Polyphenol oxidases in plants-recent progress

Phyrochrmutry. Vol 2b. No I. pi

I I-20. 1987

0031 -9422 87 s3 no + 0 00 & 1987 Pcrgmoa Joumh Lid

mntcd I” cmc &r~n

REVIEW ARTICLE

POLYPHENOL

OXIDASES

NUMBER

22

IN PLANTS-RECENT

PROGRESS

ALFRED M. MAYER Dqmnmm

OT Botany, Hebrew Univcrs~ty of Jcruclkm

(Rccerurd 23 Jun Key W& me&anti

I&x--Higher plan& polypkol physiological function.

Jcrusakm, Israe

1986)

oxideuc. catachol oxiduc; Layc;

propertia; structure; r*Icllon

Aktract-Progress in the plant polyphcnol oxidascs in the period 1978-1986 is summarized. Methodology. occurrence. properties and physiological function of laccascs and catcchol oxidases art critically reviewed. The advances in the understanding of reaction mechanisms are cited. The plaot polypbcnoloxidascs remain enzymes in search of a function. -

MEll4ODOLOGY

ISTRODUCnON

USED IN ASSAY ASD STUDY OF EKZYM): PUOPUTIES

Since we last reviewed the plant polyphcnol oxidascs [ 1J the international nomenchture has again been changed, monophenol monooxygenau (tyrosti) being referred IO as 1.14.18.1, diphcnol oxidase (catcchol oxidasc. diphenol oxygen oxidorcductasc) as 1.10.3.2. and laozase as I. 10.3.1. This new classification is no improvement since it differentiates bctwoen IWO reactions of the same enzyme, formerly referred IO ascrcsolasc and catccholascactivities. At the same time the ckarly distinct laocases from fungi and higher plants are still given the same number. We will therefore retain the general terms ofcatcchol oxidasc and laccasc for the sake of simplicity. A large number of papers have been published sinoz the last attempt 10 review the topic extensively and it is incvitabk that we will be sckctivc in our coverage; yet an attempt will be made IO deal with various aspazts of polyphenol oxidasc biochemisLry and physiology. A number of reviews covering certam aspects have appeared in recent years. Laocasc has been reviewed with particular stress on the properties of the enzyme and the nature of the catalytic site [Z-6]. These bring surveys of the reaction mechanism up IO date almost to the time of writing of this review. Catcchol ox&se has also been reviewed, but kss comprehensively. Its reaction mechanism has been considered 17-91 and the mokcular properties of tyrosinasc rcvkwcd [IO, 1I] and attention given to its overall biological function and subcellular location [ 17 131. As will be seen in the following saclions, program has ban impressive in unravclling the basic structure of several of the polyphcnol oxidascs and in understanding some of the basic mahanisms of the oxidation-reduction process. However, in the understanding of the biological function, progress has ban very slow and this problem remains csscnlially unsolved.

In the determination of laozasc, syringaldazinc has been succasfully used for the quantitative assay of the fungal enzyme [ 14-16J. thus extending the use as first described 1171. The use of L6dimcthoxyphcnol as a laccasc substrate is also worth mentioning [IS]. The number of substrates reported IO be attackal by laccasc seems to increase steadily. The location of both laccasc and catechol oxidase by clearon microscopy using specific substrates has been attemptai. These have located catcchol oxidase quite ddinitely in phstid thylakoids [ 19-21 J, in accord with previous reports [l]. In the c8x of lactase definite assignment of its locatlon using the EM is uncertain because its location is based on the assumpuon that failure to oxidize tyrosinc and the ability to oxidlzc DOPA can be regarded as proof of la-se activity and prccludacatcchol oxidase activity [22 J. Thecytochemical localization of catccbol oxidasc outstde plastids (e.g. In fungal cells) and of laccasc still presents serious problems. HPLC has ban used to follow enzyme actlvlty and is obviously useful m order to separate the products of enzyme ac[ivlty, many of which arise by secondary reactions [23-251. Some success has been obtained usmg antibodies raised agamst either laocase [26, 271 or catechol oxidase 12%301. Most of the purifications razently reported for catcchol oxidase follow known procedures. but hydrophobic gel chromatography sams to have distinct advanlagcs [3 I, 32],and its use should be extended. The separation of enzymes by ckctrophoresis is now a standard proccdurc. Improvements have been reported by purifiortion of the enzyme in the presence of inhibitors of proteolysis which reduces the numbers of apparent lsozyma on the gel [ 331. Elcctroblotting of catcchol ox~dasc [W] and SDS tech-

II

12

A. M. MAYen

niqucs [ 351 have also been succzssfully applied. lnhtbrtors contmuc to bc used extensively m studies of the physrological function of both laccasc and catechol oxidase. The two activtttcs can be differentiated by the use of cinnamrc acid dcrrvativa to inhibit catcshol oxidasc and cationic detergents such as CTAB to inhibit laccasc [36]. Tropolone acting as a Cu’ ’ chelator is very effective in Inhibiting catcchol ox&se. at concentrations in the PM range 1373. Its cffcct on laccasc has not yet ban reported Often inhibitors are used in order to establish the function of enzymes. It cannot bc stressed too strongly that inhibitor studies alone cannot prove the participation of an enzyme in a physiological process, a fact often ignored. An mtercsting if somewhat exotic use for laccasc has been suggested. A lacing membrane ekctrodc has ban constructed and has been used to measure tpincphrinc using a quinolquinone coupk [38]. &case has also been used to measure glucosidasc or amylax activity, by couplmg oxidation of a phenol and 2&dibromo-4aminophcnol [39]. The method was based on the rekasc of a sugar from a phcnolic glycosidc followed by the cnzymic oxidation of the phenol rckascd. The possible uses of laccasc or catechol oxidasc for analytical studies on the one hand or the immobilization of the enzymes in order to use them to carry out controlkd oxidation reactions continued to arousc interest, although until now success has been rather limited. The most impressive ux of modern methodology IS in the analysis of the reaction mechanism of laccasc and catcchol oxidasc. IIK full array of biophysical tcchmqucs has been applied to this problem. Thus EPR [40.41]. ctrcular dichrotsm measurements [42], Raman spcctroscopy [43] and ckctron nuclear resonance spectroscopy 144.451 have all been used in recent work on laaasc. A large number of studies have utilized the technique of partial copper depktion of laaasc [46.47] including puke radiolysis [48]. laser radiation of the depleted enzyme [49] and X-ray absorption studtcs [SO]. Metal substitution by, for exampk, Hg’ [Sl J or removal and rcsubstitution of Cuzs [52] have been used to distinguish between some of the different copper sites of &case. Another approach has ban the titration of the copper l

sites using reagents such asazidc [53]. nitrite [54] or nitric oxide [ 551. Stopped flow kinetics have been used to follow the catalytic reactton sequence of tree lactase 1561. The results obtained usmg these methods will be discussed in the section on structure and reaction mechanism. There have ban fewer reports on catcchol oxidase using these techniques, bccausc of the known EPR silena of copper m the enzyme and because very few really pure preparations of this enzyme are available. The assay of competitive binding of inhibitor has been used to study Meurosporo catcchol oxtdase (tyrosinase) [57.511]. OCCLR~~C.SCL:OF LACCASE ASD CATECHOL OXIDASE Reports continue to appear which support the widcspread occurrena of these enzymes and which mdicatc the almost universal appearance ofcatechol oxidasc. Only a few ofthc more recent reports will becited. Almost every basidiomyccte examined appears to contain l.acca% cxtraand/or intracellular, at least at some stage of its dmelopment. Among the fungal laccascs recently described. that of Srerewn has been partially purified and chara~t~izcd [S9]. Extracellular laccasc appears to be present in many mycorrhizal fungi 1603. In this rapact the observation that most cctomycorrhual fungt do not produa catecho

