1969 ABSTRACT A pathway for the biosynthesis of tropic acid from tryptophan in Datura Stramonium has been proposed by Goodeve, and was supported by Hamon. The purpose of this investigation was to determine quantitatively the percentage of incorporation of tryptophan into tropic acid. This was attempted using vacuum infiltration and sterile root culture techniques with auto-radiography being utilized to identify the radioactive metabolites. Tryptophan was not found to be converted to tropic acid under the experimental conditions used in this investigation. The purpose of this investigation was also to extract crude enzyme preparations which would convert certain postulated intermediate compounds into tropic acid. The conversion of a-phenyl-3-aminopropionic acid to atropic acid and the conversion of atropic acid to tropic acid was attempted. The enzyme extracts of Datura Stramonium root tissues were prepared from acetone powders, from fresh tissue, and from freeze-dried tissues. These extracts did not show any activity in vitro. It is concluded that some differences existed between the tissues used in the present investigation and those used in previous work. Signature of examiners i i TABLE OF CONTENTS LITERATURE REVIEW 1 INTRODUCTION TO THE PROBLEM 19 METHODS AND MATERIALS 20 RESULTS 42 DISCUSSION OF THE RESULTS 55 CONCLUSIONS ' 63 REFERENCES 64 i i i LIST OF TABLES Table Page 1. Standard Compounds Used to Identify Radioactive Metabolites 25 la. Data on Chromatography for Attempted Identification of Germination Inhibitors 39 2. Data on the Root Tissue Culture Experiments 40 3. Data on the Vacuum Infiltration Experiments 41 4. Results of Indole-acetic acid-2-C^ Incubation with Enzyme Extract 5^ 5. Results of a-phenyl-3-aminopropionic acid-3-C^ Incubation with Enzyme Homogenate 54 LIST OF FIGURES iv Figure Page 1. Postulated pathway for the biosynthesis of tropine 5 2. Formation of hyoscine from hyoscyamine 9 3. The proposed pathway for the biosynthesis of tropic acid from phenylalanine 12 4. Examples of intramolecular rearrangements 14 5. The proposed mechanism for the biogenesis of tropic acid by a carbon monoxide insertion reaction.... 15 6. The mechanism for the in vitro formation of tropic acid from phenylalanine 16 7. The proposed pathway for the biosynthesis of tropic acid from tryptophan 18 8. The mechanism for the synthesis of a-phenyl-3-amino-propionic acid 31 9. Absorption spectra of tropic and atropic acids.... 38 10. Standard curve for the determination of atropic acid... 38 11. Composite diagram of autoradiograms from indoleacetic- 43 acid-2-C^4 sterile root eulture experiments 12. Composite diagram of autoradiograms from tryptophan-(2-indolyl)-Cl4 sterile root culture experiments 45 13. Composite diagram of autoradiograms from phenylalanine-(U.L.-ring)-C-^ sterile root culture experiments 47 14. Composite diagram of autoradiograms from a-phenyl-P-aminopropionic acid-3- ci ^ sterile root culture experiments 49 15. Composite diagram of autoradiograms from indole-acetic acid-2- C14 vacuum infiltration experiments 50 16. Composite diagram of autoradiograms from tryptophan-(2-indolyl)-Cl4 vacuum infiltration experiments 52 V ACKNOWLEDGEMENTS The author would like to thank Dr. A.M. Goodeve for his guidance and encouragement which was contributed during this investigation. Thanks are extended to the Graduate awards selection committee for the University of British Columbia Graduate Fellowship Awards. Financial assistance from the Medical Research Council of Canada is also gratefully acknowledged. The author would also like to thank Miss M. Harrison, X-ray technician at the University of British Columbia Health Services, for her technical assistance in developing the X-ray films. 1. LITERATURE REVIEW 1-Hyoscyamine is the most commonly occurring alkaloid in plants of the family Solanaceae. The alkaloid is an ester consisting of a tropine and a tropic acid portion. Atropine is the racemic form of the alkaloid, but this is thought to be an artifact due to the extraction procedures, and is not thought to occur naturally in the plants^-. Interest in the biosynthesis of hyoscyamine probably arises from the alkaloid's medicinally useful mydriatic and anti-spasmodic properties. The biogenesis of the tropine portion of the molecule has been reviewed by Hamon2 up to 1964. At this point i t had been shown that ornithine is a direct precursor for the pyrrolidine portion of the tropane skeleton. Ornithine-2- specifically labels only one of Hyoscyamine the two bridgehead carbon atoms (carbon one or five). Carbon atoms six and seven of the pyrrolidine ring are assumed to also be derived from ornithine. Carbon atoms two, three, and four of tropine are derived from acetate. Position three is labelled i f acetate-l-C^ is fed, and positions two and four are labelled when acetate-2-C-^ is fed. The mechanism for this incorporation of acetate into tropine is thought to involve the condensation of two acetate units to form acetoacetic acid. The acetoacetic acid must be in the activated form of aceto-acetyl-CoA (acetoacetyl-Coenzyme A). In fact, one of the acetate molecules must fi r s t be converted to malonyl-CoA before acetoacetyl-CoA can be formed^. The acetoacetyl-CoA molecule then becomes attached through its carbon atom number two to the intermediate formed from ornithine. Following this condensation the carboxyl group of the acetoacetic acid is eliminated, leaving carbons two, three, and four derived from acetic acid. A recent review article by Fodor^ " gives an excellent account of the biosynthesis of the tropane alkaloids up until 1967* Cuscohygrine is an alkaloid which is found in Atropa Belladonna and has a structure consisting of two pyrrolidine rings joined with a three carbon bridge. Cuscohygrine It has been recently shown-3 that acetate-2-C-L4' labels only carbons one and three of the bridge, while acetate-l-C^ labels only carbon two of the bridge^. This would support the theory that the bridge carbons are formed from acetoacetic acid. The hypothetical pathway that leads to cuscohygrine is closely related to the one for tropine and probably involves a double Mannich reaction between N-methyl-dl-pyrrolidine and acetoacetic acid. At the present time, tracer work has not been reported which demonstrates that the origin of the pyrrolidine rings of cuscohygrine is ornithine. Putrescine, which is the decarboxylation product of ornithine, was suggested as a possible precursor of tropine. Putrescine-1,4-C-^ was fed to whole Datura Stramonium plants in hydroponic culture, but Q no incorporation was found in the tropane skeleton. Kaczkowski showed that putrescine-1,4- was indeed incorporated into tropine using Datura metel sterile root cultures. Degradation was not done to deter-mine which carbon atoms were labelled, but i t is to be expected that both bridge carbons would be labelled. In this case i t appears that there are some differences between tissue culture metabolism and whole plant metabolism. It is suggested9 as unlikely that the biosynthetic route from ornithine proceeds via putrescine since the symmetry of putrescine would cause the label from ornithine-2- to be distributed equally between carbon one and carbon five of the ultimate tropine. But i t is possible that putrescine can be utilized by tissue cultures which represent an abnormal situation for the metabolizing tissues. Neumann and Schroter-^ in 1966 showed that a-N-raethyl-C-^-ornithine was incorporated into tropine better than ornithine. This demonstrates that N-methylation probably occurs before ring closure. IAebisch and Schutte^ used double labelled ornithine-2-and double labelled ornithine-2-cl\oc-N15 and found that the heterocyclic nitrogen of the tropane nucleus is derived mainly from the ef—NE2 group of ornithine. Neumann and Schroter^ used a-N-methylornithine and J-N-methylornithine labelled with carbon^ on the N-methyl groups and found that the tropine was labelled on the N-methyl group only when 12 a-N-methylornithine was administered. Gilbertson and Leete have resolved this apparent conflict of results with a criticism of the syntheses of Neumann and Schroter. The suggestion is that the syntheses of Neumann and Schroter may not have yielded authentic products. Gilbertson and Leete suggest that the method used for synthesizing a-N-methylornithine may have actually yielded ^-N-methylornithine, while the method for synthesizing ^-N-methylornithine may have produced a product heavily contaminated with a-N-methylornithine. Leete and Nelson^ have devised a hypothetical scheme for the bio-synthesis of tropine to account for the tracer results of the various groups which have been actively working on the problem. This scheme is shown in Figure 1. 