Plant Physiol. (1983) 71, 300-3020032-0889/83/7 1/0300/03/$00.50/0

Endogenous Inactivators of Arginase, L-Arginine Decarboxylase,and Agmatine Amidinohydrolase in Evernia prunastri Thallus1

Received for publication May 12, 1982 and in revised form October 4, 1982

MARIA ESTRELLA LEGAZ AND CARLOS VICENTEDepartment of Plant Physiology, The Lichen Team, Faculty of Biology, Complutense University,Madrid 3, Spain

ABSTRACT

Arginase (EC 3.5.3.1), L-arginine decarboxylase (EC 4.1.1.19), andagmatine amidinohydrolase (EC 3.5.3.11) activities spontaneously decay inEvernia prunastri thalli incubated on 40 millimolar L-arginine used asinducer of the three enzymes if dithiothreitol is not added to the media.Lichen thalli accumulate both chloroatranorin and evernic acid in parallelto the loss of activity. These substances behave as inactivators of theenzymes at a range of concentrations between 2 and 20 micromolar,whereas several concentrations of dithiothreitol reverse, to some extent,the in vitro inactivation.

When lichen thalli are floated on media containing the inducersof certain enzymes, induction of each of these proteins is followedby a loss of its activity after a time of incubation. This fact is truefor urease of Lobaria pulmonaria (18), Parmelia roystonea (19),Cladonia verticillaris (20), and Evernia prunastri (2). Addition ofDTT to the culture media prevents the decay of the enzymeactivity. It is not possible to explain this effect by an accumulationof the end product of reaction inasmuch as DTT does not protecturease when ammonia is supplied to the thalli at inhibitoryconcentrations. It has been postulated that the use of urea by thelichen thallus promotes, through photosynthetic fixation of CO2and carbohydrate translocation from the alga to the fungus, anaccumulation of acetyl-CoA which could be used by an aromaticsynthetase (12) to produce lichen acids which could inactivate theenzymes by blocking their essential-SH groups (2,5).

In the present paper, we attempt to explain the loss of activityof several enzymes of L-arginine catabolism on the basis of theaccumulation of certain lichen acids in E. prunastri thallus.

MATERIALS AND METHODSEvernia prunastri (L.) Ach., growing on Quercus rotundifolia

Lam., was used throughout this work. Samples of 1.0 g of air-dried thallus were floated on 40 mM L-arginine in 0.1 M Tris-HClbuffer (pH 9.15) for arginase induction, or pH 7.5 for simultaneousinduction of both L-arginine decarboxylase and agmatine amidi-nohydrolase. When it is indicated, 1.0 mm DTT was added to themedia. All the incubations were carried out in the dark at 26C.At different times, the samples were washed with distilled H20

and then macerated with sufficient volume (10-15 ml) of theadequate buffer (pH 9.15 for arginase and pH 7.5 for bothdecarboxylase and hydrolase) to obtain a cell-free extract contain-

' Supported by a grant from the Comisi6n Asesora Cientifica y Technicade Presidencia del Gobierno (Spain) No. 3768-79.

ing 1.0 mg protein/ml. The homogenates were centrifuged at27,000 g for 20 min at 4C. Supernatants were filtered throughMillipore GS filters (0.22 ,um pore diameter) and then dialyzedovernight at 4C against 4.0 L of the adequate buffer. Protein wasestimated in the cell-free extracts by the method of Potty (13)using BSA as a standard. Arginase was assayed according toGreenberg's method (6) modified by us (7). A unit of specificactivity is 1.0 ,umol urea produced/mg protein min. L-Argininedecarboxylase was assayed by the conventional Warburg tech-nique (16). A unit of specific activity is 1.0 ,ul CO2 produced/mgprotein-min. Agmatine amidinohydrolase was assayed as indi-cated by Morris and Pardee (9). A unit of specific activity wasdefined as 1.0 ,umol urea produced/mg protein -min.To identify lichen acids, samples of thallus were superficially

washed off with acetone to remove the cortical phenols. Theextracts, dried in vacuum at 37C, contained evernic acid, atra-norin, and chloroatranorin but no D-usnic acid (3). Samples ofthis mixture were chromatographed on a thin layer of Silicagel G-60 using acetone:chloroform (1:1, v/v) as solvent (14) and thespots were located by UV fluorescence by exciting at 360 nm.Characteristics of isolated substances are shown in Table I.The quantitative determination of lichen acids was achieved by

