Citric Acid Cycle during Alkaloid Production in Claviceps purpurea

r Biochem. Physiol. Pflanzen l'i4, 660-671 (1979)

Citric Acid Cycle during Alkaloid Production in Claviceps purpurea G. GLUND, D. SCHLEE and H. REINBOTHE Pflanzenbiochemische Abteilung der Sektion Biowissenschaften, Martin-Luther-Universitat Halle Key Term Index: citric acid cycle, titrate synthase, fluoroacet.'Ite, peptide alkaloids, Claviceps pttrpurea.

Summary Alkaloid production in saprophytically ergotoxine producing C. purpurea strain pepty 695 appears to be bvoured by supplementation of the culture broth with citrate or some other interme" diates of the citric acid cycle as well as with some metabolically related compounds, such as L-asparagine. The role of citrate and of compounds which can replace citrate in supporting growth and alkaloid production was studied. Inhibition of growth by fluoroacetate has been found to be dependent upon the concentration of the analog. In short time experiments, kinetics of 14COa release from [I-UC]and [2-14C]- acetate as well as from [2-14C]- and [3- t4 C]- pyruvate has been followed. Results obtained confirm the presence of an intact CAC in C. purpurea. Citrate synthase (CS) is specifically regulated by ATP. In the course of alkaloid production, CS drastically declines when measuring specific enzyme activity, but is birly constant and at a high level under non-producing conditions as well as in a Claviceps strain uncapable to produce ergot alkaloids.

Introduction

Numerous strains of the fungus, C. purpurea, exhibit in submerged culture a characteristic carbon source requirement for both growth and alkaloid production. Besides a sugar, an organic ammonium salt is required for optimal growth and alkaloid synthesis. The anions used are common intermediates of the CAC, such as citrate and succinate, or are metabolically related to the CAC, such as L-asparagine (ARCAMONE et al. 1961, 1970; REHACEK et al. 1977). The added sugar is metabolized via the glycolytic sequence or via the oxidative pentose phosphate cycle reactions (McDoNALD et al. 1960; KIRSTAN 1977). Relatively little is known about the importance of the organic acids usually taken to supplement the culture media. A simple buffering effect (MORTON and McMILLAN 1954), a substrate function (TABER 1968,1971) as well as a siphoning off of an "unusual" CAC by the carbon skeletons of the organic acids have been proposed (REHACEK et al. 1974). The accumulation of acetyl coenzyme A and of phosphoenolpyruvate, respectively, by simultaneous utilization of sucrose and citrate as carbon sources has been suggested, too (A~IICI et al. 1967). The present investigation was started to get a deeper insight into the role of the CAC in ergot alkaloid production of C. purpurea. The results so far obtained suggest that CAC intermediates are necessary for alkaloid production through providing acetyl coenzyme A. Abbreviations used: CAC citric acid cycle, CS citrate synthase, dr. ",t. dry weight, m. wt. molecular weight.

Citric Acid Cycle

601

Materials and Methods Organisms and culture conditions Strain pepty 695 of C. purpurea produces ergotoxin alkaloids under saprophytic growth conditions. Strain pur 218 of C. purpurea produces ergotamine under parasitic growth conditions, but is unable to produce alkaloids in saprophytie culture. The composition of the fermentation media 720 and lU 10 were described elsewhere (MAIER et a!. 1971). The inoculum was taken from a 7-day old culture grown in medium 720. Each flask was inoculated to a final concentration of mycelium up to 1.5 mg/ml (dr. wt.). The pH of the fermentation broth was adjusted to 5.2-5.4 using NaOH/HCI. Analytic procedures Determination of dr. wt. and the assay of alkaloids were reported elsewhere (SCHMAUDER and GRGGER 1978; l\Lm-:R et a!. 1972). The concentration of citrate in the culture filtrate was estimated according to SAFFRAN and DEXNSTEDT (LOWENSTEIN 1969). Acetyl coenzyma A prepared from coenzyme A
Biochcm. Physiol. Pflanull. Bd. 17-1

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ml (dr. wt.). The procedure of monitoring uptake and 14C02 release was a modification of that reported by Wasternack (1975). The uptake was measured in 1 ml portiom which were transferred into 5 ml ice-cold medium 720. 14C0 2 production was terminated and 14C0 2 was quantitatively released by adding 1 ml 2 N H 2S0 4 , The dried filters were placed in vials without any addition of trichloroacetic acid.