13 Schutte et a l J have shown that administration of N-methylputrescine-1-C1^ .N"'"-' and N-methylputrescine-l-H^ to Nicotiana rustica plants resulted in a specific incorporation of label in position 5' of the pyrrolidine ring of nicotine. They used wick feedings as well as root absorption feedings in their studies. Their data indicate that N-methylputrescine was incorporated unsymmetrically into nicotine and that methylation was possible prior to ring closure to give the pyrrolidine ring. 3! V Nicotine Figure 1. Postulated pathway for the biosynthesis of tropine. (Leete and Nelson) Gilbertson and Leete^2 administered DL-a-N-methyl- -ornithine-2-and DL-cf-N-methyl-Clif-ornithine-2- to Nicotiana tabacum plants. The </-N-methylornithine yielded radioactive nicotine which was labelled solely at C-2 1 and on the N-methyl group, and the ratio of activity at these two positions indicated that the precursor was incorporated intact without any prior cleavage of the N-methyl group. The a-N-methylornithine was a much poorer precursor and the nicotine was labelled at C-21, C-5' and on the methyl group, the distribution of activity being consistent with demethylation of the a-N-methyl-ornithine prior to its incorporation into therpyrrolidine ring. It would appear at the present time that the formation of the pyrrolidine ring of nicotine and of the tropane alkaloids seems to follow a similar pathway. The biosynthesis of hyoscyamine is known to occur principally in the roots of Datura Stramonium and other plants which produce the alkaloid^. From the roots, the alkaloid is transported to the leaves where i t is stored. Jindra and Staba^ have shown that Datura Stramonium sterile root tissue cultures possess esterase activity. They showed that an enzyme preparation from the tissue cultures was able to catalyze the formation of hyoscyamine from tropine and tropic acid as substrates. Esterases from microorganisms^0 and animals have also been discovered. Atropine-esterase activity has been demonstrated in rabbit serum-'-'7, and this would be responsible for the lack of toxicity of atropine for rabbits. Cosson and Paris-1-0 have extracted enzyme preparations from Datura  tatula plants which are capable of degrading hyoscyamine and hyoscine into tropine and scopine respectively plus tropic acid. £ H 3 W Scopine The enzyme preparations obtained from the roots were the most active, while those obtained from the aerial parts showed the greatest activity just prior to the time of flowering. This would seem to imply that some turnover of the alkaloids must be occurring in the plants. Firmin and Parish have provided evidence that the bases tropine and scopine are found to occur free in Hyoscyamus muticus and Hyoscyamus  aureus during the course of development of the alkaloids hyoscyamine and hyoscine. Il'in et a l ^ synthesized nicotine singly labelled with carbon^ in different positions and introduced the precursors individually into different Nicotiana tabacum plants. As a result of metabolic changes, biosynthesis of radioactive protein occurred and the individual amino acids were labelled with carbon^ with different degrees of intensity. In the course of the splitting of the nicotine pyrrolidine heterocycle, the highest radioactivity can be observed in leucine, and after splitting of the pyridine heterocycle, in serine. These results confirm the relation between the transformation of alkaloids and the plants amino acid metabolism. 21 . Fairbairn has evidence for turnover of atropine in Atropa  Belladonna. He found distinct changes in the alkaloid content through-out the growing season when samplings were taken at short time intervals. Samplings were taken of l aves, ovaries or fruits, and calices. 8. The results showed a marked variation in atropine content in some parts as the season advanced and a peak of atropine concentration in other parts. 9 jh, ~~ „^—... administered ornithine-2-C to three month old Datura meteloides plants. The meteloidine which was isolated was found to be labelled on one or both of the bridgehead carbons of the teloidine moiety of the alkaloid. This result is consistent with the hypothesis that teloidine is formed by the hydroxylation of tropine. (teloidine is 6,7,-dihydroxytropine). Meteloidine (6,?,-dihydroxytropinetiglic acid ester) The in vivo interconversion of tropane bases has been discussed by Fodor . A Datura ferox branch grafted onto a Lycopersicon esculentum (tomato) root, unable to synthesize tropane type alkaloids by itself, was fed hyoscyamine and the plant was able to convert this to hyoscine. It was also shown by feeding carbons-labelled hyoscyamine to Datura  Stramonium sterile root cultures that 6-hydroxyhyoscyamine and hyoscine were found to be labelled. Hence, hyoscine would seem to arise from hyoscyamine via 6-hydroxyhyoscyamine. This scheme is shown in Figure 2. Several compounds have been considered as possible precursors for the tropic acid moiety of hyoscyamine. These are reviewed by Fodor\ The precursor of tropic acid which has been given the most attention is phenylalanine. Leete^ fed phenylalanine-3- sodium formate-and formaldehyde- to two month old Datura Stramonium plants grown in nutrient solution. He found that phenylalanine-3-C-^ labelled only 9 0-Tropic Acid 6-hydroxyhyoscyamine Figure 2. Formation of Hyoscine from Hyoscyamine. 10 carbon two of the tropic acid while sodium formate-C-^ and formaldehyde-labelled only the tropine portion. From this work he concluded that phenylalanine is a direct precursor of tropic acid. Leete and Louden2-^  later showed by a similar feeding technique that phenylalanine-2-24 labelled the hydroxymethyl carbon of tropic acid. Louden and Leete then administered phenylalanine-1- to Datura Stramonium plants by a wick feeding method and found that the isolated tropic acid was specifically labelled in the carboxyl group. These feeding experiments may be summarized as follows: / VcHg-CH-COOH <( V c H - C O O H ^ ' NHg ' CH20H 24 These results are explained as being the result of an intramolecular carboxyl shift or as a carboxylation reaction utilizing the carbon 14 dioxide produced by decarboxylation of the phenylalanine-l-C . This decarboxylation and recarboxylation reaction would not be expected to produce specific labelling of the tropic acid molecule. Therefore 24 feeding experiments were performed on Datura Stramonium plants with sodium bicarbonate- The tropic acid which was isolated was indeed found to be randomly labelled. None of the normal one-carbon sources such as carbonate, formate, formaldehyde, methionine, or serine have been found to significantly label the carboxyl group of tropic acid. Underhill and Youngken2^ further substantiated the finding that phenylalanine-3- specifically labels carbon two of tropic acid. They also found that phenylacetic acid-l-C^ labelled the tropic acid, and the radioactivity was thought to be in the hydroxymethyl carbon. They found that lactate-2- and propionate-2-C^ were poor precursors, 11. thereby refuting the possibility of condensation of shikimic acid with lactate or propionate. These workers proposed a hypothetical pathway for the biosynthesis of tropic acid which represented a logical sequence of events which might explain the experimental findings. None of the intermediates in this pathway have been isolated. This pathway was mod-ified in a further paper by Gibson and Youngken . The pathway is out-lined in figure 3* Gibson and Youngken administered phenylalanine U.L.-C^, phenylalanine-l,3-C-^, and shikimic acid-U.L.- to Datura innoxia by a wick feeding method. Since the ratio of the specific activity of carbons one and two of tropic acid was found to equal that of carbons one and three of the phenylalanine precursor, i t was concluded that the carboxyl carbon of phenylalanine is a precursor of the carboxyl carbon of tropic acid. Phenylalanine-U.L.- was found to label a l l the carbon atoms of tropic acid and shikimic acid only labelled the aromatic portion of tropic acid. These results strongly favor the pathway shown in Figure 3. If a decarboxylation-recarboxylation reaction occurs, then i t must occur via enzyme bound intermediates. Schutte and Liebisch^ fed phenylalanine-l-C to sterile cultures of isolated roots of Datura metel. They found that the isolated alkaloids were labelled almost entirely in the tropic acid portion with carbon^. This loss of tritium would indicate that a transamination step is part of the biosynthesis of tropic acid from phenylalanine. Intramolecular rearrangements have been shown to occur in several species of plants and animals. These rearrangements involve migration of different groups, but in most cases a coenzyme form of vitamin Bj_£ has been shown to be an absolute requirement for the reaction to occur*10 in animal metabolism. The biosynthesis of 7-hydroxy-4*-methoxy-isoflavone in red clover^ involves the migration of an aryl group to form a 2-phenyl-propionic acid structure from phenylalanine. The conversion of glutamate Phenylalanine transamination^ COOH <( \-CH-CH20H ^ ' COOH Tropic acid NADH Shikimic acid Pyruvic acid <^~^)-CH2-Cj-C00H Phenylpyruvic acid C0?-complex -^^-CHgCHO Phenyl ac et aid ehyd e r H-CHO COOH Figure 3. The proposed pathway for the biosynthesis of tropic acid from phenylalanine. (Gibson and Youngken) 13. to (3-methylaspartate in Clostridium tetanomorphum required vitamin B £^ and involves the intramolecular transfer of a glycine group from the f3-to the a-carbon of the propionic acid moiety of glutamic acid. The con-version of succinyl-CoA into methylmalonyl-CoA in animal tissues also required vitamin Bj^ and involves the shift of a thioester group from the 3- to the a-carbon of the propionic acid moiety of the molecule. These structures are shown in Figure 4. It has been shown that phenylalanine can be chemically converted in vitro to tropic acid by a nitrous acid deamination procedure. Ghosal has provided a possible mechanism to explain the in vitro reaction which may be involved with the in vivo mechanism. This mechanism is shown 31 in Figure 5« Yamada et al^ studied the stereochemistry of the in vitro conversion of phenylalanine into tropic acid. These workers have also proposed a mechanism for the non-enzymatic transformation which is shown in Figure 6. This mechanism involves a shift of a phenyl group. Leete32 used phenylalanine-3- to non-enzymatically produce tropic acid and he found that the hydroxymethyl carbon was labelled on tropic acid. This would substantiate the mechanism of Yamada et a l . Leete suggests that the in vitro formation of tropic acid must differ from the in vivo formation because of the observation that 1-phenylalanine was converted to R(+)-tropic acid in vitro whereas S(-)-tropic acid is formed from 1-phenylalanine in vivo. Therefore, while the non-enzymatic conversion of phenylalanine is interesting, i t is probably not particularly pertinent to the problem of tropic acid biosynthesis in vivo. Tryptophan has also been shown to act as a precursor in the bio-synthesis of tropic acid. Goodeve and Ramstad33 fed tryptophan-to whole Datura Stramonium plants grown in hydroponic culture and found the isolated tropic acid was labelled on carbon two. The plants were 14. D O A -CHo-CH-COOH Phenylalanine Formononetin OCHo COOH CHNHo CH2-CH2-COOH Glutamic acid ®12 COOH I CHNHo I 2 CH3-CH-COOH 3-methylaspartic acid < S-Coenzyme A CH2-CH 2-C00H B l 2 S- Coenzyme A CH3-CH-COOH Succinyl-Coenzyme A Methylmalonyl-Coenzyme A Figure 4. Examples of intramolecular rearrangements. Figure 5. The proposed mechanism for the biogenesis of tropic acid via a carbon monoxide insertion reaction. 