Plant Physiol. (1983) 71, 822-8270032-0889/83/7 1/0822/06/$00.50/0

Changes in the Activities of Pyrrolooxygenases during theGermination of VVheat Grains'

Received for publication September 22, 1982 and in revised form December 6, 1982

ADRIANA R. SBURLATI AND ROSALIA B. FRYDMANFacultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Junin 956, Buenos Aires (1113) Argentina

ABSTRACT

Porphobilinogen oxygenase, skatole pyrrolooxygenase, and tryptophanpyrrolooxygenase were found in the different parts of germinating wheat(Triticum aestiwum) grain seedlings. In the embryos of grains germinatedfor 24 hours, the activities ofPBG oxygenase and skatole pyrrolooxygenasewere inhibited by a labile inhibitor. Tryptophan pyrrolooxygenase activitywas not inhibited. Embryos of grains germinated for 48 hours showedhigher activities for the three enzymes. The latter were also present in theradicles and coleoptiles of 96-hour germinated wheat grains. A DEAE-cellulose anaylsis of a crude enzymic preparation from embryos allowedthe separation of two molecular forms of the three pyrrolooxygenases. Themore cationic forms of porphobiinogen oxygenase and skatole pyrroloox-ygenase were associated with the inhibitor. This form of porphobilinogenoxygenase had allosteric kinetics while the more anionic form had Mi-chaelis kinetics. Both forms of skatole pyrrolooxygenase had Michaeliskinetics. The activity of tryptophan pyrrolooxygenase was highest inseedling roots and was found to be inhibited in seedling young leaves. Thisenzyme oxidized tryptophanyl dipeptides, as well as a nonapeptide, to N-formylkynurenine-containing peptides. The pyrrolooxygenase also oxidizedthe tryptophanyl residues of lysozyme, chymotrypsin, and trypsin.

Pyrrolooxygenases are mixed function oxidases that oxidizepyrrole derivatives as well as the pyrrole ring of indoles (3). Threeenzymes were identified within this group: PBG2 oxygenase, tryp-tophan pyrrolooxygenase, and skatole pyrrolooxygenase. PBGoxygenase oxidizes PBG to give 2-hydroxy-5-oxoporphobilinogenas the main product and 5-oxoporphobilinogen as a minor product(Fig. 1) (10). This enzyme was isolated and purified from wheatgerm (10, 17) and it was found to be a cationic iron-sulfur protein.It was also isolated from pepper, poinsettia, and Swiss chard leaves(8), as well as from rat liver (16), rat blood reticulocytes (18), andhuman erythrocytes (7). It was shown in mammals that theoxygenase is involved in the control of heme biosynthesis (9, 18),and it is very likely that in plants the enzyme is also involved inthe control of heme and Chl biosynthesis, since PBG is theprecursor of both.Trp pyrrolooxygenase was isolated only from plants (2, 8). The

oxidation of Trp by the enzyme produces N-formylkynurenine(Fig. 1) (5), the same product that is formed by Trp pyrrolase inmammals. In contrast to the pyrrolase, Trp pyrrolooxygenase ofwheat germ also oxidizes tryptophanyl-containing peptides andproteins (6). The oxidation proceeds without cleavage of thepeptide bond.

1 Supported by the Secretaria de Estado de Ciencia y Tecnologia(Argentina).

2Abbreviations: PBG, porphobilinogen; Trp, tryptophan.

Skatole pyrrolooxygenase was isolated from plants (2, 8) andwas also detected in certain animal tissues (3). It oxidizes skatoleas well as IAA to 2-formamidoacetophenone (Fig. 1). Its possiblephysiological role in plants is still to be assessed.