Radioactive compounds: [P4C]-acetate (spec. radir<1.ctivity 24 mCi/mmole, [2-14C]-acetate (8 mCi/mmole), [2_14C]_pyruvate (4 mCi/mmole), and [3-14 C]-pyruvate (2 mCi/mmole) were pm chased from ROTOP, Dresden, GDE. [1.5-14 C]-citric acid (20 mCi/mmole) was obtained from the Radiochemical Centre, Amersham, England.

Results

Variation of the carbon source in the culture medium Sucrose of the common fermentation medium was replaced by some other sugars and sugar alcohols (Table. 1). As is seen from Table 1, a mixture of glucosejmannose a,s well as sucrose resulted in greatest amount of alkaloid with respect to growth. Lactose, arabinose, ribitol, and sorbitol were not so effective. It seems of special interest that glycerol instead of sucrose favours growth and alkaloid production, too. Citric acid as a component of fermentation medium 720 can be replaced by some other organic (tcids as well as by some amino acids. With regard to efficacy, citrate is, however, the most effectiYe of any added compound. Interestingly, the application of some "uncommon" compounds, such as oxalate, y-aminobutyrate, and CaC03 , respectively, results in growth and alkaloid production too (Table 2.). In contrast to this, neither growth nor alkaloid production could be achieved with tlcetate (see below), propionate, and ethanol, when added instead of citrate in the concentration indicated. Table 1. Effect of different carbon sources on growth and alkaloid production. The sucrose of the culture medium 720 is replaced by other sugars as in.dicated. Their concentrations are given in brackets (g/l) Sugar

SUl'rose (200) laetose (200) glucose (200) glucose (100) mannose (100) glucose (100) + mannose (100) arabinose (100) ribitol (100) sorbitol (100) ribitol (100) + sorbitol (100) glycerol (24)

1-!d

Fermentation

Dry weight (mg/ml)

Alkaloid (flg/ml)

Alkaloid (flg/mg dry weight)

33 24 39 33 35

524 120 459 425 438

15.8 5.0 11.7 12.8 12.4

44 21 2G 31

734 li2 176 248

IG.7 3.0 G.7 8.1

43 27

248 275

11.4 10.2

663

Citric Acid Cycle

Table 2. Effect of the replace/llent of citric acid in medium 720 by otlier organic acids and by amino acids on gro!cth and alkaloid production of pepty 695. All compounds weTe C-equimolar to citric acid. A constant amount of NH4 0H according to the nitrogen content of medium 720 was used. Volatile acids weTe filter-sterilized (n. d. not determined) Compound

II'I

citrate succinate mahLtc pyruyatc oxalate acetate propionate ;'-aminobutyrate glutamate aspartate glyeine ahtninc serine proline valine histidine CaCO a (2 ~~) ethanol

14. d Fermentation Dry weight (mg/ml)

Alkaloid (flg/ml)

Alkaloid (/1g/mg dry weight)

29.7 26.2 26.8 28.3 9.0 2.0 1.13 17.7

778 351 390 514 225 22 23

31.1 13,3 14.5 19.4

400

37.0

583 417 310 480 290 375 430 35 300 31

22.9 15.5 16.4 13.1 12.8 8.7 9.6 12.0

25.4 23.6 37.3 33.8 39.0 30.6 23.0

n. d. 3.7

20.0

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The metabolic activity with respect to growth and alkaloid synthesis of the ergot fungus depends on the concentration of citrate. Without an.'- citrate in the fermentation broth, growth and alkaloid production is not possible .•-\t concentrations lower than that used in medium 720, both growth and alkaloid production are markedly repressed. ::\0 enhancement could be observed, however, at concentrations more than 12.6 gil citric acid (Fig. 1. A + B). Citrate is takrn up and metabolized during growth (Fig. 1.