16. NaNCb ^CH2-CH-C00CH2CH 3  phenylalanine ethyl ester ^ / C H 2 - CH—COOCH2CH3 0"Ae ^-$0 Ac O-CH2-CH-COOCH2CH3 HOCHg-CH-COOH tropic acid Figure 6. The mechanism for the in vitro formation of tropic acid from phenylalanine. 17. harvested at various time intervals and this allowed the determination of the metabolites between tryptophan and tropic acid. A pathway was proposed which is shown in Figure 7. In the elucidation of this pathway, all of the proposed intermediate compounds were isolated, including the unusual amino acid, a-phenyl-f3-aminopropionic acid. This amino acid is actually an isomer of phenylalanine, differing only in the position of the carboxyl group. Leete^ attempted to explain the incor-poration of tryptophan by suggesting that the radioactive tryptophan was metabolized in the plant yielding radioactive carbon dioxide which was then incorporated into tropic acid by a hypothetical carboxylation of phenylpyruvic acid or phenylacetic acid. This theory would not probably 33 2 allow for such specific labelling as was found by Goodeve^. Hamon^  has verified that tryptophan-3- a precursor of tropic acid. He also showed that indoleacetic acid-2- C1^ and tryptophan-(2-indolyl)-C1^ were incorporated into tropic acid. At the present time no further work has been published regarding the incorporation of tryptophan into tropic acid. The incorporation of labelled a-phenyl-3-aminopropionic acid into tropic acid would be required to definitely show that i t is an intermediate in;the proposed scheme. ha It has been recently reported by Hamon, Hamon and Tashiro 7 at the 1969 American Society of Pharmacognosy meeting, that Serine- has been successfully incorporated into tropic acid in Datura innoxia using a vacuum infiltration technique. Upon decarboxylation, the tropic acid yielded styrene which was found to contain 100 per cent of the radioactivity. This would seem to indicate that some mechanism other than a simple carboxylation of a C6-C2 compound must be involved. The incorporation of serine may support the tryptophan pathway hypothesis since serine is known to be involved in the formation of tryptophan. C H - C O O H tryptophan C H O indole-3-aldehyde COOH - C H f H 2 NHo C H o r c l - C O O H indolepyruvic acid CH2-C00H indole-3-acetic acid COOH I _C I! C H 2 a-phenyl-g-aminopropionic acid atropic acid C O O H tropic acid C H I CH20H Figure 7. The proposed pathway for the biosynthesis of tropic acid from tryptophan. (Goodeve and Ramstad) INTRODUCTION TO THE PROBLEM Two approaches to the problem were contemplated: (i) an attempt to quantitate by means of liquid scintillation counting the carbon^ labelled intermediates of the tryptophan-tropic acid biosynthetic pathway in Datura Stramonium using both sterile root culture and vacuum infiltration techniques. (ii) an attempt to prepare and test crude enzyme isolates from Datura Stramonium root tissue for their ability to carry out the metabolic transformations necessary to produce the intermediate compounds as set out in the scheme of Goodeve and Ramstad. A description of the procedures used will be given in the following chapter. METHODS AND MATERIALS  Sterile Root Culture Experiments The need for conditions which could be easily maintained, constant, and reproducible necessitated the use 'of sterile root tissue cultures. Root tissues were chosen since the site of alkaloid synthesis in Datura Stramonium is generally considered to be in the roots. The culture 2 3k medium chosen was that used by Hamon and outlined by White^. The liquid culture medium consists of the following parts (a) Inorganic stock salt solution. Ca(N03)2'4H20 2.88 gm. MnSCv^HgO 0.0665 gm. Na2SC% 2.0 gm. ZnSO^HgO 0.0267 gm. KC1 0.8 gm. H3BC>3 0.05 gm. NaH2P04«H20 0.19 gm. KI 0.0075 gm. The above compounds were dissolved, one at a time, in sufficient distilled water to produce 800 ml. of solution. Then 7.37 gm. of MgSC2j,'7H20 were dissolved separately in 200 ml. of distilled water. These two solutions were then mixed and stored under refrigeration. The solutions are ten times the concentration which is used in the final culture solution. (b) Stock vitamin and hormone solution Thiamine 300 mg. Nicotinic acid 50 mg. Pyridoxine 10 mg. These compounds were dissolved separately in 100 ml. of distilled water and the solution was stored under refrigeration. (c) Carbohydrate and iron source. Sucrose 20 gm. per Liter of final culture solution Fe2(304)3 2*5 mS* P e r Liter of final culture solution. 21. To prepare one liter of complete nutrient solution, the following were mixed:-20 gm. of sucrose dissolved in 500 ml. of distilled water 2.5 mg. of Fe 2 ( S Q l L ) 3 100 ml. of stock salt solution 10 ml. of stock vitamin and hormone solution q.s. to 1000 ml. with distilled water. The nutrient solution was then placed in 125 ml. erlenmeyer culture flasks, with each flask containing fifty milliliters of solution. The flasks were stoppered with cotton plugs. The radio-isotopes were added at this point when feeding experiments were being done. The flasks were then autoclaved at one hundred twenty-one degrees centigrade and fifteen pounds pressure for thirty minutes. The root tissues were prepared from seeds taken from plants grown in the field. At maturity the seed capsules were removed from the plant and surface sterilized by immersing them in a two per cent sodium hypochlorite solution for thirty minutes. The capsules were then broken open and the seeds removed aseptically. The seeds were then surface sterilized by shaking them in a stoppered erlenmeyer flask with thirty per cent hydrogen peroxide for ninety seconds. The seeds were then removed and placed in previously autoclaved petri dishes with three layers of filter paper and ten milliliters of sterile distilled water in each. Approximately fifty seeds were placed in each petri dish. The petri dishes were then placed in a dark incubator at twenty-eight degrees centigrade and they were allowed to germinate. After seven to ten days, the largest roots were excised and placed in the culture flasks containing White's medium. About ten ro.ot sections were placed in each flask. (All transfers of seeds and roots were 22. carried out in a "Baker Sterishield" utilizing a positive pressure of filtered air). The flasks were then placed in the dark incubator which was set to maintain twenty-eight degrees centigrade. The culture flasks with their root tissues were allowed to grow for seven days and were then transferred to fresh medium. If feeding experiments were being done, the fresh medium contained the radio-isotope which was to be employed. Samplings were carried out at various intervals. When the feeding period extended beyond seven days, the roots were trans-ferred to fresh medium but the fresh medium did not contain more radio-isotope. It was felt that more isotope would not facilitate detection of those compounds which were produced in more than seven days. Any compounds labelled in less than seven days would appear from the early samplings. After the desired metabolism period, the roots were removed from the flasks, rinsed with distilled water, blotted dry on filter paper, weighed, and placed in a glass mortar. A small amount of sand and ethanol were added and the root tissues were then ground to a smooth paste. The paste was then placed in a soxhlet extraction thimble and extracted in a micro-soxhlet apparatus with twenty milliliters of ethanol for six hours. The ethanolic extract was then dried down in a stream of air at room temperature. The dried extract was redissolved in fifty microliters of ethanol and ten microliters of this were spotted on eight inch square pieces of Whatman #1 filter paper. The paper was purchased with corner punched holes for use on Smith's Universal Apparatus-^. The spotted sample was then subjected to two-dimensional ascending chromatography. The first solvent system used was isopropanol, ammonium hydroxide, water (20:1:2), which was run overnight for fifteen hours. The paper was then allowed J to dry for thirty minutes at room temperature in a stream of air. Then the paper was placed in the second solvent of n-butanol, acetic acid, water (12:3:5) until the solvent front reached the edge of the paper (approximately six hours). The chromatograms were removed from the tank and dried in an air stream for twenty-four hours. The prolonged drying is necessary to remove all traces of solvent before producing autoradiograms. Autoradiograms were prepared by placing the chromatogram in contact with a sheet of Kodak No-screen Medical X-Ray Film. In some cases, Kodak Royal Blue Medical X-Ray film was used for greater sensitivity and thus a shorter waiting period. The film and