Plant pyrrolooxygenases exist in multiple forms which differ intheir ionic, molecular, and kinetic properties (2, 8, 15). Pyrroloox-ygenase activities in both, plant and mammals, is controlled andsometimes completely masked by the presence of an inhibitor ofproteic nature which was isolated from preparations of wheatgerm (10). The interaction between the inhibitor and PBG oxy-genase was extensively studied during erythropoietic stress con-ditions in rat blood and bone marrow (18). The inhibitor was animportant factor in the regulation ofPBG oxygenase activity bothin rats and humans (7, 16). It was therefore of interest to examineif during germination and early seedling growth there was also aninteraction between the inhibitor and pyrrolooxygenase activities.Inasmuch as the physiological significance of pyrrolooxygenasesin plants was not so extensively examined as in mammals, andbecause Trp pyrrolooxygenase was only isolated from plants, it isof interest to follow the variations of the enzymic activities underthe dynamic conditions of germination and seedling growth. Weobserved that Trp pyrrolooxygenase activities from wheat germvaried greatly with the variety of the wheat grains and the storageconditions of the latter. Since this enzyme could be a useful toolfor Trp oxidation in proteins, it was also of interest to learn moreabout the relation of germination and Trp pyrrolooxygenaseactivity. These considerations led us to the present study whoseaim was to establish the variation in pyrrolooxygenase activitiesduring the processes of germination and seedling growth.

MATERIALS AND METHODS

PBG was obtained by synthesis (4). L-Trp, N-acetyltryptophan,L-Trp methyl ester, tryptophanyl dipeptides, a-chymotrypsin, ly-sozyme, and trypsin were obtained from Sigma. Skatole wascrystallized from ethanol. The nonapeptide 5-carboxy-2-pyrroli-done-Trp-Pro-Arg-Pro-Gln-Leu-Pro-Pro was a generous gift fromSquibb and Sons (New Brunswick, NJ). Sodium dithionite was acommercial sample of analytical grade. DEAE-cellulose was ob-tained from Sigma and was used after treatment by the method ofPeterson and Sober (14).

Plant Material. The experiments were carried out with wheatgrains (Triticum aestivum, var Klein Rendidor). The grains weresurface sterilized by washing with a 0.02% HgCl2 solution andthen washed free of HgCl2. They were then placed in glass beakerswith distilled H20 and shaken at 26°C under aerobic conditions.The water was changed every day. For germination periods longerthan 72 h, the seedlings were grown hydroponically under illu-mination.Enzyme Preparation. The plant material was thoroughly ground

with a pestle in an ice-cold mortar with three volumes of 10 mMTris-HCl (pH 7.4) buffer, (3 ml of buffer/g of fresh weight). Thehomogenate was filtered through several layers of nylon cloth,

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PYRROLOOXYGENASES IN GERMINATING WHEAT GRAINS

:02H -ftH2'WI~ ~ ~ O~N2 HO

H

N- FORMYLKYNURENINE

0

FICH3go HOH

2 -FORMAMIDE-ACETOPHENONE

FIG. 1. Products formed by the action ofpyrrolooxygenases on pyrrolesand indoles.

and centrifuged at 20,000g for 30 min. All operations were per-formed at 4°C. The supernatant was filtered through two layersof cheese cloth and used as an enzyme source either for directactivity measurements or for further enzyme purification. Proteinwas estimated by the method ofLowry (12) using BSA as standard.

Separation of Different Forms of Pyrrolooxygenases. The sep-aration of different forms of pyrrolooxygenases was performed byDEAE-cellulose chromatography using a stepwise elution proce-dure. When the 48-h germinated grain embryo preparation wasanalyzed (Fig. 2), 1 ml (18 mg ofprotein) was applied on a DEAE-cellulose column (1.1 x 18 cm) previously equilibrated with 10

mm Tris-HCl buffer (pH 7.4). Fractions of 1 ml were collected.The column was first eluted with the same buffer which eluted themore cationic form (peak A), and then with 50 mm NaCl in thesame buffer which eluted the less cationic form (peak B). Whenthe preparations from radicles of 96-h germinated grains wereanalyzed (Fig. 3), 3 ml of the extract (13 mg of protein/ml, 1 gfresh weight) were applied to a DEAE-cellulose column (1.5 x 18cm) and 2-ml fractions were collected. The column was eluted asdescribed above.Enzyme Assays. When PBG oxygenase and skatole pyrroloox-

ygenase were assayed, the reaction mixture contained in a finalvolume of 100 psl: 10 ,umol of sodium phosphate buffer (pH 7.4),25 nmol of PBG or 42 nmol of skatole, 0.1 yImol of sodiumdithionite (which was used as the reducing agent), and enzyme.The amounts and protein concentrations of the latter were thoseindicated in each case. Incubations were run for 30 min at 370C.Blanks omitting either the enzyme or the reducing agent were runsimultaneously. Enzymic activity was assayed by measuring sub-strate consumption with Ehrlich's reagent (2% w/v ofp-dimethy-