C + D). From these findings is was of interest to get more insight into kind of CAC present in the fungus and its regulation in relation to alkaloid biosynthesis. This was achieved by three independent ways: (1) to study the influence of inhibitors of the CAC, (2) to follow by radiorespirometry the metabolic fate of labellrd precursors of CAC intermediates, and (3) to determine the properties of citrate synthase initia,ting the CAe by condensing acetyl coenzyme A and oxaloacetate to form citrate. The effect of GAG inhibitors. Application of malonate known to be a competitive inhibitor of succinic dehydrogenase (W:EBB 1965) to fermentation broth in the concentration range from 0.01 mM to 1.0 mM, did not affect growth and alkaloid production. The reasons for that are unknown. Fluoroacetate known to be converted in vivo to yield fluorocitrate which in turn is inhibitory against aconitase (for review sec KUN 1969) causes CAC inhibition resulting in the accumulation of citric acid in fluoroacetate

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Fig. 1. Effect of different concentrations of citric acid at constani nitrogen content on gron'lll (A) and alkaloid production (B). eoncentmtion of citrie acid in the culture medium 720 during fermentation of pepty 695 (D). Uptake of [1.5-14 C]-citmte and 14e02 release (e). The concentration of the labelled citmte was 0.2 !lei/m!.

intoxicated animals and plants (SPENCER and LOWEN'STEIN 1962; WARD and HUSKISSON 1969). The concentration of fluoroacetate necessary to inhibit the CAC depends UPOD the capability of acetyl coenzyme A synthase and citrate synthase to accept the fluoro compound, the sensitivit~- of aconitase towards flu oro citrate as well as on the possibility that fluoroacetate might be dehalogenated (CHAPMAN and GRAHAlIl 1974). As we have shown, the inhibitory action of fluoroacetate on growth of C. purpurea strain

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665

Citric Acid Cycle

pepty 695 strongly depends upon the concentration of the analog (Fig. 2). At concentrations of more than 0.1 mM, inhibition of growth occurs. At concentrations of 0.1 mM and 0.01 mM, fluoroacetate was without any effect on growth.

M elabolic fate of labelled GAG precursors. The release of 14C0 2 from labelled acetate or p)'Tuvate was used to obtain knowledge about the mode of CAC in different living beings (BULLA et al. 1970; CONNET and BLUM 1972; HAGEMAN and RINNE 1976). Kinetics of 14C0 2 release was followed in pepty 695 after application of [l_14C]- and [2_14C] -acetate as well as of [2_14C]_ and [3- 14 C]-pyruvate, respectively. In the CAC, a subsequent production of 14C0 2 from these precursors is implicated. A preponderance of [1- 14 C]-acetate as compared with [2- 14 C]-acetate is assumed in the time sequence. In analogy,14C0 2 release from [2_14C]-pyruvate is assumed. to be earlier than from [314C]-pyruvate against time. A mathematic calculation (GLUND 1978) based on the assumption of steady state conditions revealed that the kinetics of 14C0 2 release from both acetate and pyruvate must be linear and parallel in an appropriate time interval when an intact CAC is operative. The parallelity of 14C02 release is independent on any drainage away from the cycle or other reactions utilizing acetyl coenzyme A or pyruvate. Furthermore, it becomes clear that if no other reactions are inclosed the distance against time between the two curves goes against zero. The formation of 14C02 from both the specifically labelled precurS(}fS according to the prediction made (acetate C-1 > acetate C-2, p)'Tuvate C-2> pyruvate C-3) is shown in Fig. 3. The experimental curves obtained are parallel to each other. Hence, an intact CAC operates in Glaviceps. 'i -u