chromatogram were then placed in a shielded holder for a suitable period of time. Some of the chromatograms were sprayed with "Cmnispray" intensifier which shortened the waiting period for exposure. The "Omnispray" is a solution of anthracene which is available in a pressurized container from New England Nuclear Co. This material causes a secondary emission of light when in close contact with radioactive isotopes. The light then exposes the film much more efficiently than the original radiation would have done. The use of "Omnispray" yields a ten to fifteen times sensitivity improvement factor with paper chromatograms. When "Cmnispray" is used i t is necessary to expose the films at dry-ice temperature for maximum effectiveness. After a suitable exposure period, the films were removed from the holders and developed. A second solvent system was employed to confirm the identity of selected acidic compounds which were eluted from the original chromato-grams. The first solvent was ethanol, ammonium hydroxide, water (16:1:3) which was run for seven hours. The sheets were dried for thirty minutes and then run in the second solvent which was composed of n-propanol, eucalyptol, formic acid, water (5:5:2 sufficient to saturate). The chromatograms were run in the second direction for five hours. Numerous reference compounds were chromatographed in order to identify the radioactive metabolites which were produced from the labelled precursors. These compounds are listed in table 1. The reference compounds were dissolved in ethanol to produce a concentration of ten milligrams per milliliter. I f the solubility in ethanol was too low for this concentration, another more suitable solvent was used. These solutions were stored under refrigeration. The reference compounds were then spotted on paper or thin layer sheets in a similar manner to the plant extracts. The amount spotted was usually ten microliters which would equal one hundred micrograms of material. Visualization reagents are shown along with Rf values in Table 1. The method of preparation and use of these reagents is given by Smith^ -'. All of the above reference compounds were purchased from chemical manufacturers with the exception of atropic acid, 3-aminohydrocinnamic acid, and a-phenyl-3-arainopropionic acid. Thin layer chromatography was also utilized in this investigation is some cases in place of paper chromatography. The samples were spotted on eight inch square prepared "Chromagram" sheets purchased from Eastman Kodak Co. The sheets were run in Eastman Kodak "Sandwich type" chambers. The solvent systems used were: vertical direction - ethyl acetate, isopropanol, ammonium hydroxide (45:35:20) horizontal direction - chloroform, acetic acid (95:5) Atropic acid was prepared from atrolactic acid by N.W. Hamon using the method of McKenzie and WoodJ . At a pressure of ten to fifteen milliliters of mercury, 500 milligrams of atrolactic acid 25. Table 1. Standard Compounds Used to Identify Radioactive Metabolites. Organic Acids *Solvent Rf Values one **Solvent two Detection Reagent amygdalic acid 0.44 0.80 U.V. (Ultra-Violet) atrolactic acid 0.62 0.94 bromophenol blue atropic acid 0.56 0.96 bromocresol green benzoic acid 0.51 0.98 bromocresol green caffeic acid 0.10 0.76 U.V. chlorogenic acid 0.07 0.54 U.V. cinnamic acid 0.54 0.90 U.V. citric acid 0 0.27 aniline-xylose o-coumaric acid 0.32 0.93 U.V. p-coumaric acid 0.29 0.93 U.V. ferulic acid . 0.15 0.83 U.V. glutaric acid 0 0.78 aniline-xylo s e glycolic acid 0.19 0.59 aniline-xylose p-hydroxycinnamic acid 0.27 0.91 U.V. 5-hydroxyindole-3-acetic acid 0.09 0.74 Ehrlich 5-hydroxytryptophan 0.15 0.23 U.V. p-hydroxyphenylac etic acid 0.26 0.97 bromocresol green indole-3-acetic acid 0.29 0.96 Ehrlich indole-3-glycolic acid 0.16 0.93 Ehrlich indole-3-glyoxylic acid 0.29 0.83 Ehrlich indole-3-lactic acid 0.31 0.93 Ehrlich indole-3-propionic acid 0.37 0.96 Ehrlich •Solvent one = isopropanol, ammonium hydroxide, water (20:1:2) ••Solvent two = n-butanol, acetic acid, water (12:3:5) Table 1. (Continued) 26. Organic Acids Rf Values •Solvent one ••Solvent two Detection Reagent kynurenic acid 0.30 0.64 U.V. malonic acid 0.02 0.41 bromocresol green mandelic acid 0.56 0.88 aniline-xylose oxalic acid 0 0.21 acridine phenylacetic acid 0.53 0.90 aniline-xylose a-phenyllactic acid 0.57 0.95 acridine 8-phenyllactic acid 0.60 0.96 acridine phloretic acid 0.44 0.975 bromocresol green potassium fumarate 0 0.74 aniline-xylos e protocatechuic acid 0.03 0.80 U.V. quinaldic acid 0.53 0.89 aniline-xylose shikimic acid 0.04 0.43 U.V. sinapic acid 0 0.87 U.V. succinic acid 0.02 0.77 bromocresol green tiglic acid 0.15 0.90 bromocresol green tropic acid 0.35 0.87 bromocresol green vanillic acid 0.07 0.84 U.V. veratric acid 0.43 0.92 bromocresol green Amino Acids 8-alanine 0.08 0.38 ninhydrin-HAc L-alanine 0.15 0.33 ninhydrin 8-aminohydrocinnamic acid 0.30 0.67 ninhydrin-HAc L-arginine HC1 0.05 0.23 ninhydrin •Solvent one = isopropanol, ammonium hydroxide, water ( 20 :1 :2 ) ••Solvent two = n-butanol, acetic acid, water ( 12 :3 :5 ) 27. Table 1. (Continued) Amino Acids •Solvent Rf Values one ••Solvent two Detection Reagent aspartic acid 0.02 0.19 ninhydrin glutamic acid 0.03 0.21 ninhydrin glycine 0.07 0.20 ninhydrin and U.V. L-histidine HC1 0.45 0.24 ninhydrin L-isoleucine 0.33 0.64 ninhydrin L-leucine 0.30 0.69 ninhydrin L-lysine HC1 0.04 0.21 ninhydrin DL-ornithine HC1 0.06 0.23 ninhydrin a-phenyl-3-amino-propionic acid 0.34 0.83 ninhydrin-HAc phenylalanine 0.27 0.59 ninhydrin DL-serine 0.05 0.24 ninhydrin DL-threonine 0.12 0.28 ninhydrin tryptophan 0.15 0.45 Ehrlich L-tyrosine 0.14 0.39 ninhydrin DL-valine 0.20 0.53 ninhydrin Miscellaneous Compounds atropine sulfate 0.95 0.74 Dragendorffs hyoscyamine 0.97 0.77 Dragendorffs indole-3-aldehyde 0.99 0.52 2,4-DNPH indole 0.98 0.57 Ehrlich kynurenine sulfate 0.15 0.49 Ehrlich skatole 0.95 0.95 Ehrlich tryptamine 0.85 0.71 Ehrlich •Solvent one = isopropanol, ammonium hydroxide, water (20:1:2) ••Solvent two = n-butanol, acetic acid, water (12:3:5) 28. Table 1. (Continued) Compounds •Solvent Rf Values one ••Solvent two Detection Reagent phenethylamine 0.91 0.71 bromocresol green N, N,-dimethyltryptamine 0.99 0.59 Ehrlich tryptophol 0.94 0.94 Ehrlich tyramine HC1 0.89 0.76 bromocresol green •Solvent one = isopropanol, ammonium hydroxide, water (20:1:2) ••Solvent two = n-butanol, acetic acid, water (12:3:5) were distilled. The vapour distilled at one hundred and eighty-degrees centigrade and quickly condensed to form a white crystalline solid. This solid was dissolved in warm seventy per cent ethanol and hot water was added to this solution until i t became turbid. Upon cooling, crystals of atropic acid separated which had a melting point of one hundred seven degrees centigrade. 8-aminohydrocinnamic acid was prepared by Dr. A.M. Goodeve according to the method of Johnson and TAvak^?. Twenty grams of benzaldehyde, twenty grams of malonic acid, and thirty grams of ammonium acetate were mixed with forty milliliters of ethanol and the mixture was refluxed on a steam bath for five hours. After this period, the 8-aminohydrocinnamic acid separated from the alcoholic solution and was reerystaliized from hot water. The melting point was 231 degrees centigrade. a-phenyl-8-aminopropionic acid was synthesized by the method 38 of Mannich and Ganz . Ten grams of diethylphenylmalonate were hydrolyzed by heating at reflux temperature with forty milliliters of a fifteen per cent aqueous sodium hydroxide solution for two hours. The material was then placed in an ice bath until i t began to freeze and cautiously neutralized with concentrated hydrochloric acid. The mixture was made slightly acidic (pH 2) and extracted three times with ether. The ether was allowed to evaporate spon-taneously, leaving behind crystals of phenylmalonic acid. 1.8 grams of phenylmalonic acid were chilled in an ice-bath and made faintly alkaline with a twenty-eight per cent solution of ammonium hydroxide. Then 1.8 grams more of phenylmalonic acid were added and the mixture was stirred until i t was homogeneous. Then 2.0 milliliters of formaldehyde solution were added to the mixture which was stirred for fifteen minutes at zero degrees centigrade. The mixture was allowed to stand at room temperature. Decarboxylation began after thirty minutes and crystals began to separate after eighteen hours. The reaction continued for seventy-two hours. Following this period the crystals of a-phenyl-8-aminopropionic acid were filtered off and recrystallized in ninety-five per cent ethanol. The observed melting point was one hundred and eighty-four to one hundred and eight-five degrees centigrade. a-phenyl-8-aminopropionic acid was also 14 synthesized with carbon on the 8-carbon position. The synthesis involved similar quantities to that of the unlabelled material. The formaldehyde which was added contained one hundred microcuries of 0 . The yield of a-phenyl-B-aminopropionic acid was 5 0 9 « 2 mg. The material was recrystallized three times to obtain a constant specific activity. The compound was counted in a "Picker Liquimat" liquid scintillation counter in a scintillator solution composed of PPO (2,5-diphenyloxazole) and bis-MSB (p-bis-(o-Methylstyryl)-benzene) in toluene. The a-phenyl-8-aminopropionic acid was run two-dimensionally on paper and thin layer chromatograms as a test for purity. The compound was found to be homogeneous. The final activity was found to be 5 9 t 5 3 6 counts per minute per milligram of material. The mechanism of the Mannich reaction which is involved in this synthesis is seen in the following outline-^ i n Figure 8 . The radioactive compounds used in tissue culture