EcqC%

-

w

z

Go0C)CDm

C')aw(1)-I

-4

0z(AcKrna

0

_ Tris OmM _ NMaCI 50 mM

FRACTION NUMBERFIG. 2. DEAE-cellulose elution profile ofPBG-oxygenase, skatole, and

Trp-pyrrolooxygenase of the embryos from 48-h germinated wheat grains.Conditions ofcolumn run and enzyme assays were described in "Materialsand Methods." The protein content was detected by its 230 am A, sincepyrrolooxygenases show little 280 nm A. Activity was measured immedi-ately after elution using 50 id of enzyme. Incubations were carried out at37°C for 30 min. A, Protein (0) and PBG oxygenase (A) elution profiles.B, Trp-pyrrolooxygenase (A) and skatole pyrrolooxygenase (0). Thedashed lines show the enzymic activities measured 48 h after elution.

laminobenzaldehyde in glacial acetic acid-perchloric acid (84:16,v/v). The colored reactions were measured at 552 nm for PBGand at 540 nm for skatole, after previous addition of HgC12.When Trp pyrrolooxygenase was assayed, an incubation mix-

ture as described above was used, except for the substrate whichwas either L-Trp (100 nmol), or substituted tryptophanyl deriva-tives with the equivalent Trp content, or tryptophanyl-containingenzymes (2 mg/ml, 25 or 50 ,ul). Blanks were run simultaneouslyomitting either dithionite or enzyme, which were added after theincubation was completed. The reaction was stopped by dilutingthe reaction mixture with 1 ml of water. Trp consumption wasestimated either at 280 nm (e = 5,500), or by its fluorescence at350 um (excitation at 280 nm).Analysis of the Oxidation Products of Trp and Tryptophanyl

Derivatives. The incubation mixtures were analyzed by TLC onsilica gel (Merck; thick layer, 0.25 mm) using as developing solvent

PBG

N NHITRP

XH H3

H

SKATOLE

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SBURLATI AND FRYDMAN

Ec0rf)Nl

IA~~~~~~~~~~~~C

0 0.5

4~~~~~~~~~~~~~co

2

z

0. 0020 3 0 5

1)

0 32

II

I0~

0 10 20 30 40 50Tris 10mM _ NoCI SOmMM

FRACTION NUMBERFIG. 3. DEAE-cellulose elution profile of PBG-oxygenase and skatole

pyrrolooxygenase of the radicles of 96-h germinated wheat grains. Theconditions of the column run and assay were those described in "Materialsand Methods." Activities were assayed using 50 pl of enzyme, PBG-oxygenase (A) and skatole pyrrolooxy.enase (0) were assayed immediately(--) and 48 h after (---) eluting the column.

the upper layer of butanol:acetic acid:H20 (4:1:5, v/v). The plateswere run overnight and the spots were located either by theirfluorescence under UV light or by spraying with Ehrlich's reagent.Trp or Trp derivatives gave bluish spots, while N-formylkynuren-ine, kynurenine, and their derivatives gave yellow-orange spots.The analysis ofthe oxidized tryptophanyl derivatives and peptideswere performed as described elsewhere (6).

RESULTS

Pyrrolooxygenase Activities in Wheat Grains at DiferentStages of Germination. During the germination process of wheatgrains it was possible to measure pyrrolooxygenase activities bothin the embryo and in the endosperm. All the three known activi-ties: PBG oxygenase, skatole pyrrolooxygenase and Trp pyrro-looxygenase were present in the tissues of the germinating grains.From our previous studies with crude extracts from wheat germ,it was known that pyrrolooxygenase activities decreased when

Table I. Comparison of the Activities ofPyrrolooxygenases in the ExtractsPreparedfrom Wheat Seedlings at Dfferent Stages of Germination

The incubation mixtures were those described in "Materials andMethods." The crude enzyme preparations were used. The protein contentwas 16 to 18 mg/ml for the embryos and 11 to 13 mg/ml for the coleoptilesand roots. Values in parentheses were obtained after a 48-h storage of theextracts at 4°C.