oxidasc is perhaps sigmficant 161). The spcc~s Lclcrorius is an exception to this gencralizatron and further invcstigations are therefore needed. Other reports do indicate a quite general presence of catcchol oxidasc in mycorrhiza [62]. Roth kccasc and catcchol oxidase are sufficiently stable that they can survive in the decomposing plant residues in soils. In this state they may even become part of the humus complex [6-S]. An extremely new interesting laccasc has ban shown to lx excreted into the growth medium of cells of Acer pseudoplafanum (sycamore) in suspension cultures [66]. This enzyme is of considerabk interest as providing an additional rare exampk of a higher plant laccasc. It is secreted in quite large amounts and appears to be a typical glycoprotein laccasc wrth a very high carbohydrate content. TIK carbohydrate moiety of this enzyme has ban investigated and fully characterized. It has been shown to be an %-linked oligosaccharidc with a recurrent xylosccontaining structural unit [67]. The oligosaccharrda in it are of the biantcnnary complex type. It contains xylostmannox and fucosc-N-acetylglucosamme linkages. An intrrguing enzyme, described as a phenoloxidasc. has been shown to be present in the pathogenic fungus Cryprococcus neojvrmans, which can cause meningitis [68 701. nK presence of the enzyme appears to correlate with the virulence of the pathogen, as shown by expcriments using mutants. Although the enzyme oxtdizcs phenok [68], it seems to be debatable whether it is a catcchol ox&se. smce its substrate specificity and inhibitor response differ considerably from those normally associated with catbchol oxidasc. Further charactertzation is nadcd in this case. There are far more reports on catcchol oxidase both in fungi and plants. Interest m wheat catcchol oxidase has revival because of its possible significance in baking [ 7 I-731. since its presence may cause darkening in dough. The attempt to further the cultivation of yam has led to an investigation of the properties. distribution and amount ofcatcchol oxidasc m various yams, e.g. Dioscorea sp. [74 761 as well as in Xantho.wma [77]. The yam enzyme appears to be a typical catcchol oxrdasc with all thecharacteristics of these enzymes. Thccatcchol oxidascs In grapes continues to arouse interest because of its apparent contribution to the quality of wines [78-801. Tissue culture of plants for secondary product formatton continues to be a topic which is extensively researched. Catechol oxidascs have been invattgatcd in suspcnston cultures of tobacco [2 I, Sl] and of MUCUM, which has not been previously studied 125.821. Some other spccics whose catcchol oxidases have not been previously dcscribed are okra seeds (Ablmoschus) 1831 and guava (Psi&m) fruit [84]. Enzyme activity has ban followed in date. Phoenix dacryhjero, fruit [SS]. Perhaps not surprisingly, galls formed in a number of plants appear to contain elevated kvck ofcatcchol oxidasc as compared to normal, non-infected tissue [87-891. Since gall formation may be regarded as a kind of wound reaction, this observatron is therefore in line with reports on tissue response to injury. LOCATlOf’i AFiD COMXOL

OF ENZYME ACTIVITY IN V/V0

L4JCCC.W The site at which the enzymes are present continues to raise a variety of problems. In the case of laccaru this is closely lurked with the question of its function during

Polyphcnol oxidua

development. Whether kccasc is specifically formed during fruiting of various fungi is still not ckar. The evidcrke is quite contradictory. An extracelMar laccasc from Lunfinus appeared in fruiting bodies; the increase in enzyme activity was associated with the rapid growth of non-pigmented mycelium and the formation of pigtncnted primordia [90]. In Aspergilltu parasiticus kaasc was found only in conidiating cultures, but LaccaJe formation and conidiation did not seem to be correlated. The kvel of enzyme production was partially controlkd by the composition of the growth medium-(NH.)sSO. suppressing and glutamate promoting laccasc formation [91]. The laocase of A. nidulans has also been studied and in rather great detail. This kccasc was present in conidia and showed all the typical characteristics of fungal laozascs. Laccasc isolated from white spore and green spore strains differed somewhat in characteristics. but tbc differences arose after pigmentation had occurred. The difference in colour of the strains may have ban due to lack of substrate rather than due IO differences in their laccasc or its amount [92]. Further studies of this fungus showed that two distinct kccascs occur which differ immunologically and ckctrophoretically. One was associated with conidial pigment formation and the other with clcistothcca formation. Cytcchemkal methods to definitely localize the enzyme were not entirely convincing, because the reagent used also reacts with catcchol oxidase [93]. In Cop&us a somewhat similar situation was observed. A clear correlation between localized laa~~ formation and the development of primordia for carpophora was observed, but laozasc was not apparently involved in the actual developmental process, nor was there a critical kvel required for differentiation [WI. In Agaricus an extracellular laccasc is produced, which has been fully characterized. Antibodies have been raised against it [95]. In this case thcrc seemed to be no induction of enzyme formation by components of the culture medium. The enzyme closely resembled other fungal laaascs in mokcular properties. During fruiting body development the kvel of this extracellular kccasc dacreascd due to both inactivation and protcolysis. making a function in fruiting body formation very unlikely [%]. In Pudospora anwrinu, hxase actually formod during cell lysis and it was demonstrated that this was a developmentally regulated process, involving de IU)IJO synthesis. Here, thercforc,extracellular enzyme formation is correlated with lysis and its control [973. The induction of lactase formation in various fungi has been documcnted in many cases. Thc inducers can be quite different In Polyporus rcsorcinol acted as inducer [98]. During the testing of induction of a variety of laccases, it was noted that whik 2,S-xylidinc induced txtreccllukr kccase formation in Fames, Pholiota, hawwtes and Pleurotus it faikd to do so in Bottyris cinerea and Rhizactonia [W]. Induction of laccasc formation in Borrytis has been studied in detail in our laboratory [ 10&104]. This work showed a very compkx situation with regard to the control of the induction of this enzyme in Bottytis. The enzyme is produced in very small amounts or is absent entirely in the absence of inducer. In the presence of inducer it is formed, but its molecular properties such as MW. ekctrophorctic b&&our, substrate specificity. beat inactivation and isockctric point all change depending on the nature of the inducer. Among the effective inducers were gallk acid, coumaric acid and grape juice. Not only did the properties change, the amino scid and sugar composition of the enzyme also changed depending on

in plxnts

13

the inducer. In the prauna of two different inducers, two distinct enzymes formed, indicating thaw different genes are activated by different inducers. Induction is developmental in nature; the induar must be present long before the enzyme is actually prod& in quantity. Very small amounts of baasc may be present constitutivcly. Most interesting. tbc actual amount of leccasc produced in the medium was regulated by a second inducer, which was pectin or a derivative or breakdown product of it [ 1041. Tbcsc findings make it ckar that it will be quite difficult to establish unequivocally the dmlopmcntal rok of kccasc in fungi. Ttk lack, at present, of a really satisfactory method of localizing it cytochcmkally or under the EM adds to this difficulty. Perhaps immunobglcal techniques linked to ekctron microscopy will be able to resolve at kast this question. Fungal cute&o/

oxidme

The oaxrrcnce of catcchol oxidasc seems to be correlated with thcappcamnce of fruiting bodies in a number of basidiomycetcs [ IOS]. but there does not appear to be any causal relationship. The isozyme pattern of catcchol oxidasc extracted from basidiomyceta at different stages of tbcir development ako shows no important differences [ 1061. A novel highly effective inducer ofcatcchol oxidasc has ban found in the case of Coprinw [107]. Enzyme formation was induced by Zdcoxy-c+glucosc, but fruiting body formation was inhibited by it. Nevertheless, regulation of the two procases was distinct and no evidence for combined regulation was found. Again the corrclations seemed to be fortuitous In Neurospora a low mokcular weight inhibitor accumulated, but this is pnvented if the sulphate ion supply is limited [IOS]. The mechanism of regulation appeared to be that factors causing a reduction of the inhibitory -SH compound, such as osmotic stress or phosphate starvation, daeprcss catcchol oxidasc formation. The complicated fashion in which the amount of catcsbol ox&se in fungi may be regulated is illustrated by the situation in Aspergillus oryzae [ 1091. In this species a protyrosinasc (procatcchol oxidasc) has been found, which can be activated by exposure to pH 3.0. The pro- and active forms of the enzyme differ in their protein conformation. In addition, as isolated. the proenzyme appeared IO be firmly associated with proteinases. Theactivity of these protcinascs was responsible for the multiplicity of bands observed following ckctrophorcsis of the enzyme. Rapid acid acitivation is strongly reminiscent of the bchaviour of grape catcchol oxidasc [I IO]. Tbc association of a protcinasc with the enzyme throughout purification should serve as a warning to all who do not pay sufftcicnt attention to this point and may well be a common phenomenon [see also 331. Plant calechol oxidases