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666

K. GLUND, D. SCHLEE and H. REINBOTHE

As is seen from Fig. 3., the curves of the 14C0 2 release from [l-14C]-acetate and [2_14C]pyruvate cut the ordinate at the point zero. Failure of lag period in product formation from a radioactive precursor fed excludes the existence of non-labelled precursor pool (KARLIN et al. 1975) as was determined for CAC intermediates as well as for acetate and pyruvate. In contrast to the mathematical calculation, a great distance exists between the curves of 14C0 2 release from [1- 14 C]-acetate and [2- 14C]-acetate (12 min) and from [2_14C]-pyruvate and [3- 14 C]-pyruvate (20 min), respectively. At present, we are out of experimental evidence for an explanation. It is suggested that another entrance into the CAC for acetate and pyruvate might exist as that catalysed by citrate synthase only.

Citrate synthase (EC 4.1.3.7,) Citrate synthase is the key regulatory emzyme of the CAC in many organisms. In vitro, CS is affected by several inhibitors which might be also involved in enzyme regulation in vivo (SRERE 1974). As proposed by LUCAS and WEITZMAN (1978), different regulatory properties of the enzyme may reflect different reactions of the CAC realized. In a complete CAC, CS is regulated by NADH and ATP being some type of end-pro-

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5 4 Fig. 4. Double reciprocal plots to Shall' the effect of acetyl-CoA and oxaloacetate on the reaction velocity of CS from pepty 695 after partial purificatioll. (B) shows oxaloacetate as variable substrate, concentration of lIcetyl-CoA 1 mM. (A) shows vllriation of the initial velocity as 11 function of the acetyl-CoA concentration, without lind with addition of ATP in the concentrations indiclIted at fixed concentration of oxalollcetllte of 1 mM. Fig. 5. lIfolecular weight determination of CS on a calibrated Sepharose 6B column ClIlibration proteins were obbined from Boehringer, FRG. The reference proteins lire 1) cytochrom c, 2) chymotrypsinogen, 3) albumin, 4) 1Ilbumin, 5) 1Ildolllse, 6) clItllJase, 7) ferritin, lind 6) CS from pepty 695 lifter ammonium sulfate preeipitation

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Citric Acid Cycle

667

ducts. In an incomplete ("split") CAC or-curing in anaerobically grown Escherichia coli and in blue-green algae its reactions only fulfil anabolic demands. Then, CS is regulated either by lX-oxoglutaric acid or by the combined action of ex-oxoglutarate and succinyl coenzyme A, but not by NADH and ATP. Citrate synthase of pepty 695 was purified as has been described in materials and methods. The main enzyme activity appeared in the eluate at 0.25 mM NaCl. The enzyme preparation possessing a purification factor 11 and the specific activity 1.2 ,umoles CoASH/min/mg was free from malate dehydrogenase and deacylase. Optimum enzyme activity was at pH 8.0--8.2 in Tris/HCI buffer. Potassium and sodium ions do not activate the enzyme. Excect Ca2+ and Mg2+, divalent cations, such as Mn2+, Cu2+, C02+, and Zn2+, inhibit the reaction rate. With respert to both the substrates, the enzyme displays a hyperbolic kinetics (Fig. 4.). The Km values for oxaloacetate and acetyl coenzyme A are 0.007 and 0.14 mM, respectively. The molecular weight of the enzyme from pepty 695 was determined as 100.000 by gel filtration on Sepharose 6B (Fig. 5.). On Sephadex G-150, CS from C. purpurea strain pepty 695 cochromatographed with CS from pig heart (m. wt.100.000, obtained from Boehringer, Mannheim, FRG). Therefore it belongs to the citrate synthases of the "small" type (WEITZMAN and JONES 1975). This is in accordance with the regulation of the enzyme by ATP which has been shown to be a strong and specific inhibitor of the CS from pepty 695. Other nucleotides, such as ADP, AMP, and NADH, only weakly inhibit the enzyme. Table 3. lnflttence of metabolites on the reaction velocity of partially purified CS (as per cent of reaction rate without any addition) from pepty 695. The concentration of acetyl coenzyme A was 0.021 and that of oxaloacetate 0.025 mM, respectively, metabolite concentrations used are indicated Metabolite