experiments were: Source 1 . indoleacetic acid -2-C 1^ Radiochemical Centre 2 . tryptophan-(2-indolyl)- Calbiochem 3 . phenylalanine-(U.L. r i n g ) - ^ New England Nuclear 14 4. a-phenyl-8-aminopropionic acid-3-C Laboratory synthesis H + Nl NH4OH R - • C-H -OH Figure 8. The Mechanism f o r the Synthesis of a-phenyl-g-aminopropionic Acid. During the course of the tissue culture experiments, difficulty in seed germination was seen until the seeds were aged several months. Only about ten per cent of the freshly harvested seeds were seen to germinate in contrast with about seventy per cent of the old seeds. Washing the new seeds overnight under running cold tap water was found to increase the germination to about fi f t y per cent. It was found that fluorescent materials were extracted into water when the seeds were 40 soaked. Koves and Varga have reported the extraction of fluorescent substances from the pericarp of Datura Stramonium fruits. Those fluorescent materials were identified as various phenolic compounds and were found to be responsible for germination inhibition. These previously identified germination inhibitors were chromatographed along with the unknown fluorescent substances in an attempt to identify the substances extracted from seeds. Descending one-dimensional chroma-tography was utilized on Whatman #3 paper, eighteen inches by twenty-two inches in size. The solvent system was n-butanol, acetic acid, water (12:3:5). The results are seen in Table la. Vacuum Infiltration Experiments. Administration of radioactive materials was also carried out using a vacuum infiltration method. The root material was obtained from whole Datura Stramonium plants growing in a liquid culture solution. The plants were aerated with sintered glass bubblers. The culture solution consisted of the following: (a) The following solutions were made up: 1.0 Molar MgSO^RgO 24.65 gm/100 ml. 1.0 Molar Ca(N03)2'4H20 23.61 gm/100 ml. 1.0 Molar KH^ PO^  13.61 gm/100 ml. (b) A solution of Fe-EDTA was prepared containing five milligrams of iron per milli l i t e r . 33. (c) the following micro-element solution was prepared: H3BO3 2.86 g. MnCl2'4H20 1.81 g. ZnCl2 0.11 g. CuCl2'2H20 0.11 g. Na2Mo04»2H20 0.025 g. Distilled water q.s. 1 Liter. (d) to prepare the complete nutrient solution, the following solutions were added to each liter of water: 1.0 Molar MgSO^  solution 2.3 ml. 1.0 Molar Ca(N03)2 solution 4.5 ml. 1.0 Molar KH2PQij. solution 2.3 ml. Fe-EDTA solution 1.0 ml. Micro-element solution 1.0 ml. The root tissues used were the fresh white tips removed from plants which appeared to be healthy. The weight of tissue used in each tube was approximately 400 milligrams. The root tissues were placed in micro test-tubes and a solution of the isotope was added so that the liquid just covered the roots. The tubes were then placed in a vacuum desiccator. The air in the desiccator was then pumped out until bubbles of gas formed in abundance in the solutions contained in the test tubes. This partial ' vacuum was then held for a period of fifteen minutes. The vacuum was then released to allow the solution with radio-isotope to be forced into the tissues. This was considered as zero time. The tissues were then removed from the tubes at various intervals. The tissues, were then ground, extracted, and spotted on Whatman #1 eight inch square chromatography paper in a similar fashion to that of the sterile root cultures. The isotopes used were a-phenyl-8-aminopropionic acid- indole-3-acetic acid-2-and tryptophan-(2-indolyl)-Cli*'. (The sources were noted previously). 34. Attempted Enzyme Assay Experiments. In an attempt to veri fy certain steps of the pathway postulated by Goodeve, i n v i t ro enzyme assays were attempted. The ul tra-violet absorption spectra of atropic acid and tropic acid seemed to be suff ic iently different which would be favourable for a spectrophotometric assay. The absorption spectra are shown i n Figure 9. Atropic acid shows a peak i n absorption at 245 millimicrons which i s not shown by tropic acid. Therefore, one should be able to observe the enzymatic conversion of atropic to tropic acid i n v i t ro by monitoring the change i n absorbance at 245 millimicrons. The enzyme extraction was attempted using fresh root tissues from whole plants grown i n l iqu id culture media as previously described. Several methods of extracting enzymes were tr ied and these are described below. The preparation of an acetone powder was carried out according to 41 42 the methods of Ochoa et a l and Loomis . The plant material was removed from the plant and rinsed with ice-cold d i s t i l l e d water. Then the tissues were placed i n a chi l led Waring blendor and homogenized for two minutes with ten volumes of acetone chi l l ed to minus ten degrees centigrade. The suspension was then f i l tered by suction i n a Buchner funnel. The residue was then washed i n the funnel twice with cold acetone, once with acetone at room temperature and twice with ether at room temperature. The powder was allowed to dry at room temperature and the dry powder was stored i n a t ight ly stoppered bottle under refrigeration. The powder was extracted for use i n an assay by grinding 100 o milligrams l i gh t ly i n a mortar with ten m i l l i l i t e r s of 0.2 Molar phosphate buffer at pH 7.4 for ten minutes. (Overnight extraction with buffer was attempted also). This was then centrifuged and the supernatant was used as the enzyme solution for the assay. The reaction mixture consisted of the following: 1.0 ml. of atropic acid solution (0.05 mg. /ml. in water) 2.0 ml. of enzyme solution 2.0 ml. of phosphate buffer pH 7.4 The times of incubation were one, two, four, and eight hours at thirty degrees centigrade. The reaction was stopped by heating the tubes. The mixtures were filtered with a fine sintered glass f i l ter before reading. Some readings were also made of ether extracts with appropriately altered blanks. A l l readings were taken on a Bausch & Lomb Spectronic 505 recording spectrophotometer. An enzyme solution was prepared by extracting fresh root material also. 11.0 grams of fresh Datura Stramonium root material, were ground with sand in a chilled mortar. This was then made to 40.0 ml. with ice-cold disti l led water and centrifuged for ten minutes at 13,000 revolutions per minute at two degrees centigrade. The supernatant was used as the enzyme solution. The reaction mixture used was the same as previously described. Grinding the fresh roots with buffer at pH 7.4 was also tried. The extraction of enzymes from plant tissues poses more problems 43 than extraction from animal tissues. Loomis et al have shown that phenolic substances such as tannins which occur in many plant tissues are rapidly oxidized and polymerized when the tissues are disrupted. Phenols combine with proteins reversibly by hydrogen bonding and irreversibly by oxidation followed by covalent condensation. Polyclar AT (an insoluble form of polyvinylpyrrolidone purchased from General Aniline and Film) has been used as an additive for removal of these phenolic compounds since i t contains groups similar to the amide bond in proteins. Ten grams of Polyclar AT were added to fifty milliliters of water forming a slurry which was allowed to hydrate for twenty-four hours. During this period, the water was decanted several times to remove any water-soluble impurities. 0.025 Molar ascorbic acid was added as an anti-oxidant to the slurry. Ten grams of fresh Datura Stramonium root tissues were then excised and ground in a chilled mortar with sand and the chilled Polyclar-ascorbic acid slurry. The homogenate was then centrifuged for twenty minutes at 12,000 revolutions per minute at three degrees centigrade. It was found that the ascorbic acid interfered with the spectrophotometry assay at 245 millimicrons. Therefore the ascorbic acid was deleted when spectrophotometric assays were attempted. Indoleacetic acid-2-C^ was incubated with an enzyme extract prepared as above from fresh root material with Polyclar ATsadded to inhibit browning of the preparation. The reaction mixture consisted of the following:-1.0 ml. of indoleacetic acid-2-Cl^ (containing 1.0 microcurie) 1.0 ml. of enzyme solution 1.0 ml. of phosphate buffer pH 7.4 The time of incubation at thirty degrees centigrade was fifteen minutes. The reaction was stopped by heating the tube. Then a twenty-five microliter aliquot of the reaction mixture was spotted on thin layer sheets and developed as previously described. A mixture of compounds, involved in the tryptophan pathway to tropic acid, was co-spotted with the aliquot from the reaction mixture. After two-dimensional development of the chromatograms, the standard spots were located with ultra-violet light. The spots were cut out and scraped into scintillation vials and counted in the Picker Iiquimat scintillation counter. The scintillator solution was the same as that previously described. The results are shown in table 4. Root tissues from whole Datura Stramonium plants grown in liquid culture were freeze-dried. 13.0 grams of fresh roots yielded 0.92 grams of freeze-dried material. 200 milligrams of the dried material were mixed with five milliliters of pH ?A phosphate buffer with 0.25 molar ascorbic acid added to make an enzyme homogenate. This preparation was then used in incubations with a-phenyl-B-aminopropionic acid-The reaction mixture was as follows :-0.5 ml. of a-phenyl-3-aminopropionic acid-3- C 1 4 (10.0 mg./ml.) 0.5 ml. of enzyme homogenate 1.0 ml. of phosphate buffer pH 7A The incubation times were for twenty and forty hours. After the required incubation period, ten microliters of the mixture were spotted on thin layer sheets and developed one-dimensionally in a solvent system consisting of chloroform, acetic acid (95s5)» Standards of a-phenyl-3-aminopropionic acid, atropic acid, tropic acid, and hyoscyamine were co-spotted along with the reaction mixture aliquot. The spots were then cut out, scraped into scintillation vials and counted as previously described. The results are shown in table 5. Wavelength mu Figure 9. Absorption spectra of tropic and atropic acids. 