That catactml oxidases are present in thcchloropksts of plants is by now fairly widely accepted [ 13,1921]. Ditfcrcnces have been reported in the catcchol oxidase content of bundk sheath or meJophyll cell of Sorghum, it being absent from the former [ I1 I]. Its location within the thylakoids has also been confirmed in Aegopudiwn [ 1121. Further evidence of the chbropkstk nature of catcchol oxidase comes from work with the enzyme from Yicia /&a [ 1131. Poly A mRNA was translated in rirro and the enzyme formed was identified by immunoprecipi-

I4

A. M. MAYER

tation and shown lo have an M, of 45 000. This is identical with the native enzyme. Moreover it appears that the enzyme was transported into the chloroplast without processing, an unusual and important finding, if confirmed. Almost all reports on the transport of protein into chloroplasts indicate that they are procased during passage into the chloroplast. It may be noted that evidence was brought in this work thaw the Vicia f&a enzyme contains a small amount (%4 %) of carbohydrate. From work with Nicoriwur [I I43 it is quite char that the chloroplastic catcchol ox&se is nuclear coded, and supporting cvidcna comes from the work on Vicia and Aegopcdiwn [ I 12, 1I3 3. The highly convincing cvidcna for the chloroplastic location of catcchol oxidase has been challenged by work on ca~echol oxidasc in carrot cultures [115.-l 171. A procatcchol oxidase was isolated, which was activated by Ca’ * or Mg’ * at 1 mM and also by trypsrn. The authors claim that this enzyme IS soluble in nature and becomes activated and then associates with various suballular fractions thus accounting for the appcarana of the enzyme in different organelks, including perhaps chloroplasts. Although these findings might account for the widespread reports of the association of catcchol ox&se with organelks [I], it is difficult to acocpt that it accounts for its presence in chloroplasts. The evidence for the presence of the enzyme in thylakoids sctms to be compclling [I, 12 13, 19-211. In addition, the work oncarrolall cultures addresses the question of the time of appearance of catcchol oxidasc. It appears that il is restricted lo embryogcnic cultures only and might xryc as a marker for a finite dcvclopmrntal stage. Indirect support for this view comes from the finding that r-amanitin is oxidized only by differentiating carrot cells and that this oxidation is mediated by cat&o1 oxidasc [ 1181. However, although there isan indication ofdcvclopmcntalcontrol ofcatcchol oxidasc appearana. studies using in rirro translation of mRNA indicated that in Vicio at least it is possible to get translation products corresponding to catechol ox&se at many stages of development [ 1lY]. Since calcchol oxidasc is often activated in plant tissues the factors responsible for activation have been studied. In Viciu /ah activation of the latent catcchol ox&se is obtained either by acidification or release of a free fatty acid [ 1201. In spinach the inactive catcchol oxidasc can be isolated from thylakoids and linolcnic acid can bring about 100% activation [121]. This suggests that indeed natural activators of catcchol oxidasc do occur. This might be related to the often observed activation of latent catachol oxidasc durmg SenesceIIcc, which has been ascribed IO release of the enzyme from binding IO the thylakoids [ 122. 1231. Senescence may well involve release of fatty acids during membrane breakdown. Natural inhibitors of catcchol oxidasc have also been described a number of times. Among more recent partially identified ones are oxalatc in spinach kavcs [ I241 and qucratin and leucoanthccyanin in tea kavn [ 1251. Although the latter inhibitors were said to be active at quite low conantralions in no case has it been convincingly shown that such inhibitors regulate enzyme activity in cicc. Stress conditions can also affect catcchol oxidasc activity. Salt stress caused increases in enzyme activity in wheat and barley with differences in the response of tokrant and resistant varieties, uvzreascs being greatest in thesalt sensitive ones [ 1261. Mineral nutrition such as Ca or P deficiency [ 1277 seemed to result in decreased

enzyme activity and boron ddiciency appeared to result in increases in its activity in dicotykdons, but not in monocotykdons such as Sorghum [l28]. These results should all be considered together as showing that intcrfercnce in the normal metabolism of the plant results in a multitude of responses including changes in the kvel of catcchol oxidase activity. No really spacihc cffocts have been demonstrated. In the same light the effect of ventilation during storage of avocado must be analysed. Poor ventilation dunng storage or water stress during growth resulted in ckvated kvels of enzyme, apparently due to rekasc from membranes [ 1291. Since copper is an integral part of catcchol oxidasc. attempts IO relate copper supply and enzyme activity have been made. Copper deficiency reduces enzyme activity in subterranean clover in its aerial parts, except in very old parts [ 1301, as is also the case for C+unfhemwn [ 1311. More detailed analyses in clover attempted lo resolve the question of when the enzyme is actually synthesized [ 1321. Leaves of plants grown under conditions of copper deficiency failed to develop enzyme activity even if Cu’ was supplied at a later stage. Apparently the holocnzyme can only be formed during an early developmental stage. It is not yet ckar whether the apocnzyme can be formed and fails to incorporate copper or whether it loo is not formed during copper deficiency and its subsequent formatton later in development 1s repressed [l IS- 1193. The difference in pattern of catcchol oxidascs between organized and unorganized tissues of Solanum melongenc was said IO reflect changes of gene activity, which oavrrcd during development [ 1331. The question of the stage al which catcchol oxidase is formed in a given tissue appears to be central when trying IO understand the control of its activity in the plant. The overall evidence is still full of cootradictions which are difficult to reconcile and insuflicient data are availabk. The initiation of fibre formalion in cotton can be regarded as an early or late stage of development, depending on which plant organ is being considered. the plant or the fibre. In cotton fibrcs, formation is associarcd with an initial increase in enzyme activity [ 1341. Does this support the idea of its formation early in development? Changes in catcchol oxidasc activity in suspension all cultures have ban studied using cthioninc or norkucinc as specific developmental inhibitors of enzyme formation [2l. 81,821. In cultures of MUCUM ptur~en.scatcchol oxidase activity rose during 8 days of culture, the rise bcingalhzctcd by the kvcl of nitrogen in the basal medium. Enzyme activity peaked lust beforeall growth reached its maximum [82]. Very low levels of cthioninc added IO the growth medium (lO&lOOO PM) inductd considerable increases in enzyme activity, 10 contrast to earlier reports which showed that higher concentrations repressed activity [135]. The differcna betwan the effect of this methioninc antagonist may be due IO differences in mcthioninejmhibitor ratios in the culture medium. The use of a different methioninc antagonisl, norlcucmc. provided further cvrdcna that enzyme activity can lx regulated by manipulating the ratio of antagonist lo amino acid [81]. It also provided convincing proof that enzyme activity can be repressed without in any way effecting the viability or growth rate of the tobacco cultures. The only structural change induced by norleucinc was appearance of amyloplasts instead of plastids. Whether this should be regarded as a regulatory mcchanism seems doubtful, since it could be shown that tnxyrr~ l

Polyphenol 0xrda.s~~ rn phnts wrth reduced activity was synthesized into which the amino acrd antagonist had been incorporated. Thlhc~e results do not therefore answer the question whether or not developmental events are among those controlling enzyme synthesis in the plant. They also do not provide an unequivocal answer IO the probkms about location with the tissue, which we discussed above.

P~~YSI~LOGI<‘AI. FUF;(TION

OF POLYPHESOI.