Concentration

ATP ADP

0.8 0.8 1.6 0.8 1.6 0.5 1

AMP NADH

isocitrate
Per cent of control

2

19 90 75 91 75 104 91 75 98 83

4 8 1 2 1.6 1.6 1.6

66 100 100 100 87 100

2 citrate

(m~l)

1

77

Except citrate, intermediates of the CAC as well as L-glutamate are not inhibitory_ Citrate inhibits in relatively high concentrations (Table 3). Other strains of C. purpurea

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differing from pepty 695 in ergot alkaloid synthesis show the same inhibition pattern of their CS. The specific activity of CS was followed in dependence on growth phase (Table 4.). In alkaloid producing C. purpurea strain pepty 695, CS activity declines in the course of discontinuous cultivation, whereas in non-producing culture medium CS activity of the strain is nearly constant. Wild-type strains of C. purpurea not able to produce ergot alkaloids have a fairly constant high enzyme level (Table 4.). We conclude that alkaloid production might be related to a decrease in citrate synthase. Table 4. Course of specific activity of CS in tu'O strains of CZaviceps purpurea alld ullder different cultivatioll conditions. Tests were carried out in crude cell extracts after disruption by ultrasonification and dialysis Days

Specific activity (,umoles X 1O-3/min/mg) pepty 695 in 720

pepty 69,) in .M 10

223

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177

10 13

54 ±9

263 175 209 2:?6

pur 218 in 720 196 117 163 120

Discussion

Growth and alkaloid production of Claviceps purpurea strain pepty 695 in submerged culture depend upon the presence of two different carbon sources in the medium. The efficacy of different compounds seems species-specific. The mostly used carbon sources in the cultivation of ergot fungi are sugar alcohols and sucrose (ARCAMONE et al. 1961; MAIER et al. 1971) as well as a mixture of sucrose and galactose (TABER and VINING 1958). The finding that glycerol can replace sucrose (Table 1.) indicates that pepty 195 must be able to carry out at least partially gluconeogenesis and strongly points to the occurrence of fructose-1.6-biphosphatase. The influence of organic acids on growth and alkaloid production in strains of C. purpurea is different and appears to be species-specific, e. g. in a strain capable to produce lysergic acid derivatives, succinate, malate as well as tartrate were most effective, whereas citrate was much less effective. No growth could be achieved with lactate, acetate, and oxalate (ARCAl\IONE et al. 1961). ARCAMONE and his associates reported a fermentation of ergotamine(1970). The most suited organic acid was found to be citrate. In a mixed fermentation of two strains of C. purpurea producing ergotamine, ammonium oxalate was used a nitrogen source (KOBEL 1975). A strain of C. fusiformis producing clavine alkaloids was supplied with ammonium sulfate and 2 per cent calcium carbonate which were more suited than L-asparagine, L-glutamate, succinate or citrate (BANKS et al. 1974). Asparagine was ruled out as in nitrogen source in a strain of C. purpurea producing clavine alkaloids