Germination Enzyme Seedling Substrate ConsumedTime Used Tissue PBG Skatole Trp

h ,u nmol/30 min5 7.8 (6.7) 14.0 (14.7) 13.5

24 10 Embryo 6.6 (8.1) 11.0 (17.6) 17.025 4.3 (11.7) 4.0 (19.6) 23.0

5 12.8 (11.5) 21.0 (21.0) 25.048 10 Embryo 11.8 (13.5) 23.0 (25.0) 27.0

25 11.2 (15.7) 17.0 (27.0) 30.5

5 13.8 (13.3) 28.0 (27.3) 23.772 10 Radicle 13.0 (16.7) 26.0 (30.0) 30.6

25 7.8 (19.2) 20.0 (32.5) 32.0

5 5.0 (5.3) 22.0 (21.6) 3.472 10 Coleoptile 10.5 (10.0) 23.5 (27.0) 10.0

25 10.0 (15.0) 15.0 (36.0) 23.0

higher concentrations of the extracts were used in the assays. Thiswas due to the presence in the extracts of a protein inhibitor whichwas isolated during the purification of the enzymes (10). Thisinhibitory effect obtained by increasing the concentration of thecrude extracts disappeared after a storage of 48 h at 4°C, due tothe lability of the inibitor (5, 10, 16, 18). This simple assay of thepresence of a pyrrolooxygenase inhibitor was also used when theenzymic activities were measured in the different tissues. Theenzymic activities increased in the embryo during the first 48 h ofgermination and the largest increase was found in the activity ofTrp pyrrolooxygenase (Table I). PBG oxygenase and skatolepyrrolooxygenase activities decreased when higher enzyme con-centrations were used for the enzymic assay, indicating that theinhibitor was present in the preparations. The highest inhibitionwas observed in the embryo preparations of 24 h germinatedgrains, and the inhibitory effect decreased with longer germinationtimes (Table I). This type of inhibition was absent when Trppyrrolooxygenase activity was measured. The presence of thelabile inhibitor was confirmed when the activities ofPBG oxygen-ase and skatole pyrrolooxygenase were assayed after 48 h storage.Under those conditions, the activities increased with enzymeconcentration. The enzymic activities measured after a 48-h stor-age period indicated that there was less inhibitor present in theembryos of the 48-h germinated grains than in those of the 24-hgerminated grains.

Although the activities of the purified pyrrolooxygenases in-creased as expected with enzyme concentration, the values ob-tained with the crude extracts departed from linearity at higherenzyme concentrations (Table I). With enzyme concentrations upto 5 p,1. a linear increase in activity with enzyme concentrationwas obtained; but, at the higher enzyme concentrations used todetect the presence ofthe inhibitor, the pyrrolooxygenase activitiesin the crude extracts were not linear. This effect was also evidentin the preparations obtained from 48-h germinated seedlingsstored during 48 h, and was therefore unrelated to the presence ofthe inhibitor (see also activities of Trp pyrrolooxygenase). Thereare many possible explanations for this effect; one of these may berelated to the existence of multiple molecular and oligomericforms of the enzymes.

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PYRROLOOXYGENASES IN GERMINATING WHEAT GRAINS

ChymotrypsinC

III' %

I %I/

320 340 360

WAVELENGTH380 400

(nm)

320 340 360 380 400

WAVELENGTH (nm)FIG. 4. Fluorescence emission spectra of: A, lysozyme (25 !d); B, trypsin (25 pl); and C, chymotrypsin (50 ld) plus Trp-pyrrolooxygenase (---) and

of the proteins after enzymic oxidation (--). Excitations were made at 280 nm. Trp-pyrrolooxygenase crude extracts (25 ,il, 0.34 mg/prot) of the rootsof 96-h germinated wheat grains were used. Excitations were made at 280 nm. The fluorescence spectra of either lysozyme, trypsin, or chymotrypsinwere not affected by incubation in the presence of dithionite.

Table II. Distribution of Trp-Pyrrolooxygenase Activity among the Tissuesof 96-Hour Light Germinated Wheat Seedlings

The incubation mixtures were those described in "Materials andMethods." Crude enzyme preparations were used (25 ,l of the enzymewere equivalent to 7 mg of fresh tissue weight). Values in parentheses wereobtained after a 48-h storage at 4°C.