OXIDASFS

Laccuse Consrdcrabk attention has been focusscd on the function of the extracellular laccax in the decomposition of lignin. The probkm has ban approached on the one hand by studying the possible mechanism by which &case causes lignin breakdown and on the other hand by attempts to inhibit laccasc or rcduoc its kvel in wood rotting fungi. In addition IO the well recognized oxidativc activity II is clear now thaw laccasc also GUI demethylatc methoxy groups [ 1361. The mechanism ofdemethylation has ban studied in some dctarl [ 137, I3111 and indirect cvidencz indicates that this function is important in lignin degradation. This dcgradativc actlvily is closely associated with the ability of lactase IO affect the degree of polymerization of the substrate. High mokcular weight lignosulphates were decomposed whik low molecular sub stratcs were polymerized [ 1393. The presence of lignosulphatcs in the growth medium stimulated lactase production [ 1401 and mutants of Pleurorus lacking kccasc also degraded lignin poorly [ 141, 1421. The dchgnification of cotton straw required the preliminary action of laccasc [ 1411. The wood decaying ability of Hcrerobasidium ( = Fome~)was shown to be strongly oxygen dependent, and this mdkatcd a K, 02 of the fungal laccasc of IO.20 9,. However, wood degradation was not absolutely dependent on O2 [ 1431. In contrast IO the results showing a function of laccasc m wood decomposition. in Daeduleo there was no correlation between fungal mass and laozasc production. Additional evidence against the function m lignin breakdown is also r~rtcd. Mutants of Phunerochoere which lacked the abrlity to oxidize oanisidine was still abk IO degrade lignin, but II was not shown that the ability IO oxidize o-anisidinc is charactcristk only of laccasc [ 1451. Antibodies raised agamst Coriolus laccasc were used to inhibit the enzyme but the fungus was nevertheless abk IO degrade hgnin 1271. Similarly in thecax of Fames inhibition of laccasc did not prevent lignin degradation 11461. These apparent contradrctions may bc related IO factors controlling laccasc formation. The enzyme is partly constitutive and m part produced only m the presence of suitable inducers (see above). The role of phcnolic compounds in controlling lactase formation has been studied in Hererobasidium and in I;omes. Phenols added to fames in liquid culture inhibited its growth [ 1471. The inhibitory phenols could be oxidized by the fungal laccasc. Moreover the same phenols when added IO sprua wood faikd to inhibit fungal growth. The phenols were abk IO induce laaasc formation. Exposure of tfererobusidium to lignosulphatc induced de now formation of an extraallular laa-ase. The hyphae appeared IO contain specific receptor sites for phcnolic inducers. These sites could be blocked by synthetic macromolecules which were not degraded by the fungus. As a result. induction of laaz~~ formation by

IS

phenols was blocked [ 1481. The apparent multipk action of lactase in lignin degradation must also be taken into account. Laccasc can oxidize. dcmcthylatc, polymerize or dcpolymcriac. Thus it is possibk that its importance lies not in lignin oxidation ppr se, which perhaps can be affected in other ways, but in its ability IO polymerize oxidation products of lignin which arise during lignin oxidation. A gcnetrc analysis of laozasc formation in Pleurorus is the only detaikd study of its kind [ 1493. Multipk laccases were found; there was a high degree of genetic variation in isolates of wild strains of Pkurorus. one isozymc being spccitically associated with fruiting body formation. Roth sympatric and allopatrk spcciation occurs in basidiomycetcs as determined in this investigation. Thus when considering the function of laccasc in a specific physiological process it will be necessary to identify which of the multipk forms is studied and IO ensure, when malting comparisons, that a well defined specks of the organism under investigation is used. Tbc possibility that laccasc is involved in the infection process during pathogen&s has ako ban considered. Rigidoporus infects the roots of the rubber tra, Hewa. hca.u activity in the roots showed a clear gradient with high activity in the infected tissue, near the progress of the fungal infection, while it was low in old infbctcd tissue. Healthy tissue was much better abk to induce laccasc formation than old infected tissue [I So]. Similar findings have ban brought with regard to the infection of cucumber by Borryrir [ 1041. In this work it was found rhat laccasc activity showed a gradrent from the infected area to the edge of the uninfected rcgron of the fruit. It was hypothesized that the laccasc is involved in the initial process of infection. Perhaps it overcomes some of the dcfence reactions of the host plant by oxidizing and polymerizing cndogcnous plant phenols, thereby rtndering them nontoxIc. Although the results are suggat~e that the above mechanisms are operating many more experiments will be needed before the hypothesis can be confirmed.

Fun&

and plum carechol oxkfaw

Most of the recent papers have pursued the dca that in some way catechol oxidase is involved in fungal and plant interactions. Research has attempted to show correlations between kvck of enzyme activity or location and the degree of infection which is achieved. Rauhs arc very difficult to interpret. Lack of correlation between kvel of enzyme and lccali.xed necrosis has been reported [ ISI- 1533. but in other cases correlation between infcction and catcchol oxidase has ban reported to exist [ 1541. It has been claimed that catechol oxidase activity in the host actually drops as a result of infection [ 1551. Fungal catcchol oxidase can cause oxidative polymerization of

host phenol& making them less effactivc [156]. This recalls similar roks assigned lo laccasc. Overall corrclations between catcchol oxidasc kvel and spread of infectron are probably not very rekvant, localized increase of host enzyme activity infection might limit spread [ 1573. whik production of fungal enzyme activity

since even a very

at the cdgc of the the very localized

might suBa to overcome a host reaction. The results of catechol oxidasc action might be very indirect. As already shown in the past products of the oxidative activity of catechol oxidasc can inhibit artain fungal enzymes, such as endo-

16

A. M. MAYER

polygalacturonascs [ lSg]. Coupled oxidation reactions can inactivate alkaloids, for example in the capsule of Popover [1593. This may a&cl infective processes, if the alkaloids have a protective function. which is by no means certain. The ability of fungal catcchol oxidax IO oxidize tyrosine residues of proteins IO DOPA followed by dopaquinone formation [ 1601 constitutes an important addition IO the known activities of the enzyme. Oxidation of a protein might kad IO inactivation, and therefore this activiIy of the enzyme might be related IO its function, a point which may be worthwhik pursuing Catcchol oxidasc aclivily in Sorghum during grain development decreased while at the same lime appreciabk amounts of Iannms were formed. Thus kd to the suggation that the initial protective rokof theenzyme was being taken over by the tannin [ 1611. Although this suggestion is interesting. at this stage II strll must bc regarded as speculative. Renewed attempts have ban made IO relate levels of growth substances with enzyme activity. As in the pas1 the results have been negative or unconvincing. Changes in the grafting ability of peach shoot aptc+s, which show seasonal variations, were not correlated wtth catcuhol oxidase activity [ 1621. The ability of appkcuttings to root did correlate with increases ofcatachol oxidase activity in the xylem sap. Oxidation of phloridzin by the enzyme resulted in the formation of compounds with some activity of induction of rooting of the cuttings [ 163J. but iI seems at Ims~ questionable that this IS the cause of variability in the abiltty IO rooI. Transition of plants to the flowering slage was found to be conelated with enzyme activity [ 1641, as have kvels of IAA [ 1651. Such correlative studies arc at best the basis for continuing experiments. AI prcscnl they are not sufficiently convtncing to assign a definite function to catcchol oxidasc. Functions have also been sought at the biosynthctlc level. The enzyme from Agorkus oaatalyscsthe formation of 2-hydroxy-4-iminoquinone, which IS an SH reagent. Steps in the reaction have been identified. Since the quinone is metabohcally active, this constttutcs the first demonstration of the involvement of catecho oxidasc in a definite biosynthetic pathway [ 1661. A pcoumaric acid hydroxylasc from spinach has been described. This is a Cu enzyme, apparently a catechol ox&se, whose activity was regulated by IighI. as far as its hydroxylasc activity was concerned [ 1671. Whether II is a genutnc catcchol oxidax and whether it actually functions in CM in the hydroxyhtion ofpcoumaric acid remains IO be established, since a family of noncopper hydroxylascs is known to exisr [U]. Hydroxylation of carbon 3’ of compounds such as kacmpferol has been shown to be associated with catcchol oxidase from Zea and its possibk sigrnficance in Ravonoid biosynthesis has been discu.sscd [ 16tt]. However, tissues in which catcchol oxidase acrivity in the chloroplasts was repressed by the USCof tentoxin, which causes the loss of plastidk enzyme. contained a normal compktncnt of gavonoids [1703. Consequently the involvement of the enzyme in Ihc formation of phcnolics is at besl open lo dcbatc. The apparent absence of hydroxylativc acuvny in many enzyme preparations is probably at kasr partly the result of methods of isolation and extraction. Hydroxylasc activity may bc detcctcd by more scnsiIivc means such as by trittum release [171]. Summing up, one is kft with the uncomfortabk conclusion that catcchol oxidasc remarns an enzyme tn search of a function. Among the possible roles might bc an