Citric Acid Cyrle

669

(REHACEK et al. 1977). Several authors did show that the organic acids supplied are taken up by the ergot fungus and are metabolised (A:mcI et al. 1967; TABER 1968, 1971; see also Fig. 1.). From our data it must be emphasized that without any citrate in the culture medium glucose and ammonium ions do not support growth (Fig. 1.). All those acids which can replace citrate in pepty 695 are either intermediates of the CAC or metabolically related to it (Table 2.). This might be true also for oxalate (DIJKHUIZU et al. 1977) as well as for calcium carbonate which could be introduced in the CAC by anaplerotic carboxylations. In Pepty 695 a complete CAC occurs which functions in energy production and in anabolism. This statement is based in three different lines of experimental evidence: (a) the inhibition of growth by fluoroacetate, (b) the evolvement of 14C0 2 from specifically labelled acetates and pyruvates and the kinetics of 14C0 2 release, (c) the speciiic inhibition of citrate synthase by ATP. Obviously, alkaloid production is accompanied by a decline in activity of the citric acid cycle. This conclusion is based on the finding that the specific activity of citrate synthase constantly declines after onset of alkaloid production. Under non-producing culture conditions as well as in strains incapable to produce alkaloids, CS activity is not changed. This is in accordance with REHACEK (for review see REHACEK 1974) who r;howed that in contrast to a non-producing Claviceps strain, a procuder strain had a very low specific activity of citrate Sy nthase. One could speculate that ergot alkaloid production might be analog to ketone-body formation in animals. Ketogenesis is the result of an overflow of acetyl coenzyme A under certain circumstances (LOPES-CARDEZO et al. 1975). Interestingly, ergot alkaloid production of Claviceps is paralleled by other acetyl coenzyme A consuming processes, such as fatty acid biosynthesis and formation of a type of pigments which are ultimately derived from acetyl coenzyme A (GRiiGER 1972). Experiments performed to increase the intracellular level of acetyl coenzyme A by feeding of acetate were, however, unsuccessful because of the toxic effect of acetate against pepty 695. Acetate applied to mycelia at concentrations ranging from 1 gil up to 10 gil culture broth (medium 720) caused a total inhibition of growth and alkaloid production. Toxicity of acetate against microoorganisms was reviewed by HUETING and TE1[PEST (1977). Acknowledgements We wish to thank D. GROGER and his assoriates, Institute of Plant Biochemistry, Halle, for kindly providing the inoculum of the strains used and many technical support and them as well as C. WASTERNACK from our laboratory for helpful discussions.

References AMICI, A. :,\1., :'\iINGHETTI, A., SCOTTI, T., SPALLA, C., and TOG NOLI , I.: Ergotamine Production in Submerged Culture and Physiology of Claviceps purpurea. Appl. :.\Iierobiol. 15, 597-602 (1967). ARCAMONE, F., CASSINELLI, G., PERNI, S., PENCO, S., PENN ELLA, P., and POL, C.: ErgotamineProduction and Metabolism of Claviceps purpurea Strain 275-F1 in Stirred Fermenters. Can. J. Microbiol. 16, 923-931 (1970).