reverse-phase HPLC from lichen samples floated on 40 mM L-arginine as indicated. Chromatographic conditions used were asfollows: column, 300 x 4 mm packed with Micropack MCH-10;mobile phase, acetic acid:water (2:98, v/v): methanol (20:80, v/v)according to Strack et al. (15); flow rate, 1.0 ml min-'; tempera-ture, 20C; range of absorbance on the detector, 0.05; detector,UV set at 154 nm; internal standard solution, gallic acid (SigmaChemical Co.), 0.1 mg -ml-' in methanol; External standard so-lution, evernic acid (Sigma Chemical Co.), 0.1 mg ml-' in meth-anol.

Extractions were carried out after superficial washing of thesamples for 15 min at room temperature with acetone to removethe cortical phenols. The washed thalli were then macerated inacetone and centrifuged at 27,000g for 30 min at 4C. Thesupernatants were dried in vacuum and dissolved in methanol (0.1mg -ml-) to be chromatographed.To test the effect of lichen acids on enzyme activities, 5.0 ,ug of

purified enzyme was incubated for 5 min at optimal pH andtemperature, before the addition of the substrate. When it wasindicated, DTT was included during the preincubation.

RESULTSWhen lichen thalli are incubated on 40 mM L-arginine, enzyme

activity rapidly increases to stabilize (arginase) or decrease (L-arginine decarboxylase) later, whereas this effect is not observedwhen 1 mm DTT is added to the incubation media (Fig. 1).Agmatine amidinohydrolase activity does not appear when DTTis not included in the medium.

Figure 2 shows a chromatogram obtained from a wash of a300

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INACTIVATION OF ENZYMES OF UREA PRODUCTION

Table I. Characteristics of Lichen Acids Isolatedfrom E. prunastri by TLC and HPLC

Lichen Acid TLC UV Fluores- Ama E at A. Relative Retention TimecenceinHLRF x 100 nm min

Atranorin 83 Orange 265 6.37 0.3Chloroatranorin 53 Orange 272 4.79 1.1Evernic acid 8 Dark violet 265 27.64 3.5Unknown substance 14 Light violet 270 8.92 8.8

08 or~~~~~~Q8C

6 0.~~~~~~~~~~~6

4X = 0.248~~~

2 4 6 8hours

FIG. 1. Time-course ofenzyme activity in E. prunastri thallus incubatedon 40 mM L-arginine (0,A,EI) or 40 mM L-arginine + mM DTT(0,A,U). (0,0), arginase; (A,A), L-arginine decarboxylase; (El, U), agma-tine amidinohydrolase.

E

I. ~ ~~~~~I.(

u~~>

c

0~~~~~~~~~~~.0

oC

2 4 6 8 10 12 14 16min utes

FIG. 2. Chromatogram in HPLC oflichen acids extracted from a lichensample freshly collected.

thallus sample freshly collected. All peaks shown could be fullyresolved in less than 15 min. Retention times, shown in Table I,are extremely reproducible. Evernic acid from Sigma ChemicalCo., D- and L-usnic acids from Laako (Finland), and the phenolsisolated and characterized by TLC have been used as markers. D-Usnic acid, described by Culberson (3) as a product ofE. prunastri,has not been identified in the thallus. The peak which shows arelative retention time of 8.8 min has not been identified.The occurrence of substances in the thallus samples incubated

on 40 mM L-arginine is shown in Figure 3. Chloratranorin is the

,,8.0 _680

2 4 6 8hours

FIG. 3. Time-course of lichen acids accumulation in E. prunastri thallusincubated on 40 MM L-arginine at pH 9.15 ( .....) or pH 7.5 ( )(A,A), chloroatranorin; (A), evernic acid; *, unknown substance.