involvement in photosynthesis, by acting as an oxygen buffer or scavenger [I] or via effects on the Mehler reaction [ 133. II cannot be overstressed that investigations in this respect must become much more rigorous and should lccatc both enzyme and substrate cytologically by much more sophisticated means and must assign its function at a finite and defined developmental stage. Proof of function must eventually be accompanied by studies in which mutants and absolutely specific inhibitors, such as antibodies. are used. With the increasing use of tissue suspension and callus cultures, some of these aims should now be attainable.

I.OCC0.W

The entire problem of laccasc structure and reaction mechanism has been rcviewcd authoritatively and in great detail in a number of articles [2 7. 172. 1731 whrh cover the toprc very comprehensively up to about 1982. Although a number of laccascs have been rather fully characterized from the point of view of MW. amino acid composition, multiplicity of subunits and their carbohydrate moiety [90,92,95. W, IO1 103, 174, 1751. work on the reaction mechanism has been confined to one higher plant enzyme. that from Rhus, and two fungal ones. from Polporus and Pdosporo. Much of the rectnt work has focussed on the three types of copper SIIC present in the enzyme. The type 2 copper site. which is the site of substrate binding [see for cxampk 473 has ban tnvatigatai. using the Icchnique of depktion of type 2 Cu2’, which can be restored. permitting the observation of changes during interaction with substrate. However, not only the site 2 Cu can be depktcd C47.523. Type I Cu has been removed and can tx party replaced by Hg’ ’ Replacement of type 1 Cu2 ’ by Hg * resulted in greatly reduced activtty [ 5 I]. The type I site is directly aaessiblc IO solvent including water 144. 1761. The coordination of the different types of Cu site doffers, type I being apparently linked to cystctne. histidine and probably methtoninc L4.45. 1771 in Polypurus. The studies on type 2 Cu show II IO be ligandcd by two to three nitrogens and one or two oxygens. Type I and type 2 sites interact. The type 3 and ~ypc 2 sncs can be bndgcd for example by andc. The coupled btnuckar site in nattvc laccasc seems to be very dtffercnt from the binuckar site of catcchol ox~dasc [ 1781. How type 3 Cu is affected by dcplctton of type 2 is still being debated [40,53]. In this respect the observations that in the nativeenzyme. as isolated, part of the type 3 SIIC remained reduced (about 2596) is important. This may mean that some of the results of the studies using oxidants such as Hz02 may be due to oxidation of these sites rather than as prevtously interpreted [ I7Y]. Electrons appear IO enter the lactase system via site 1 and are transferred to SIIC 2 at least as far as fungal lactase IS concerned. In Rhus lactase, at certain pHs entry may also be via s~tc 2. However, type 2 Cu’ . depletion may enttrcly prevent electron transfer between sites I and 3 [48]. Types 2 and 3 appear IO be the Cu sires responsible for O2 reduction [ ISO]. The two sites arc apparently coopcrartve [48]. but much IS still ut&ar in this respect. A number of recent papers cmphastu the complcxtty of all interpretations. Laser Irradiation does not reduce rcductton of copper, but induces changes which may be

Polyphmol or&se5 in plants due to breakage of a cysteine-

type 1 Cu’ ’ bond. This irradiation also affects site 3 Cu. The native enzyme was found to be more stabk to laser irradiation than type 2 conforcul* depleted enzyme [49]. Enzymcprotcin mation plays a role and changes as the type 3 Cu’ * centre bccomcs reduced and these changes at site 3 lcad to changes in site I Cu’ * 1561. The oxidized enzyme actually exists in two kinetically different forms, an active and an inactive one [ 1811, which are not in equilibrium in the oxidized state [S6]. Changes at site 2 do not appear to afkct site 1 Cu’ - [44]. Thcclox relationship bctwan the copper sites is indicated by the fact that the binuckar Cu* site is disturbed if type 2 Cu’ is removed, perhaps by increasing the Cu Cu distance in theccntre. Not only is this observation important in itself, it may also make it necessary to reinterpret some of the results obtained using the copper depktion technique [50]. It is becoming ckar that the type 2 and type 3 centrcs are not separated. The type 3 antrc loses tts functional integrity when type 2 Cu’ - is dcpktcd [ISZ]. The pH dcpcndcna of the reduction step above pH 8.0 has ban described and indicates that they occur after oxygen binding Protk qutlibna appear to be involved, if it is assumed that the rate limiting rcductant step is reduction of type 1 Cu’ * Recently it has been proposed that a trinuckar Cu stte exists [ 1781 which is abk to bind and bridge small mokcuks. Oxygen bmding may involve thra ckctron rcducad dioxygen, and depends on the type 2 and type 3 sites being close together. Clearly it ts very difficult to summarize these new results on thecatalytic mechanism of laccasc. The reviews already mentioned present a synthesis of many findings [S,6] which will, however, rquire modification in the light of new results. Differences of interpretation of known results still exist. It IS not absolutely clear whether the m&k whtch apply to Rhus laazasc can be applied without modification to the fungal kaase. An ekment of doubt still exists on this point 143. 551. Certainly it must be remembered that the two types of laocase diRer appreciably in their substrate specificity [I]. Subtle differences exist in the protein coupling of type I Cu” between lac tra and fungal laccasc. Coupling is kss in the tra Laocaseas is the reduction potential at this site [45]. There are apprcciabk differences in the carbohydrate content of the various hocases. The significance of this part of the molecule has not yet ban considered in the analyses of reaction mechanisms. It may be that the carbohydrate IS concernad with enzyme solubrlity or excretion, but at present evidence is Lacking. l

l

17

made on fungal catcchol oxidase. A series of papm by Lerch and co-workers have unravelkd the compktc amino acid scquena of Neurospora tyrosinase (catcchol oxidasc) [ 57,58, 184 1891. A model of the site of interaction with the phenohc substrate, mono- or diphenol, has been proposed [84] based on their binuckar antre of the type 3 copper. This closely resembles the model for hacmocyanin [SS]. Photooxtdation of the apoenzymc leads to an inability to reactivate it with Cu’ l. Histidinc residues appear to ligand at Icast one of the two active site copper atoms. A thiocster between cysteinc and histidinc residues may be concerned in the regulation of enzyme activity [ 1851, although the ester does not actually bind Cul’ . The full description of the primary structure of the enzyme from Neurospora [ 187-1891 is undoubtedly a major step in understanding its mechanism of action. Inhibitors such as azide and mimosinc displaa peroxide from oxytyrosinc and this reaction has been used as a probe for the enzyme mechanism [58]. Suggesttons that superoxide might be involved in the hydroxylation rcaction have been shown to be incorrect 11901. It has also ban shown that copperchelators protect catcchol oxidase against inactivation by peroxide [I911 and apparently cu” is necessary for enzyme inactivation by pcroxidc. which is not mediated by an OH radical. Tht overall reaction mechanism of at kast Neurospora catcchol oxidase has been described in models by Lcrch [8] and Solomon [7], which are now generally accepted. These models include a binuckar centre and copper hgandcd in part by histidine. Monophenols bind to one of the cuz - atoms, while diphenols bind to both of them. The reaction mechanism appears to be similar to that of hacmocyanin, while that of &case rcsembks that of ceruloplasmin. Despite very significant progress. some major questions remain. The structure of only one catcchol oxidase has ban established. Agaricus catechol oxidase is composed of two dissimilar subunits as first reported by Mason [I921 and this has been confirmed [l93]. In contrast the Neurospora enzyme, whose structure is now known. is composed of similar subunits [IW]. The question then arises whether the mechanism described for Neurospora, the copper binding and various other features also apply to the Ayaricus enzyme. Furthermore, since the subunit structure of htghcr plant catcchol oxidase is unresolved, it cannot yet Lx said with certainty whether the reaction mechanism dcscnbed ako applies to them.