Enzyme Source Enzyme Trp Consumed

,ul nmol/30 min

Roots ~~~10 31.6 (29.5)Roots 25 36.5 (37.3)

Coleoptiles10 22.0 (22.0)Coleoptiles 25 31.0 (32.2)

10 32.7 (27.4)Leaves 25 14.5 (32.7)

After 72 h of germination, the radicles and the coleoptiles ofthe wheat seedlings were separated and the enzymic activities weremeasured in both of them. When PBG oxygenase was measuredusing small amounts of enzyme (5 pl), the enzymic activity wasmuch higher in the radicles than in the coleoptiles. Skatole pyr-rolooxygenase activity was also higher in the radicle extracts, andan inhibitor of the enzymic activity was present both in theradicles and the coleoptiles. Trp pyrrolooxygenase activity washighest in the radicle extracts, but the labile inhibitor associatedwith the other two activities was not detected. However, the lackof correlation of Trp pyrrolooxygenase activity with enzyme con-centration (Table I) could still be attributed to the presence of an

Table III. Synthetic Tryptophanyl Derivatives as Substrates of Trp-Pyrrolooxygenasefrom Wheat Seedlings

The enzyme used (25 1d, 7 mg fresh weight) was the preparation ofseedlings of the 96-h germinated wheat grains. Trp (110 nmol) was used,or its equivalent in Trp content when the Trp derivatives were used. Trpconsumption is expressed as the decrease (%) of its maximum fluorescencepeak at 350 nm. Incubations were run for 15 min at 37°C.

Substrate Trp Consumed

L-Tryptophan 39N-Acetyltryptophan 37Tryptophan methyl ester 37Trp-Gly 38Gly-Trp 55Trp-Phe 53Trp-Leu 555-Carboxy-2-pyrrolidone-Trp-Pro-Ary-Pro-Gln- 70

Leu-Pro-Pro

unidentified inhibitor.Multiple Forms of Pyrrolooxygenases Isolated at Different

Stages of Wheat Grain Germination. We have already reportedthat PBG oxygenase and skatole pyrrolooxygenase from wheatflours and from commercial germ could be separated into twoforms by DEAE-cellulose chromatography (2, 15). When a similaranalysis was performed on the enzymic preparation isolated fromembryos of wheat grains germinated during 48 h, it was foundthat all the three pyrrolooxygenases exist in two forms; a strongly

a

7

4

.0%

° 21I

C-

5

w

h-)

3

L 1

OR I I1 * a a.1

10 Lysozyme101A

10

0 60~~~~~~~~0

:0,~~~ ~ ~ ~ ~ ~~~~4

0 0

0

10 20-

0 -

s 0 a I n a 0 0 L-

825

6

ll.

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SBURLATI AND FRYDMAN

charged (peak A) and a less strongly charged form (peak B) (Fig.2). In the case of PBG oxygenase, peak B was the predominantform, while in the case of skatole and Trp pyrrolooxygenase, peakA was the predominant form. The relative proportions of bothforms for each enzyme varied with the germination stage of thewheat grains. The inhibitor, when present, was always associatedwith the more cationic peak A. The inactivation of the inhibitorafter a 48-h storage at4°C resulted in slight increase in the activityof peaks A of both PBG oxygenase and skatole pyrrolooxygenase.The activity ofTrp pyrrolooxygenase was not increased by storageat4°C, due to the absence of an inhibitor of this enzyme.The presence of an inhibitor of PBG oxygenase and of skatole

pyrrolooxygenase associated with the A forms of both enzymeswas evident in extracts from radicles obtained after 96 h ofgermination (Fig. 3). Fraction 13 (Fig. 3, peak A) was highlyenriched in the inhibitor, and could be used as a good inhibitor ofthe active purified preparations of both enzymes. The activities ofthe enzyme forms B were not affected by storage, and weretherefore free of the inhibitor. The data of Figures 2 and 3indicated that the two molecular forms detected for pyrrolooxy-genases in extracts from wheat germ and wheat flours (15) arealso present in the tissues of the germinating grains. Their relativeproportions were dependent of the stage of germination and ofthe tissue from which they were isolated.Form A of PBG oxygenase showed sigmoidal kinetics, regard-

less of the stage of germination of the wheat grain. This type ofkinetics has been described for PBG oxygenase of other sources(10, 17, 18). Form B had Michaelis-Menten kinetics which weredetermined on a purified enzyme isolated from whole seedlingsafter 96 h of germination. Both forms of skatole pyrrolooxygenaseisolated from the same source had Michaelis-Menten kinetics.Trp Pyrrolooxygenase from Wheat Seedling: Distribution and