Table II. Effects of Lichen Acids on the Activity of the Enzymes of L-Arginine Catabolism

% of Activation % of InactivationEnzyme Lichen Acid

100 0 50 100/AM

Arginase Chloroatra-norin 7. la 8.3 10.0 11.7

Evernic acid 5.6 7.9 15.8L-Arginine de-

carboxylase Chloroatra-norin 3.9 25.1

Evernic acid 1.2 1.7 7.6

Agmatine amidi-nohydrolase Chloroatra-

norn 2.8 11.2 14.1 17.8Evernic acid 11.2 19.2 22.4 35.5

a Numbers represent concentration of lichen acid that produces theindicated % of activation or inactivation.

only substance accumulated in thalli maintained at pH 9.15.However, evernic acid accumulates in thallus samples when theyare maintained at pH 7.5 for 8 h of incubation, and both chlora-tranorin and the unknown substance are also retained in thethallus at relatively low amounts.Our hypothesis is that lichen substances are endogenous inhib-

itors of the enzymes assayed here (Fig. 1) when a protector of-SHgroups is not added to the medium. We studied the effect of thesesubstances in the three enzymes. For this purpose, 160-fold puri-fied arginase (7), 117-fold purified L-arginine decarboxylase (16),and 460-fold agmatine amidinohydrolase (17) have been used.Arginase is strongly activated by chloratranorin at a range ofconcentrations varying from 1 to 8 ,UM but it is completely inacti-vated by concentrations higher than 12 ,UM. Evernic acid totally

301

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LEGAZ AND VICENTE

Table III. Reversal by D77 of the Enzyme Inactivation by Lichen AcidsAddition to the Incubation Mix- Enzyme Activity

ture

L-Arginine AgmatineLichen acid DT1 Arginase decarboxyl- amidinohy-

ase drolase(M) mM %

100 100 100Evernic acid (18.5) 41.5 0.0 23.6Evemic acid (24.0) 0.0 0.0 0.0Evernic acid (15.8) 1.0 71.1 68.7 57.1Evernic acid (24.0) 2.0 0.0 121.4 35.7Chloroatranorin (12.6) 0.0 63.7 0.0Chloroatranorin (18.6) 0.0 0.0 0.0Chloroatranorin (12.6) 2.0 0.0 129.0 56.4Chloroatranorin (12.6) 5.0 29.6Chloroatranorin (18.6) 2.0 0.0 49.3 56.4Chloroatranorin (18.6) 5.0 11.8

inactivates the enzyme at concentrations higher than 16 ,M (TableII). A similar behavior is shown by agmatine amidinohydrolase,which is activated by both chloroatranorin and evernic acidbetween 1 and 10 uM and strongly inactivated above 14 lsM,whereas L-argmiine decarboxylase is always inactivated by bothphenolics at the assayed concentrations (Table II).

Inactivation of these enzymes can be partially reversed byincluding adequate concentrations of DTT in the incubationmixtures (Table III).

DISCUSSIONE. prunastri has been defined as a usnic acid-producing species

(3), and even a catabolic usnic acid dehydrogenase has beencharacterized in this lichen (4). Thus, the substance called here'unknown' had been described (1 1) as D-usnic acid on the basis ofits RF value in TLC, micro-crystallization reactions, and UVfluorescence. However, we have concluded that, in HPLC, thereis no substance in lichen extracts that behaves as the standard. Inaddition, Strack et al. (15) did not find D-usnic acid in E. prunastriextracts by the same procedure.

Nevertheless, atranorin has been identified as a substance whichshows a relative retention time of 0.3 min, but it is not found inthallus extracts. This fact can be explained in terms of an abso-lutely extracellular occurrence of atranorin which is totally re-moved by the acetone washing previous to the maceration (dataare not shown). Thus, atranorin has been excluded from theenzyme analysis because it is not an intracellular metabolite.As deduced from the in vitro studies, arginase, L-arginine decar-

boxylase, and agmatine amidinohydrolase are inactivated by bothchloroatranorin and evernic acid at the concentrations shown inTable II. Density of E. prunastn thallus has been calculated as0.36 g cm3. On this basis, chloroatranorin concentration in thal-lus incubated at pH 9.15 is about 5.3 mm after 4 h of incubation.When the thalli are floated at pH 7.5, chloroatranorin concentra-tion decreases from 1.76 mm at 4 h incubation to 0.35 mM at 8 h.At this time, concentration of evernic acid in the thallus (Fig. 3)is about 8.56 mm, whereas this compound is practically absentduring the first 6 h of incubation. These values can explain theloss of enzyme activity found after 4 or 6 h of culture (Fig. 1).

Inasmuch as the inactivation is prevented by including DTT inthe media as well as in the incubation mixtures, both chloroatra-norin and evernic acid can clearly be related to the in vivoinactivation of the enzymes.