CONCLUSIONS

Carechol

oxidase

There have been very few significant additions to the study of the structure and mechanism of the reaction of higher plant catechol oxidascs in recent years. Only a few enzymes have ban compktcly purihcd and characterized [ZS. 32,33,75. 77,781. The results have in general confirmed the previous knowkdge of the structure and properties of the enzyme. The most detailad study 1321 has kd to the preparation ofa very stabk enzyme with few ekctrophoretic bands, indicating that most of the multiplicity, so often observed. is due to secondary reactions (tanning. ctc) as also suggested by others 1333. In contrast to the rather poor progress on higtkr plant catcchol oxidasc. very signitkant advances have bun

Catcchol oxidasc continues to be the subject of extcnsive research There are striking differences in the dcgra of sophistication in the methdology used to tackk the biochemical and biophyskal aspects of research on the polyphenol oxidascs compared to those used in studying its biology and function. the former being far more advanced. Considerable progress has ban made over reant years at the biochemical and biophysical kvel From the poiot of view of biology and physiology advances have been very limited; the picture remains very unclear. and cardinal questions are as yet unanswered. These probkms provide a challenge to the plant biologist, both because of the intrinsic interest in many of the probkms and because of the continued economic importance of the enzyme.

I8

A. M. REFF.RCFiCI;S

1. Maya. h M. and Harei. E_ (1979) Phyr0chmistry Ia 193. 2. Reinhamrnar. B. (1984) In Copper Profrins Md Copprr Enz~-)mes (LonIk. R.. al.) VoL 3, p. 1. CRC Presr Boca RaIon. 3. Farva. 0. (1985) in Gas Enqmology, Proc. Symp. (Dcgn. H.. Cox. R. P.. Tortlund. H.. cds) pp. 61 78. Rcidcl. Dordrachc. 4. Reinhammar, B. (1985) in Gus Enzymolqy. Proc. Symp. (Dcgq H.. Car. R. P.. Tof~lund, H., ais) pp. 7%89. Rcidcl. Dordrccht. 5. Reintummar. B. and MalmsIrom, B. G. (1981) m Copper Proreins: Merd Icwv in Biology (Spiro. T. G.. ccl.) Vol. 3. pp. 107, 149. Wiky. New York. 6. Farvcr. 0. and PcchI, I. (1984) in Copm Prorcms aed Copper Enqmes (Lonric. R.. cd.) Vol. I. p. 183. CRC Prm. Boca Raron. 7. Solonxm, E I. (1981) in Copper Prorrim Mcrols hs in Eiobgy (Spiro, T. G.. cd.) Vol. 3. p. 41. Wiky. New York. 8. Lcrch, K. (1981) in Merd Ions UI Btologircll SysIcmc (S~gcl. H.. cd.) VoL 13. p. 143. Dckka. New York. 9. BuIc. V. S. (1985) Ann. Proc. Phyrochem. Sot. Europe 25,349 10. Robb. D. A. (1981) Ann. Proc. Phymchem Sot. Europr 19. 175 11. Robb. D. A. (1984) In Copper Prorcms and Copper Enzqrs (Lontic, R., cd.) VoL 2. p. 207. CRC Ras. Boa Raton. 12. Mayer. A. M. and Harel, L (1981) Ann. Proc. Phyrochen. sot. Europe 19. 159. 13. Vaughn. K. C. and Duke, S. 0. (1984) Physid. Pimr. 60.106. 14. PeIroskI, R. J.. Pazynska-Czech W. and Rosazzn. 1. P. (1980) Appl. Em. Microbid. a, 1003. IS. lxoniwrz. A. and Grzywnowrz. K. (1981) Enzyme Microh. rfchnd. 3. 55 16. Dubourdku. D., Gnrsm C.. Dcruche. C. and RibcrcauGayon. P. Cam. Yigme Yin 18, 237. 17. Harkin, J. M. and 0bs1. J. R. (1973) Exprricrviu 29. 3881. 18. Kovac. V. (1979) Ann. Techno/. Auric. 28. 345. 19. Vaughn. K.C.and Duke. S. 0. (198l)Proloplarmo lC@, 319. 20. WA. F.and Muelkr. W. C. (1981) Proroplaao 106.231. 21. Bar-Nun, N. and Maya. A. M. (1983) Phyrochmirrry 2T. 1329. 22. Simon, L T, Buhop, D. S. and Hoopcr. G. R. (1979) J. Bocreriol. 137. 537. 23. Badnni, M.. Fclh M., Luna, M. and Anmmr. F. (1983) An&r. Bmchm. 133. 275 24. Goodenough. F. W, Kcsr4 S.. La. A. G. H. and LocBkr. T. (1983) Phyrochenusrry 2Z 359. 25. Wichm, H. J, Pcetsma, 1.. Malingrc. T. M. and Huizmg, H. J. (1984) Pkanro I61 334. 26. Law, D. J. and Timberlake. W. L (1980) 1. Bucrrd. 144, 509. 27. Evans. C. S. (1985) fEhfS Microbial. Lerrcrs 27. 339. 28. Hutcheson. S. W., Buchanan, B. P. and MonIalbins P. (1980) Planr Physiol. 66 1156. 29. licbcrci, B.. Bkhl. B. and Voight, 1. (1981) Phyrochmsrry 20,2109. 30. Flurkey.

W. H. (1986) Planr Physiol. gl.614. 31. Jcn. J. J. and I;lurkey. W. H. (1979) How Scl. 14. 516. 32. Wisscmann. K. W. and MonIgomcry. M. W. (1985) Planr Physid.

MAYEU

36. Walker. J. R. Land 37. Kahn.

39.

40. 41 42 43.

44. 45.

46. 47. 48. 49. SO. 51. 52 53. 54. 55.

56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66.

68.

23. 2723.

E. L and Flurkey.

Andrawis.

A. W. (1985) Phpochemiwry

U.