0.8, 0.7. 0.6-Absorbance 0.5' 0.4 0.3 0.2. 0.1. 0 I . . , , , , , • 0 1 2 3 4 5 6 7 8 9 10 Concentration (rag./L) Figure 10. Standard curve for the determination of atropic acid. 39. Table la. Data on Chromatography for Attempted Identification of Germination Inhibitors. Suspected Germination Inhibitor % Value Colour under U.V. ferulic acid 0.86 dark blue chlorogenic acid 0.68 light blue cinnamic acid 0.91 brown protocatechuic acid 0.75 purple caffeic acid 0.77 light blue p-coumaric acid 0.83 blue o-coumaric acid 0.84 light blue phloretic acid 0.91 light blue p-hydroxybenzoie acid 0.86 dark blue vanillic acid 0.84 blue sinapic acid 0.80 light blue p-hydroxycinnamic acid 0.85 purple unknown compound #1 0.57 green ^ -r unknown compound #2 0.50 green unknown compound #3 0.36 blue unknown compound #4 0.31 blue unknown compound #5 0.22 green Solvent system employed - n-butanol, acetic acid, water (12:3:5) Table 2. Data for the Root Tissue Culture Experiments. 40. Isotope Amount Used Metabolism Time Activity Chromatographed Exposure Time Remarks* indoleacetie-acid-2-C1^ 5 uc. 3 days 3,000 cpm 20 :days 5 uc. 6 days 2,500 cpm 20 days 5 uc. 9 days 4,200 cpm 20 days 5 uc. 12 days 2,100 cpm 20 days tryptophan- . (2-indolyl)-C l i f 5 uc. 5 uc. 3 days 6 days 2,500 cpm 2,300 cpm 10 days 10 days R. B., omni-spray n 5 uc. 7 days 5,000 cpm 20 days R.B. 5 uc. 5 uc. 12 days 14 days 3,700 cpm 4,000 cpm 10 days 24 days R.B., omni-spray R.B. 5 uc. 21 days 3,600 cpm 17 days R. B., omni-spray phenylalanine- . (U.L. ringJ-C 1 4 5 uc. 5 uc. 1 day 2 days 17 days 17 days R.B., omni-spray ii 5 uc. 3 days 17 days it 5 uc. 6 days 17 days II a-phenyl-B-amino-propj^onic acid-10 mg. 3 days 50 cpm 20 days 10 mg. 6 days 50 cpm 20 days 10 mg. 9 days 50 cpm 20 days 10 mg. 12 days 40 cpm 20 days *R.B. = Royal Blue Film Note- The level of activity chromatographed was determined by means of a thin window Geiger Tube in direct contact with the chromatogram. Table 3. Data on the Vacuum Infiltration Experiments. 41. Isotope Amount Used Metabolism Time Activity Chromatographed Exposure Time * Remarks indoleacetic-acid-2-C1^ 5 uc. 2 hours 10,000 cpm 12 days R.B. 5 uc. 4 hours 8,800 cpm 12 days R.B. tryptophan- . (2-indolyD-C1^ 5 uc 2 hours 2,300 cpm 12 days R.B., Omni— spray 5 uc. 4 hours 6,100 cpm 12 days n 5 uc. 6 hours 6,700 cpm 12 days II tryptophan- . (2-indolyl)-C 1^ 5 uc. + 0.004 Molar phenylacetic acid 5 uc. 2 hours 4 hours 1,600 cpm 4,400 cpm 12 days 12 days R.B., Crani-spray n 5 uc. 6 hours 8,800 cpm 12 days n a-phenyl-3-amino-propi^onic acid-10 mg. 1 hour 80 cpm 7 days R. B., Omni-spray, TLC. 10 mg. 2 hours 80 cpm 7 days ti 10 mg. 4 hours 80 cpm 7 days it * R.B. = Royal Blue Film TLC = Thin Layer Chromatography Note- The level of activity chromatographed was determined by means of a thin window Geiger Tube in direct contact with the chromatogram. RESULTS The following diagrams represent composites, each from several autoradiograms of different feeding times. Any spot which is numbered represents a compound which is identified and the identification is noted on the page following each diagram. The presence of other radioactive metabolites cannot be excluded because they may not be sufficiently soluble in ethanol to be extracted or they may be present in quantities too small to be detected. Tables 4 and 5 represent the result of radiochemical assays utilizing thin layer chromatography to isolate the products and a scintillation counter to measure the activities. No results are shown for the attempted spectrophotometric assay experiments, since at no time was any change in absorbance seen when an enzyme extract was incubated with atropic acid. Figure11. Composite Diagram of Autoradiograms from Indoleacetic acid-2 r C^ Sterile Root Culture Experiments. isopropanol, ammonium hydroxide, water (20:1:2) 0 origin butanol, acetic acid, water (12:3:5) See following page for identification of numbered compounds. Figure 11. (continued) Compound Number Identity 1 malonic acid 2 fumaric acid 3 aspartic acid k indole-3-acetic acid 5 p-hydroxycinnamic acid 6 leucine 7 valine 8 citric acid 45. Figure 12. Composite Diagram of Autoradiograms from Tryptophan-(2iindolyl)-cS Sterile Root Culture Experiments. <3 isopropanol, ammonium hydroxide, water (20:1:2) 5 origin butanol, acetic acid, water (12:3:5) See following page for identification of numbered compounds. Figure 12. (continued) Compound Number Identity 1 tryptophan 2 indole-3-acetie acid 3 tryptamine tryptophol 5 phenylacetic acid 6 fumaric acid 7 malonic acid 8 citric acid 47. Figure 13. Composite Diagram from Autoradiograms of Phenylalanine-.(U.L.-ring)-C1^ Sterile Root Culture Experiments. origin butanol, acetic acid, water (12:3:5) See following page for identification of numbered compounds. Fi gure 13. ( c ontinued ) Compound Number 1 2 3 5 6 Identity phenylalanine protocatechuic acid tropic acid benzoic acid phenethylamine citric acid Figure 14. Composite Diagram of Autoradiograms from a-phenyl-3-arainopropionic acid-3-Cl^ Sterile Root Culture Experiments. 49. isopropanol, ammonium hydroxide, water (20:1:2) origin butanol, acetic acid, water (12:3:5) Figure 15. Diagram of Composite Autoradiograms from Indoleacetic acid-2- Vacuum Infiltration Experiments. water (20:1:2) CD C<^> origin butanol, acetic acid, water (12:3:5) See following page for identification of numbered compounds. Figure 15- (continued) Compound Number 1 2 3 5 6 7 8 9 Identity Indole-3-acetic acid tryptophol valine fumaric acid vanillic acid glutaric acid citric acid malonic acid indole-3-lactic acid Figure 16. Composite diagram of Autoradiograms from Tryptophan-(2-indolyl)-C^^ Vacuum Infiltration Experiments. isopropanol, ammonium hyd roxid e, water (20:1:2) origin butanol, acetic acid, water (12:3:5) See following page for identification of numbered compounds. Figure 16. ( continued ) Compound Number 1 2 3 5 Identity tryptophan tryptamine a-phenyl-8-aminopropionic acid indole-3-acetic acid phenylacetic acid 54. Table 4. Results of Indole-acetic acid-2-C1^ Incubation with Enzyme Extract. Compound Counted Activity indole-acetic acid 16,208 cpm indolealdehyde 4,524 cpm a-phenyl-8-amino-propionic acid 853 cpm atropic acid 178 cpm tropic acid 1,082 cpm alkaloid 1,495 cpm Table 5. Results of a-phenyl-8-aminopropionic acid-3-CX4' Incubation with Enzyme Homogenate. Compound Counted Activity 20 hours 40 hours a-phenyl-8-amino-propionic acid 2,021 cpm 1,541 cpm atropic acid 37 cpm 42 cpm tropic acid 52 cpm 58 cpm alkaloid 35 cpm 39 cpm DISCUSSION OF RESULTS The sterile root culture feeding experiments in this investigation have not shown tropic acid to be formed from tryptophan or Indole-acetic acid. This is in direct contrast to the work of Hamon and also that of Goodeve. While Goodeve found tropic acid to be produced from tryptophan, he was working with whole plants. Hamon found results similar to those of Goodeve using sterile root tissue cultures. It is to be expected that some differences might occur between whole plant experiments and tissue culture experiments, as was found with the previously mentioned incorporation of putrescine into tropine. But, i t would be hoped that experiments utilizing similar tissue culture techniques would yield similar results. It is possible that the tissues used in the present investigation differed somehow from those used by Hamon. The tissues may have been at the wrong stage of growth for tropic acid production. It is also possible that some changes occurred in the plant so that tropic acid production was no longer possible. The possibility of inhibition of alkaloid production by some unknown factor in the culture solution cannot be overlooked. If the tropic acid were being produced at a lesser level than usual, then the autoradiographic technique used may not have been capable of detecting the acid at a reduced level. It can be seen from the indoleacetic acid-2- C^ sterile root feedings that the citric cycle acids become labelled. This would also enable various amino acids to be labelled by transamination of an a-keto acid such as aspartic acid from oxaloacetic acid. The carbon^ label of indoleacetic acid-2-C^" could easily be channeled into the citric acid cycle by splitting acetate-2-C^ from the indole nucleus. The very reactive acetate molecule would then enter the citric acid cycle by combining with oxaloacetate. An enzyme which could perform the f u n c t i o n of s p l i t t i n g i n d o l e a c e t i c ac id seems l i k e l y i n view o f the f a c t that Verzar*^ et a l have shown that the l a b e l of tryptophan-2-when fed to Vinea minor was found to be t r a n s f e r r e d to other amino acids i n the free amino a c i d p o o l . In p a r t i c u l a r , a lan ine , s e r i n e , phenyla lanine , l euc ine and v a l i n e were found to be l a b e l l e d . The authors exp la in t h i s by suggesting that an enzymatic separat ion of the i n d o l e and propion ic ac id components of tryptophan may be o c c u r r i n g . S a n w a l ^ has descr ibed a tryptophanase from E s c h e r i c h i a c o l i which converts tryptophan i n the presence of water i n t o pyruvic a c i d , ammonia and i n d o l e . This r e a c t i o n requ ired p y r i d o x a l phosphate as a c