However, the concentrations of both phenolics shown in thefirst hours of culture (Fig. 3) might promote inactivation of thethree enzymes, but this does not appear. This apparent paradoxcan be explained. Legaz and Vicente (8) report that 73% arginase,18% L-arginine decarboxylase, and 50%o agmatine amidinohydro-lase are located in the phycobiont cells in terms of total activity.Otherwise, the fungal part of lichens may perform the completesynthesis of depsides (10) and then excrete them to the cortex,outside the cells, as well as retain discreet amounts of them insidethe hyphae. In this way, algal enzymes would be protected againstactivation. Even fungal enzymes must be protected if depsideswere restricted in their own compartment isolated from the cyto-plasm, but this is now controversial. Ahmadjian et al. (1) suggestthat the concentric bodies may be involved in the synthesis andaccumulation of lichen substances. In this case, the said depsidesin the fungal partner would be inaccessible to the soluble enzymesand these would then be protected against inactivation.

LITERATURE CITED

1. AHMADJIAN V, LA RussELL, KC HILDRETH 1980 Artificial reestablishment oflichens. I. Morphological interactions between the phycobionts of differentlichens and the mycobionts Cladonia cristatella and Lecanora chrysoleuca.Mycologia 72: 73-89

2. CIFuENTEs B, MP EsTvEzZ, C VICENTE 1981 In vivo protection of urease ofEvernia prunastri by dithiothreitol. Physiol Plant 53: 245-248

3. CULBERSON CF 1969 Chemical and Botanical Guide to Lichen Products. TheUniversity of North Carolina Press, Chapel Hill, pp 346-347

4. EsTiEVz MP, ME LEGAZ, L OLMEDA, FJ PtERz, C VIcENTE 1981 Purificationand properties of a new enzyme from Evernia prunastri, which reduces L-usnicacid. Z Naturforsch 36c: 35-39

5. GARciA I, B CIFuENmTS, C VIcENTE 1980 L-usnate-urease interactions: bindingsites for the ligand. Z Naturforsch 35c: 1098-1100

6. GREENBERG DM 1955 Arginase. Methods Enzymol 2: 368-3747. LEGAZ ME, C VIcENTE 1980 Arginase regulation in Evernia prunastri. Cryptog

Bryol Lichenol 1: 407-4148. LEGAZ ME, C VICENTE 1981 Location of several enzymes of L-arginine catabo-

lism in Evernia prunastri thallus. Z Naturforsch 36c: 692-6939. MoRRIs DR, AB PARDEE 1966 Multiple pathways of putrescine biosynthesis in

Escherichia coli J Biol Chem 241: 3129-313510. MOSBACH K 1973 Biosynthesis of lichen substances. In V Ahmadjian, ME Hale,

eds. The Lichens. Academic Press, New York, pp 523-54611. ORiUS MI, MP ESTEVEZ, C VICENTE 1981 Manganese depletion in chloroplasts of

Quercus rotundifolia during chemical simulation of lichen epiphytic states.Physiol Plant 52: 263-266

12. PACKTER NM 1980 Biosynthesis of acetate-derived phenols (polyketides). In PKStumpf, ed, The Biochemistry of Plants, Vol 4. Academic Press, New York, pp535-570

13. Po=rv VH 1969 Determination ofproteins in the presence ofphenols and pectins.Anal Biochem 29: 535-539

14. SANTESSON J 1965 Studies on the chemistry of lichens. 24. Thin layer chromatog-raphy of aldehydic aromatic lichen substances. Acta Chem Scand 19: 2254-2256

15. STR-ACK D, GB FEIGE, R KRoLL 1979 Screening of aromatic secondary lichensubstances by high performance liquid chromatography. Z Naturforsch 34c:695-698

16. VICENTE C, ME LEGAZ 1981 Purification and properties of L-arginine decarbox-ylase of Evernia prunastri. Plant Cell Physiol 22: 1119-1123

17. VicENTE C, ME LEGAZ 1982 Purification and properties of agmatine amidino-hydrolase of Everniaprnnastri Physiol Plant 55: 335-339

18. VIcENTE C, M PAsI, MP EsmvEz 1978 Urease regulation mechanisms inLobaria pulmonaria Rev Bryol Lichenol 44: 83-89

19. VicENTr C, L XAvIER FILHo 1978 Exo- and endourease from Parmelia roystoneaand their regulation by lichen acids. Bol Soc Broteriana 52: 55-69

20. VIcENTE C, L XAVIER FILHO 1979 Urease regulation in Cladonia verticillaris.Phyton 37: 137-144

302 Plant Physiol. Vol. 71, 1983

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