670

K. GLCm, D. SCHLEE and H. REINBOTHE

CHAIN, K B., ~FERRETTI, :F. R. S. A., :JhNGHETTI, A., PENNELLA, P., TONOLO, A., and VERO, L.: Produetion of a l'\ew Lysergie Acid Derivative in Submerged Culture by a Strain of C!aviceps paspa!i STEYENS & HALL. Proceedings of the Royal Society, B, 115, 26-54 (1961). BANKS, G. T., .MANTLE, P. G., and SZCZYRBAI(, K.: Large Scale Production of Clavine Alkaloids by C!aviceps fusiform is. J. Gen. Microbiol. 82, 345-361 (1974). BULLA, L. A. Jr., JCLIAN, G. S., and RHODES, R. A.: Physiology of Sporforming Bacterilt Associated with Insects. III. Radiorespirometry of Pyruv,tte, Acetate, Succinate and Glutamate Oxidation. Can. J. Microbiol. 16, 1073-1079 (1971). CHAPMAN,E.A., and GRAHAM, D.: The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves 1. Relative Rates of the Cycle in the Dark and the Light. Plant Physiol. 53, 879-885 (1974). CONNETT, R. J., and BLUM, J. J.: Metabolic Pathways in Tetrahymena. Estimation of Rates of the Tricarboxylic Acid Cycle, Glyoxylate Cycle, Lipid Synthesis and Related Pathways by Use of Multiple Labelled Substrates. J. BioI. Chem. 247, 5199-5209 (1972). DECKER, L.: Die aktivierte Essigsiiure. Enke, Stuttgart 1959. DIJKHUIZEN, L., WIERSMA, M., and HARDER, W.: Energy Production and Growth of Pseudomonas OXI on Oxalate and Formate. Arch. Microbiol. 115, 229-236 (1977). GLUND, K.: Doct. Thesis, University of Halle (1978). GROGFR, D.: Ergot. In: Microbial Toxins, Vol. 8, p. 321-373 (Ed. KADIS, S., CIEGLER, A., and AJL, S. 1.). Academic Press, New York and London 1972. HAGEMAN, M. E., and RINNE, R. W.: Glucose, Pyruvate and Acetate :JIetabolism by Developing Soybean Seeds. Plant & Cell Physiol. 1 'i', 501-507 (1976). HUETING, S., and TEMPEST, D. W.: Influence of Acetate on the Growth of Candida uti!is in Continuous Culture. Arch. Microbiol. lUi, 73-78 (1977). KARLIN, J. N., BOWMAN, B. J., and DAVIS, R. H.: Compartmental Behaviour of Ornithine in Neurospora crassa. J. BioI. Chem. 252, 3948-3955 (1975). KIRST AN, K.: Diplomarbeit, University of Halle 1977. KOBEL, H.: Bildung von Ergotoxinalkaloiden in Misehkultur. In: Conference on Medicinal Plants (Abstracts), Marianske Lazne 1975. KUN, E.: Mechanism of Action of Fluoroanalogs of Citric Acid Cycle Compounds: An Assay on Biochemical Tissue Specifity. In: Citric Acid Cycle, Control and Compartmentation (Ed. LOWENSTEIN, J. M.), p. 297-339. New York and London: MARCEL DECKER 1969. LOPES-CARDEZO,1\I., MULDER, 1., VUGT, F. VAN, HERMANS, P. G. C., and BERGH, S. G. VAN DEN; Aspects of Ketogenesis: Control and Mechanism of Ketone-Body Formation in Isolated Rat-Liver Mitochondria. Moler. & Cell. Biochem. 9, 155-173 (1975). LOWENSTEIN, J. 1\1.: Chemical Methods for Citrate and Aconitate. In: Methods in Enzymology. Vol. 8 (Ed. COLOWICK, S. P., and KAPLAN, N. 0.). Academic Press Inc., New York 1969. LOWRY, O. H., and ROSENBROUGHT, N. J.: Protein Measurement with the Folin Phenol Reagent. J. BioI. Chem. 193, 265-275 (1951). LUCAS, C., and WEITZMAN, P. D. J.: Regulation of Citrate Synthase from Blue-Green Bacteria by Succinyl Coenzyme A. Arch. Microbiol. 114, 55-60 (1977). . MAIER, W., ERGE, D., and GROGER, D.: Zur Biosynthese von Ergotoxinalkaloiden in C!aviceps purpurea. Biochem. Physiol. Fflanzen 161, 559-569 (1971). - - - Uber Aktivierungsreaktionen bei C!aviceps. Biochem. Physiol. Pflanzen 163, 432-442 (1972). McDONALD, J. K., CHELDELIN, V. H., and KING, T. K: Glucose Catabolism in the Ergot Fungus C!aviceps purpurea. J. Bact. 80, 61-71 (1960). MORTON, A. G., and :J!IcMILLAN, A.: The Assimilation of Nitrogen from Ammonium S,tlts and Nitrate by Fungi. J. Exp. Bot. 5, 232-252 (1954). REHACEK, Z.: Ergot Alkaloids and Some Problems of the Physiology of their Formation. Zbl. Bakt. Abt. II, 129, 20-49 (1974).

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Citric Add Cycle

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Authors' address: Dr. KO:'lRAD GLUND, Doz. Dr. DIETER SCHLEE and Prof. Dr. HORST REIN BOTHE, Sektion Biowissenschaften, Pflanzenbiochemische Abteilung, Martin-Luther-Universitiit, Halle-Wittenberg, DDR-401 Halle (Sa ale), Neuwerk 1.