Substrate Specificity. The presence of a Trp degrading enzyme inwheat seedling is of special interest inasmuch as little is known ofTrp catabolism in plants. The 96-h germinated seedlings are agood source of Trp pyrrolooxygenase, and a study of the distri-bution of this enzyme among the different parts of the seedling isof interest to understand its physiological function. While theactivity of the enzyme is not inhibited either in the root or in thecoleoptile, a strong inhibitor of the enzyme is present in the younggreen leaves. This inhibitor was also inactivated by a 48-h storageof the extracts (Table II). Senescent leaves obtained from wheatseedlings after the 6th d of the start of the germination showed agood activity of Trp pyrrolooxygenase and were devoid of theinhibitor. Therefore, the inhibition of Trp pyrrolooxygenase inyoung growing leaves might prevent the disappearance of Trpfrom the metabolic pool. Trp pyrrolooxygenase has a specialinterest since it might be used for the specific oxidation of tryp-tophanyl residues in peptides and proteins (3, 6). As mentionedabove, the presence of this enzyme in wheat germ was stronglydependent on the source and handling of the latter. It was thereforeof interest to find out a reproducible source for this pyrrolooxy-genase. The seedlings of 96-h germinated wheat grains werealways a good source of the enzyme. It oxidized not only L-Trpbut also tryptophanyl containing dipeptides (Table III). The prod-ucts formed were in every case the N-formylkynurenyl dipeptidescontaining 10 to 20%o of the deformylated product. The latter wereseparated from the substrates by TLC methods (see "Materialsand Methods"), and afforded kynurenine by hydrolysis in acidmedia (6). N-Acetyl-Trp as well as its methyl ester were goodsubstrates of the enzyme. A nonapeptide containing a trypto-phanyl residue was the best substrate among those assayed. Theformed N-formylkynurenyl nonapeptide afforded kynurenine byacid hydrolysis (6). The oxidation of the tryptophanyl residue inthe dipeptides was influenced by the aminoacid bound to Trp,and in the case of the nonapeptide, very likely by the conformationof the latter.

Trp pyrrolooxygenase isolated from the 96-h germinated wheatseedlings also oxidized the tryptophanyl residues of a number ofproteins. The enzymic oxidation of lysozyme, chymotrypsin, andtrypsin, is shown in Figure 4. The oxidations were monitored bymeasuring the decrease in the Trp fluorescence of the oxidizedenzymes. About 50%Yo of the Trp fluorescence of lysozyme, trypsin,and chymotrypsin disappeared as a consequence of the enzymicoxidation by Trp pyrrolooxygenase.

DISCUSSION

The activities of pyrrolooxygenases increased with the germi-nation time of the wheat grains. The activities of PBG oxygenaseand skatole oxygenase were, however, diminished by the presenceof an inhibitor, whose strong inhibitory effect was exerted in thefirst 24 h of the germination. After 48 h of germination, theactivities of the pyrrolooxygenases increased, and it is still un-known if this was due to an increase in the amount of enzyme orto the disappearance of the inhibitor. The fact that Trp pyrroloox-ygenase was not inhibited, and that its activity increased with thegermination time, might explain the low amounts of Trp found incereal grains (13).The existence of multiple forms of the pyrrolooxygenases in

germinating wheat grains as well as the fact that the inhibitorappeared to be associated with one of the forms must reflect thephysiological functions of the enzymes. It is not known if themultiple enzyme forms are isozymes coded by different genes orposttranslational modifications of the former. It is known (8) thatin pepper and poinsettia leaves pyrrolooxygenases also exist inmultiple forms, whose existence depends on physiological param-eters such as fruiting, flowering, and growth conditions. Thepresence in wheat seedlings of an enzyme that oxidizes Trp tokynurenine is noteworthy, since the kynurenine degradative path-way is unknown in plants. There is a report (19) that pea seedlingslices metabolize Trp to a kynurenine derivative, but the existenceof such a pathway was not demonstrated in cereals. The oxidationby Trp pyrrolooxygenase of peptides and proteins which carryTrp residues could be of importance for the inactivation of hydro-lytic enzymes which appear during the germination process. It hasbeen recently shown (1, 1 1) that oxidation of certain proteins andenzymes accelerates their breakdown by proteolytic processes. Trppyrrolooxygenase might be involved in similar oxidative modifi-cations of plant proteins.