K.. Yao, T. and MUM. S. (1984) Nippon Kaishi 9, 1398 (Chem. Absrr. 102. 323%~). Murao. S.. Ati M, Tanaka N.. Ishikam H., Matsumoto, K. 2nd WaIanabe, S. (1985) Auric. Biol. Chem. 49,981. Reinhamma~. B. (1983) J fnorg. &ochrm. la 113. Frank. P. and PazhI. 1. (1983) Biockm. Biophys. Res. cofwnun. 114, 57. Farva. O., Goldberg. M. and PcchI. 1. Eur. 1. Biochem. 101. 71. Blair. D. F.. Campbell. G. W, Lum. V., Martin. C. T.. Gray. H. B.. Malmstrom, B. G. and Chan. S. I. (1983) J. Inorg. Blochem. 19.65. Dcsidcri, A., Morpurgo. L. AgosIinclli L. Baker. G. J. and Raynor, J. B. (1985) Bimhtm. Biophys. Acru 831. 8. Robe% J. E. Cline. J. F.. Lum. V.. Gray, H. B.. Freeman. H., Pcurh J.. Rcinhammar. B. and HolTman. B. M. (1984) J. Am. Chcm. Sot. ICr. 5324. Wynn, R. M.. KnalT. D. B. and Holwerda, R. A. (1984) Biochcmisrry 23. 241. Morm M. C.. Haucnstein. B. L Jr. and McM~llm. D. R. (1983) Brochim. Biophys. Acre 743, 389. O’NcilL P.. Fkldm. E M.. Morpurgo. Land Agosrinclli. E. (1984) Blochtm. 1. 222 71. Mwci. G.. Tou. L. Dcsdcri. A.. Morpurgo. L. and GamierSuilkroc. A. (1984) J. fnorg. Bioch. Zo. 87. Wookry. G. L, Powers. L. Pasmch J. and Spiro, T. G. (1984) Biochcirusrry 23,3428. Mark-Bcbel. M. M.. Morris, M. C.. Mentie. J. L and Mc.Millm, D. R. (1984) J. Am. Chem. !%u. lw 3677. Moms_ M. C.. Hamtcm, B. L Jr. and McMillin, D. R. (1983) Biochim. Btophys. Acw 743. 389. Morpurgo, L., Dcsidcri. A. and Rotilin. G. (1982) Biochcm. J. 207. 625. Spira. D. J.and Solomon, E. 1. (1983) Biorhcm. Bbphys. Ra. C-n. I I2 729. Maroon, C. T.. Morse. R. H.. Kannc, R. M.. Gray, H. 8.. Malmstrocm. B. G. ax! Ghan. S. 1. (1981) Biochemi.srry 20, 5147. Hansen. F. B, Nobk, R. W. and EIIingcr, M. J. (1984) Bdunuary 23, 2049. Kuipcr. H. A, Lerch K., Brunori, M. and Finazz~ Agro. A. (1980) FEBS tirers I Il. 232. Winkkr. M. E. Lcrch K. arxi Solomon E. I. (1981) J. Am. chtwt. sot. 103.7001. Miyairi, K., Shinya. M, Okuao. T. and Sawai K. (1982) Bull. Foe. Agric. Htrosoki Unrc. 37. 11. Pechkwski R. and Crusciak. E. (1980) Acre Mycol. 16.97. G~ltrap. N. J. (1982) Trans. Br. .Hpol. Sot. ?g, 75. Rams~cdt. M. and Socdcrhall. K. (1983) Trw. Br. Mycol. s4x. II. 157. Ruggko. P. and Radogna V. M. (1984) Soil Sci. IN 74. Voinova. V. N.. Taran~. L F. and Em~acv. V. T. (1980) Izc. Tiiyazvsk. S-&h Akad. I. 105 ((‘hem. Absn. 92 93224pL Voinova, V. N., Taranna. L F. and Em~scv, V. T. (1979) fzv. Tiiiryme~sk. S-&h. Akd 5.87 (Ckm. Absrr. 91.173968d). Bligny. R. and Doucc, R. (1983) Biochem 1. 289.489. Takahub N.. HOII~, T.. lshiharp. H, Mori M.. Tcjimm. S.. Bligny. R.. Akazawa. T.. Endo. S. and Arata. Y. (1986) Bialwmisiry 2!f 388. Polachadr. I.. Hear@ V. 1. and KwooC%ung K. J. (19821 1. flareriol. 150, 1212. Kwon-Chung. K. J.. Polecheck, 1. and Popkm. T. J. (1982) KG&U

W. H. and Jcn, 1. J. (1980) J. Food Biochem. 4.29. 34. Hruskocy. M. and Flurkcy. W. H. (1986) Phyrdmisrry 25. 329.

V. ad

38. Wasa. T.. Akimoto.

33. Flurkcy.

35. Angkcon.

R. F. (1980) Phyrochrmisrry

905.

67.

78.2%.

McCallion,

19. 373.

W. H. (1984) Phyrochmisny 69.

Polyphalol

oxidasa

159. 1414. J., Pobchazk. 1. and Kwon-Cbung. K. J. (1982) In~cckm Immunity 36. Il75. Lamkin, W. M, Milkr. B. S, N&on. S. W, Tnylor. D. D. and Lee. M. S. (1981) Cereal Chem. S8, 27. Inter+ F. S., Ruggiao, P.. D’AnlL. G. and Lom~arclli. F. (1980) J. Ei. Fad Agric. 31.459. lot-. F. S.. Ruggiero. P, Lpmpuclb, F. and D’Awlla. G. (1981) %. L.ttensm. Unrers Forsch. 172 100. Anosike. E. 0. and Ayecbcnc. A. 0. (1981) Phpochmulry 20. 2625. Anode. E. 0. ad Ayaebenc. A 0. (1982) Phyrcdumisrry 21. l8U9. Adamon, 1. and Abigor. R. (1980) Phyruchmirrry 19.1593. Anowkc. & 0. and O~imelukwc, P. C. (1982) 1. Exp. Bormy 134.487. Nakamura, K.. Amano. Y. and Kagami. M. (1983) Am. J. Ed. WK. 34, 122. Sapts. J. C.. Mach&. J. J. and Cordon&r, R. E (1983) Am. J. End. YlrK. W. 157. Lee.C.Y..Sm~~b,N.L.andPcnncsi,A.P.(l983)J.Sc1.Fod Agru. 34.987. Bar-Nun. N and Mayer. A. M. (1985) Phyroctirry 24. 2161. Wrhctx. H. 1.. Malingrc. T. M. and Huizing. H. J. (1985) Planru 165. 264. Mahk. C. P, BarJl N. and Vi)aykumar. K. R. (1982) Idion J. Boruny 5. 67. Augustin. M. A.. Ghazali. M. and Hashim. H. (1985) 1. Sci. Food A@. 36. 1259. Jamh. A. Z. and Benjamin. N. D. (1982) Dare Pdm 1. 1.5. Hasegawa. S. and Maicr. V. P. (1980) J. Agr. FoodChm~ Z8, 891. Joshi, S. C. and Tandon. P. (1984) Btochtm. Physiol. P@u. 179. 71 I. Tandon. P. and Arya, H. C. (1982) Biochem. Physiul. P~onz. 177. 114. Rarnawat, K. G.. Purohlt. S. D. and Arya. H. C. (1980) Sci. Culr. 46. I I I. Lcatham. G. F. and Stahmann, M. A. (1981) J. Gm. .uKrobwl. lu, 147. BEN, C. and Solberg M. (1985) FEMS M&-rob. Lrrers 27. 277. KUIU, M. B. and Champ+ S. P. (1982) J. Bacirriol. lS1, 1338. Herman. T. E.. Kurtz. M. B. and Champc. S. P. (1983) J. Eorkrwl. 154.955.

1. Bocreriol.

70. Rhodes. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 9s. W. 97.

Ross. I. K. (1982) 1. Gm. .Hicdiol. 128, 2763. Wood. D. A. (1980) J. Gm. Mtcrobiol. 117. 339. Wood, D. A. (1980) J. titn. Mccrdwl. 117. 327. Rouchmc. H.. Dupont. C. H. and Bernet. J. (198 I) Bdum. Btiphys. Acta 653, 18. 98. Sandhu. D. K. and Arora, I). S. (1985) Exptunmrua 41.355. 99. Bollag. J. M. and lxonowrr. A. (1984) Appl. Enciron. Microbial. 48. 849. 100. Gigi. 0.. Marbach, 1. and Maya. A. M. (1980) Phylachenusrry 19. 2213. 101. (;I@.

0..

chemwry 102. Marbach chcnusrry 103. Marbach. clumulry 104. Marbach.

Marbach

20. 1.. 22 I., 23. I..

I21 I. Hare1 IS35 Hard. 2713. Hare1

I. and

Mayer.

A. Y. (1981) Phyro-

E. and

Mayer.

A. M. (1983) Phyro-

E. and Mayer. A. M. (1984) PhyroE. and

Mayer.

A. M. (1985) Phyro-

chenu.ury 24. 2559.

105. Ceruti-Scum,

J.. F~usullo.

N.. Guillmo,

M. Land

Farma, F.

in

plants

I9

(1981) Allionlo 24.61. 106. Okunishi, M.. Yamadn, K. and Komagata, Appl. Micro&o/.

107. Nyunoya,

H. and

Microbial.

K. (1979) 1. Gn.

X5,329. Irhikaw&

T.

(1980)

1.

Gm.

AppL

2b. 229.