o f a c t o r . This would be a d i f f e r e n t enzyme from tryptophan synthetase which i s known to produce tryptophan from indo le and s e r i n e . Since the formation of l e u c i n e and v a l i n e s t a r t s from pyruvate^ 0 , t h i s tryptophanase would exp la in t h e i r l a b e l l i n g from t r y p t o p h a n ^ - C ^ . Alanine i s formed through a transaminat ion of pyruvic ac id and ser ine i s formed from phosphohydroxypyruvate. Phosphoenolpyruvic a c i d i s invo lved i n the synthesis of phenyla lanine . The c i t r i c a c i d c y c l e ac ids were not found to be l a b e l l e d by Hamon, and t h i s would i n d i c a t e that indeed there were some d i f ferences between h i s t i s s u e cu l tures and those used i n the present i n v e s t i g a t i o n . I t was noted that the feeding of i n d o l e a c e t i c a c i d-2 - C ^ caused some v i s i b l e changes i n the growth of the root t i s s u e s . The growth was found to resemble that of c a l l u s t i s s u e growth. Normally l a t e r a l roots were formed i n large numbers, whereas when i n d o l e a c e t i c ac id-2-cS was f e d , no l a t e r a l roots were produced. I t would appear that the concentrat ion of i n d o l e a c e t i c acid-2-C-1-^ which was added to the cu l tures was s u f f i c i e n t to act i n a growth r e g u l a t i n g capac i ty . I t was not poss ib le to place emphasis on the i d e n t i f i c a t i o n of basic compounds which were found to be labelled on the chromatograms produced from the indoleacetic acid-2-CP-4 feedings. The reason for this was the presence of spots in the same position which appeared when indoleacetic aeid-2-c£4 was chromatographed as a test of purity. From this i t can only be concluded that the indoleacetic aeid-2-Cp-^  was impure as purchased or was so unstable that i t was degraded during the normal chromatography procedure. Indoleacetic acid is known to be unstable in the presence of acid and light. Therefore the acidic solvent was always used last and the chromatograms were run in the dark whenever this was possible. The tryptophan-( 2-indolyl )-Cjl4 did n o t seem to be converted to tropic acid as judged from the autoradiograms. The tryptophan-(2-indolyl) CIA was converted to indoleacetic acid, and in the vacuum infiltration, a compound was labelled which would appear to be a-phenyl-3-aminopropionic acid. The tryptophan-(2-indolyl)-C£4 appeared to be converted into the citric acid cycle acids in the sterile root feedings, but this was not the case with the vacuum infiltration trials. This would be a further indication that some differences may be seen between isolated roots and those from whole plants. It would not be expected that labelled N-formylkynurenine would be produced from tryptophan-(2-indolyl)-Cp-^' since this a very reactive substance and the loss of the labelled formyl group would probably occur. The labelled formyl group would then be able to enter the one-carbon pool and from there could produce a wide variety of radioactive compounds. Recent papers by Macnicol ' * have shown the isolation of 6-hydroxykynurenic acid from Nicotiana tabacum, Datura Stramonium, and other species as well. Up until this finding, the known biological occurrence of 6-hydroxykynurenic acid had been restricted to mammalian and- avian urine, where i t is accompanied by other kynurenic acid derivatives. In mammals, kynurenic acid and derived compounds are formed via kynurenine as a result of the tryptophan ring opening between carbon two and carbon three, catalyzed by tryptophan oxygenase. The widespread occurrence of 6-hydroxykynurenic acid suggests that at least the early steps in the mammalian pathway of tryptophan catabolism may occur in plants. If this degradative pathway occurs in Datura  Stramonium, the kynurenic acid and anthranilic acid would not appear on the autoradiograms from tryptophan-(2-indolyl)-C-^ feedings because the label would be lost as the formyl group. When phenylacetic acid was incubated with tryptophan-(2-indolyl)-Cp-^ there was s t i l l no observable tropic acid formed. It was thought that the free formyl group from the tryptophan-(2-indolyl)-C-^ could combine with the phenylacetic acid to form tropic acid. (Leete had proposed this as an explanation for the incorporation of tryptophan-3-Cl4 into tropic acid). The results of the a-phenyl-8-aminopropionic acid-3-C^ vacuum infiltration experiments would indicate that no conversion of this compound took place under the particular conditions of the experiment. When the same compound was fed to sterile root cultures, several unidentified labelled compounds were produced. The pattern on the chromatogram seemed to correspond to a pattern of unidentified compounds on the chromatograms from tryptophan-(2-indolyl)-C-'-^ and indoleacetic acid-2-C-^ sterile root feedings. This would seem to indicate that these unidentified compounds are probably produced from degradation of the precursors and the carbon fourteen is channeled into an active one carbon pool. The variety of precursors which appear to label the same group of compounds would substantiate this proposal. The autoradiograms from the phenylalanine-(U.L. ring)-Cl4 experiments show that a compound chromatographically similar to tropic acid was formed. When this was eluted and subjected to chromatography in two new solvent systems, i t was found that there was a mixture of compounds present. Although a small amount of tropic acid was indicated the majority of radioactivity resided in an unidentified compound. Several workers have shown that the formation of tropic acid from phenylalanine involves an intramolecular carboxyl shift as previously shown. Since a-phenyl-8-aminopropionic acid is a 8-amino isomer of phenylalanine, differing only in the position of the carboxyl group, one is led to speculate whether this 8-amino acid could be formed from phenylalanine by a carboxyl shift and then converted to atropic and tropic acid. Hamon did not indicate the finding of any intermediate compounds common to both the phenylalanine and tryptophan feedings. They were not found in this investigation either. The results of the attempted enzyme assay experiments do not show any conversion of atropic acid to tropic acid. There are many possible reasons for the failure to demonstrate this enzymatic activity. These will be discussed in the following paragraphs. One of the most important reasons for this failure to demonstrate atropic acid hydrase activity could be the inability of the chosen method to extract the enzyme from the root tissues. Enzymes which are localized within compartments in the root such as mitochondria would probably not be extracted by a simple grinding technique. The majority of plant enzymes involved in secondary metabolism are probably to be found in the vacuole as soluble enzymes, but very l i t t l e information is available to verify this. The enzyme may be inactivated during the extraction process by heat 60. or some other uncontrollable factors. The grinding of a tissue to extract enzymes inevitably disrupts the organization of the tissue in which the enzyme is normally found. This may be followed by inactivation of normally active enzymes or by the activation of enzyme systems which are inactive under normal conditions. Certain inactivating agents such as phenols or tannins may be permitted to come in contact with the enzyme, whereas in the native state these agents would be separated from the enzyme. Polyvinylpyrrolidone is added in an attempt to complex the phenols and reduce this type of inactivation. The pH of the extraction medium and of the incubation mixture for most enzymes shows a definite optimum value. Values differing signif-icantly from this optimum level may completely retard the activity of a given enzyme. The sensitivity of the assay system utilizing the Spectronic 505 may not have been sufficient to detect the very small changes of substrate because of an extremely low level of enzyme in the root tissues. This problem could possibly be overcome by concentrating the enzyme solution. One possibility that cannot be overlooked is a complete absence of the enzyme in the root tissues. The enzyme may only be present at certain stages in the growth of the plant and the extraction may have been carried out at the wrong time. There may be no such enzyme as atropic acid hydrase. The conversion of atropic acid to tropic acid does not appear to be a spontaneous reaction, since solutions of atropic acid after standing several weeks do not show any changes in absorbance. The radiochemical assay utilized to measure a-phenyl-8-amino-propionic acid- ammonia lyase activity also did not show that any activity was detected. The freeze-dried enzyme homogenate should have been more concentrated than other preparations used. The type of activity being searched for here would be similar to phenylalanine ammonia lyase activity which has been found in higher plants. The substrates differ in being a-amino or B-amino isomers only. One possible reason for failure to detect this enzyme activity could be that the specific activity of the substrate was too low to allow adequate detection of the proposed product. Some conversion of the indoleacetic acid-2-Cp-^  would seem to take place when i t was incubated with an enzyme extract to yield labelled intermediates of the tryptophan pathway to tropic acid. But these results may not be entirely correct because autoradiograms produced showed that the radioactivity was smeared even though the standard compounds separated well. Although the intermediates in the pathway proposed by Goodeve have been isolated, i t is s t i l l necessary for conclusive proof of the pathway to show that the various reactions postulated can be demon-strated in vitro. The reactions are assumed to be enzymatic and it would therefore be necessary to extract enzymes capable of carrying out these reactions. It appears from the data in table la that the fluorescent materials which were extracted from the Datura Stramonium seeds were not the same germination inhibitors which Koves and Varga isolated from Datura Stramonium pericarp tissues. The Rf values of the isolated materials do not correspond with any of the materials isolated by Koves and Varga. It is not known that the fluorescent materials extracted from the seeds were definitely germination inhibitors but this is assumed because the seeds were found to germinate better after thorough washing. This washing effect would not seem to be increasing germination by allowing the seeds to imbibe water because the seeds which were not washed did not germinate well even when placed on very moist f i l t e r papers. 