LITERATURE CITED

1. FARBER JM, RL LEVINE 1982 Oxidative modification of the glutamine synthetaseof E. coli enhances its susceptibility to proteolysis. Fed Proc 41: Abstr. 3482

2. FLORES MD, ML TOMARO, RB FRYDMAN 1979 Pyrrolooxygenases from Argen-tine wheat varieties. Phytochemistry 18: 1427-1431

3. FRYDMAN B, RB FRYDMAN, ML TOMARO 1973 Pyrrolooxygenases: a new type ofoxidases. Mol Cell Biochem 2: 121-136

4. FRYDMAN B, S REIL, ME DE DEspuY, H RAPOPORT 1969 Pyrroles from azain-doles. Synthesis of porphobilinogen and related pyrroles. J Am Chem Soc 91:2338-2342

5. FRYDMAN RB, ML TOMARO, B FRYDMAN 1972 Pyrrolooxygenases: isolation,properties and products formed. Biochim Biophys Acta 284: 63-79

6. FRYDMAN RB, ML TOMARO, B FRYDMAN 1972 Pyrrolooxygenase: its action ontryptophan containing enzymes and peptides. Biochim Biophys Acta 284: 80-89

7. FRYDMAN RB, ML TOMARO, B FRYDMAN 1979 Porphobilinogen oxygenase fromhuman erythrocytes. Clin Chim Acta 97: 269-278

8. FRYDMAN RB, ML ToMARo, B FRYDMAN 1979 Pyrrolooxygenases from pepperand poinsettia leaves. Phytochemistry 18: 1119-1123

9. FRYDMAN RB, ML TOMARO, B FRYDMAN, A WANSCHELBAUM 1975 Porphobili-nogen excretion in chemical induced porphyria: reversal by induction ofporphobilinogen oxygenase. FEBS Lett 1: 206-210

10. FRYDMAN RB, ML TomARo, A WANSCHELBAUM, EM ANDERSEN, J AwRuCH, BFRYDMAN 1973 Porphobilinogen oxygenase from wheat germ: isolation, prop-erties and products formed. Biochemistry 12: 5253-5262

11. KiRscHNER RJ, AL GOLDBERG 1982 Exposure of erythrocytes to peroxide orsuperoxide stimulates the degradation of cellular proteins. Fed Proc 41: Abstr3479

826 Plant Physiol. Vol. 71, 1983

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PYRROLOOXYGENASES IN GERMINATING WHEAT GRAINS

12. LOWRY OH, NJ ROSEBROUGH, AL FARR, RJ RANDALL 1951 Protein measure-

ment with the Folin phenol reagent. J Biol Chem 193: 265-27513. NELSON JR OE 1973 Breeding for specific amino acids. In AM Srb, ed, Genes,

Enzymes and Populations. Plenum Press, New York, pp 303-31114. PETERSON EA, HA SOBER 1962 Column chromatography of proteins: substituted

celluloses. Methods Enzymol 5: 3-2715. TOMARO ML, RB FRYDMAN 1979 Pyrrolooxygenase isoenzymes from wheat

germ. Phytochemistry 18: 1433-1436

16. ToMARo ML, RB FRYDMAN, B FRYDMAN 1973 Porphobilinogen oxygenase fromrat liver: induction, isolation and properties. Biochemistry 12: 5263-5268

17. ToMARo ML, RB FRYDMAN, B FRYDMAN 1977 Purification and chemical mod-ifications of porphobilinogen oxygenase. Arch Biochem Biophys 180: 239-247

18. TomARo ML, RB FRYDMAN, A GUTrSKY, A SBURLATI 1981 Induction ofporphobilinogen oxygenase and porphobilinogen deaminase in rat blood underconditions of erythropoietic stress. Biochim Biophys Acta 676: 31-42

19. WmTsmHRE GH 1953 The oxidation of tryptophan in pea seedling tissues andextracts. Biochem J 55: 408-416

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