63. CONCLUSIONS It was not found possible to detect labelled tropic acid as a metabolite of tryptophan-(2-indolyl)-C1^ in the sterile root culture systems utilized in this investigation. The feeding of indoleacetic acid-2- led to labelling of certain compounds of the citric acid cycle. This was not noted by Hamon in his work utilizing similar techniques. The feeding of indoleacetic acid -2-C^ produced some degree of tissue abnormality of a callus tissue type growth. Tryptophan-(2-indolyl)-C-S fed to root tissues by a vacuum infiltration method led to the formation of labelled indoleacetic acid and probably a-phenyl-8-aminopropionic acid, but these were not converted to the following two compounds, atropic and tropic acids. The suggestion by Leete that the free formyl group from tryptophan could condense with phenylacetic acid to form tropic acid was not supported by our laboratory results. There was noted a similarity in the chromatographic pattern of unidentified metabolites between the tryptophan-(2-indolyl)-cl^ and indoleacetic acid -2- C^ feedings in sterile root cultures and the feeding of a-phenyl-&aminopropionic acid- 3-C^ by vacuum infiltration. It was not possible to show conversion of the utilized precursor compounds to tropic acid by the use of crude enzyme preparations. The fluorescent materials which were extracted from the Datura  Stramonium seeds into water were not identified as the germination inhibitors isolated by other workers from extracts of Datura Stramonium pericarp tissue. 6 4 . REFERENCES 1. Claus, E.P. (1961) "Pharmacognosy" fourth edition, p 307. Lea & Febiger, Philadelphia. 2. Hamon, N.W. (1966) The biogenesis of tropic acid in Datura Stramonium. Masters thesis, U.B.C. 3. Bentley, R. & Keil, J.G. (1961) The role of acetate and malonate in the biosynthesis of penicillic acid. Proc. Chem. Soc, p 111. 4. Fodor, G. in "Progress in Phytochemistry", Vol. 1 (1968) edited by Reinhold, L., & Liwschity, Y. Interscience Publishers, New York, pp 491-544. 5. Baralle, F.E. & Gros, E.G. (1969) Biosynthesis of cuscohygrine in Atropa Belladonna from sodium acetate-l-Cl^. Phytochemistry, 8:849-851. 6. Baralle, F.E. & Gros, E.G. (1969) Biosynthesis of cuscohygrine in Atropa Belladonna from sodium acetate-2-Cl^-. Phytochemistry, 8:853-855-7. Diaper, D.G.M., Kirkwood, S., & Marion L. (1951) The biogenesis of alkaloids. III. A study of hyoscyamine biosynthesis using isotopic putrescine. Can. J. Chem. 29:964-969. 8. Kaczkowski, J. & Marion, L. (1963) The incorporation of putrescine into hyoscyamine. Can. J. Chem., 41:2651-2653. 9. Leete, E. & Nelson, S.J. (1969) Biosynthesis of the teloidine moiety of meteloidine in Datura meteloides. Phytochemistry, 8:413-418. 10. Neumann, D. & Schroter, H.B. (1966) N-methylornithin als Vorstufe des Pyrrolidinringes in Tropan-alkaloiden. Tetrahedron Letters, 12:1273-1278. 11. Liebisch, H. & Schutte, H.R. (1967) Zur Biosynthese der Tropanalkaloide VIII. Borstufen des Pyrrolidinringes. Z. Pflanzenphysiol., 57.:434-439. 12. Gilbertson, T.J. & Leete, E. (1967) Biosynthesis of the Nicotiana alkaloids XII. The incorporation of a- and <f-N-methylornithine into the pyrrolidine ring of nicotine. J. Am. Chem. Soc, 89:7085-7088. 13. Schutte, H.R., Maier, W., & Stephan, U. (1968) Zur Biosynthese des Nicotins. Z. Naturforschg., 23b;1426-1429. 14. James, W. 0. (1953) Alkaloid formation in plants. J. of Pharm. & Pharmacol., 5_:809-822. 15. Jindra, A. & Staba, E.J. (I968) Datura tissue cultures: arginase, transaminase and esterase activities. Phytochemistry, 7:79-82. 65 16. Neimer, H., Bucherer, H., & Kohler, A. (1959) Uber den Abbau von Atropin durch Corynebacterlum belladonnae. Z. Physiol. Chem., 317:238-242. 17. Bernheira, F., Bernheim, M.L.C. (1938) The hydrolysis of homatropine and atropine by various tissues. J. Pharmacol. Exptl. Therap., 64:209-216. 18. Cosson, K. & Paris, R. (1967) Mise en evidence de la degradation in vitro, de l*hyoscyamine et de la scopolamine par des preparations enzymatiques obtenues a* partir du Datura tatula a* differents stades de son developpement. C.R. Acad. Sci. Paris, t265, Series D:202-204. 19. Firmin, A.S. & Paris, R. (1968) Mise en evidence du tropanol et du scopanol a' l'etat libre au cours de l'ontogenese des alcaloides chez Hyoscyamus muticus et Hyoscyamus aureus. C.R. Acad. Sci. Paris, t267, Series D:1448-1449. 20. LVin, G.S., Lovkova, M.Ya., Klimenteva, N.I. (1968) Transformation of nicotine in tobacco plants. Izv. Akad. Nauk SSSR, Ser. Biol. 1968, 6:876-881, through Chem. Abstr., 7p_:35085d(1969) 21. Fairbairn, J.W., & Wassel, G.M. (1967) Evidence for a rapid turnover of atropine in Atropa Belladonna. J. Chem. U.A.R., 10:275-285, through Chem. Abstr., 62:89l4w(1968) 22. Leete, E. (I960) The biogenesis of tropic acid and related studies on the alkaloids of Datura Stramonium. J. Amer. Chem. Soc, 23. Leete, E., & Louden, M.L. (1961) Biogenesis of tropic acid: origin of the hydroxymethyl group. Chem. & Ind. (London), pp.1405-1406. 24. Louden, M.L., & Leete, E. (1962) The biosynthesis of tropic acid. J. Amer. Chem. Soc, 84:1510-1511. 25. Underhill, E.W. & Youngken, H.W. Jr., (I962) Biosynthesis of hyoscyamine and scopolamine in Datura Stramonium. J. Pharm. Sci., 5.1:121-125. 26. Gibson, CA., & Youngken, H.W. Jr., (1967)' Biosynthesis of tropic acid in Datura innoxia. J. Pharm. Sci., 56:?.854-857. 27. Schutte, H.R., & Iiebisch, H. (1967) Zur Biosynthese der Tropanalkaloide. IX. Zum Mechanismus der Bildung der Tropasaure aus Phenylalanine1-C14,2_H3). Z. Pflanzenphysiol., 5_7_:440-443. 28. Bernhauer, K., Muller, 0., & Wagner, F. (1964) New chemical and biochemical developments in the vitamin B^ 2 field. Angew. Chem. internat. Edit., 3:200-211. 29. Grisebach, V.H., & Patschke, L. (1961) Zur biogenese Der Flavanoide. Z. Naturforschung, 16b:645-647. 66. 30. Ghosal, S. (1965) Biogenesis of tropic acid via a novel carbon monoxide insertion reaction. Sci. & Cult. (Calcutta), 31:370-371. 31. Yaraada, S., Kitagawa, T., & Achiwa, K. (1967) Non enzymic transformation of phenylalanine to tropic acid. Tetrahedron Letters, 31:3007-3011. 32. Leete, E. (1968) Observations of the non-enzymatic trans-formation of phenylalanine to tropic acid. Tetrahedron Letters, £5_: 5793-5794. 33. Goodeve, A.M., & Ramstad, E. (1961) Tryptophan, precursor of tropic acid in Datura Stramonium. Experentia, 17:124-125. 34. White, P.R. (1963) "The Cultivation of Plant and Animal Cells" second edition. The Ronald Press, New York. 35. Smith, I. (I960) "Chromatographic and Electrophoretic Techniques" Volume I, second edition. William Nelnemann Medical Books Ltd. London. 36. McKenzie, A., & Wood, J.K. (1919) The isomeric tropic acids. J. Chem. Soc. 115:part two, 834-835. 37. Johnson, T.B., & Livak, J.E. (1936) Research in pyrimidines. J. Am. Chem. Soc, 5.8:299-303. 38. Mannich, C., & Ganz, E. (1922) Berichte der Deutchen Chemischen Gesellschaft, 2:3486-3504. 39. Sykes, P. (1965) A guidebook to mechanism in organic chemistry, 2nd edition. Longmans, London, pp. 180-181. 40. Koves, E. & Varga, M. (1959) Comparative examination of water-and ether-soluble inhibiting substances in dry fruits. Fhyton, 12:93-99. 41. Ochoa, S., Mehler, A.H., & Romberg, A. (1948) Isolation and properties of an enzyme from pigeon liver catalyzing the reversible oxidative decarboxylation of 1-malic acid. J. Biol. Chem., 174:980-981. 42. Loomis, W.D. (1959) Amide metabolism in higher plants. III. Distribution of glutamyl transferase and glutamine synthetase activity. Plant Physiology, 3^:541-546. 43. Loomis, W.D., & Battaile, J. (1966) Plant phenolic compounds and the isolation of plant enzymes. Phytochemistry, 5:423-438. 44. Verzar-Petri, G., Varade, J., & Szarvas, T. (1968) Incorporation of tryptophan-2-C-^ into the free amino acid spectrum of Vinca minor L. Acta. Biol. Acad. Sci. Hung., 19:75-81. 67. 45. Sanwal, B.D. in "Modern Methods of Plant Analysis", Vol. VI. (1963) edited by Linskens, H.F., Sanwal, B.D., & Tracey, M.V. Springer-Verlag, Berlin, pp 466. 46. Sanwal, B.D., & Lata, M. in "Modern Methods of Plant Analysis", (1964) Vol. VII. edited by Linskens, H.F., Sanwal, B.D., & Tracey, M.V. Springer-Verlag, Berlin, pp 343-344. 47. Macnicol, P.K. (1968) Isolation of 6-hydroxykynurenic acid from the tobacco leaf. Biochem. J., 107:473-479. 48. Slaytor, M., Copeland, E., & Macnicol, P.K. (1968) The biosynthesis of 6-hydroxykynurenic acid in Nicotiana tabacurn. Phytochemistry, 7:1779-1780. 49. Hamon, D.M., Hamon, N.W., & Tashiro, J. (1969) Incorporation of serine-3-Cl4 into tropic acid in Datura innoxia Mill. Program Abstract 10th Annual Meeting American Society of Pharmacognosy, Corvallis, Oregon. August, I969. 

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