108. Prstdc. R. A. and Tcmui. H. F. (1982) Biocwhmc. Gene 20. 1235. 109. lchishima E. Macba. H.. Amikura. T. and Sakata, H. (1984) Buwhm. Bwphys. Acra 786, 25. I IO. Lcmer. H. R., Mayer. A. M. and Hare1 E (1972) Phyrochcmisrry 11. 2415. Ill. Vaughn,K.C.andDukc.S.0.(1981)Froropl-108.319. 112. Vaughn. K. C., M~lkr. P. D. and W&son. K. G. (1981) Cyro6ios 31. 27. 113. Flurkey. W. H. (1985) Plant Physid. 79. 564. 114. Lax, A. R.. Vaughn. K. C. and Tcmpkton, G. E (1984) 1. Herd 75. 285. I IS. Sodahall K.. Carlbc~ I. and Eriksscn, T. (1985) Plum Physwl. 78. 730.

I I6 Sodahall K. and Carlberg. 1. (1985) In Somatic Emhryogousis (Terzi, M.. P~IIO. L. and Sung. 2. R.. cds) p. 49. IPRA. 117. Carlbcrg, I, SodcrhalL K. and Enksson. T. (1985) FEBS Lcrrm

I I8

187.295.

P,!Io. L, sctrirvo. Ad.

F L and Teti

M. (1985) Pror. Sam.

SCI. U.S.A. 82. 2199.

119. Flurkey. W. H.. personal communication. 120. Hutcheson. S. W.. Buchanan, B. B. and Montalbini. P. (1980) Plant Physwl. (6 1 I SO. I21 Golbsck, 1. H and Cammarata. K. V. (1981) P/m Phpwl. 67. 977. 122. Meyer, H. ti. and Btchl. B. (1981) Phym&misrry 20.955. 123. Meyer. H. U. and Biehl. B. (1980) Phyrochemisrr~ 19.2267. 124. Sate. M. (1979) Plum Sci. hrrers 16. 355. 125. Pruidzc. G. N. (1985) FI;w/. B&him. Kufr. Rasp. 17, 162 (Chem. Absa. 102 2Cl3193b). 126. Sharma, S. K.. Bal A. R. and Joshi, Y. C. ( 1983)Curr. Agru. 71. 71. 127. Machado. M. A. and Caklas. L S. (1981) Rev. Bras. Bar. 4. 23. 128. Shkol’nik, M. Y.. Krupmkova. T. A. and Smimov. Y. S. (1981) Fawol. Rasr.. Moscow 28. 391. 129. Bower, J. P. and Van l&vckl, L J. (1985) J. HorlK. Sci. 60. 545.

Iu). Walker. C. D. and Loncragan. LA&. 48.65. 131. Grava

C. 1.. Adams.

Food Agric. 1.

J. F (1981) AM. Borany.

P. and Wmsor.

G. W. (1979) 1. Sci.

751.

132. Delhaizc. E, Loneragan, J. F. and Webb, J. (1985) Pkm Physrol. 78. 4. 133. Del Grout. E. and Ali~~ho, R. (1981) 2. PflanxnphyswL 1.2 467 I34 Nalrhani S C., Rao. N. R.. Krishnan. P. N. and Singh Y. D. (1981) Ann. Borany. Land. 48, 379. 135. Volk. R.. Harel. E. and Maya, A. M. (1979) AM. Boruny 43. 787. I36 lxonowicl, A.. Gnywnowi~ K. and Malinowska. M. (1979) Aclo Biochm. Pokmica 26. 43. 137. Lundqtust. K. and Kns~axson, P. (1985) Biockm. J. 229. 277. 138. Palmer, J. M. and Evans. C. S. (1983) Phi/. Trms. R. Ser. B3.0.293.

139. lzenowia A.. Szklarr. G. and Wo~tas-Was&w&& M. (1985) Phymckmimy 24. 393. 140. WojIas-Wakwskn. M. TroJanowski, J. and Lutcrck. 1. (1980) Acta Microbwl. Polonico 29, 353.

141. Plan. M. W., Hadar. Y. and Cha. 1. (1984) Appl. Mwrobrol. Bioreclmol. 20. 1%. 142. PLut. M. W.. Hadar. Y.. Henis. Y. and Chc~, 1. (1983) Eur. J. Appl. Mlcrobtol. Biorechwl. 17. 140. 143. Wonall J. J. and Parrnc~er, J R. Jr. (1983) Phyroparkdogy 73. 1140. 144. Arora. D. S. and Sandhu. D. K. (1985) Enzyme .Hicrob. Techwl. 7. 405. 145. Liwicki. R.. Paterson. A.. Maaionald, M. J. and Brcda, P (1985) 1. B4crtr10I. l6t 641. 146. Haars, A. and Huttarnan. A. (1980) Arch. Microbial. IX. 233. 147. Haius, A, Chct, 1. and Huc~emun, A. (1981) Eur. J. )‘orcsr Porhol. I I. 67. 148. Haan. A. and Hunaman. A. (1983) Arch. Mkrobml. 134. 309.

149. Prillingcr, H. and Molitons. H. P. (1979) Phystol. P&m. 46. 265. I50 Nicok, M. R. (1982) Physrol. Veg. 20, 465. 151. F&XX& G. (1979) Phyroparhol. 2. 95. 237. I52 Wagih, E. E. and Coutts. R. H. A. (1982) Phyropdol. 2. lD4, I. 153. Eduarda. M. and Gucdu. M. (1981) Brorrno Gcner. & 51. 154. Kupferbcrg. S. and Severin, V. (1981) An. INI. Cercer. Pm. P&W.. Ad. Sriinrr Agnc. Silcice 16, 85 (Chm. Absrr. 99. I IY471p). 155. Kaul J. L and .Mun)rl, R. L (1980) lmfran Phyroprrfhol.33, 530. 1%. Kriuman. G. and Chct, 1. (19UO) Phyropnrasirico 8.27 157. Arinzc. A. E and Stni~h, 1. M. (1982) Planr Pdwl. 31. 119. 158. Ham 1. E. and Denrus. C. (1982) AM. Appl. Biol. [email protected]. 159. Hofman. P. J. and Menary, R. C. (1984) Aus!. J. A@. Rcs. 35, 263. 160. Ito, S.. Kate. T.. Shinpu. K. and Fuji& K. (1984) Biockm J. 222,407. 161. Gknnic. C. W. (1981) A@. Feud Chm 2% 33. 162 Pocsscl. J. L. Martrncz_ 1.. Mach&x, 1. 1. and Jonard. R. PhysioI. C’tg. 18. 665. 163. Bassuk. N. L. Hunter. L D. and Howard. B. H. (1981) 1. Horric. SCI. 56 313. 164. Scrpctva L I., Konstantinova, T. N, Akscnova, N. P. and Chalhkhyan. M. Kh. (1984) Do&l. Akad. Ncutk SSSR 274. 504. 165. Jaeger-Wundera. M. (1980) Z. Pfbucnphyswl. 98, 189. 166. Bock&&c, K.. Graham, G. D.. Mizz, P. D. and JefTs, P. W. (1980) 1. Biol. C-km. 255.4766. 167. Bolwcll. G. P. and BUII.V. S. (1983) Phyrcwhcmisrry 2t 37. 168. Larson. R L and Robks. R. P. (1981) Moydwo 26. 199. 169. Vaughn. K. C.and Duke..% 0. (1981) Physiol. Planr. 53.421.

170. Duke. S. 0. and Vaughn. K. ( 1982) Physiol. Phru. 54.38 I. 171. Kahn. V. and Pomcranu. S. H. (1980) Phyrochemrsrry 19. 379. 172. Rcinhmmar. B. and MaImstrom B. G. (1981) in Copper Prorem: Merd Ions in Bio&y (Spiro. T. G.. cd.) Vol. 3. p. 109. Wiky. New York. 173. Fanner. 0. and Pccht. I. (1981) in Copprr Prormw Meral ions in Bdcgy (Spuo. T. G., cd.) VoL 3, p. 193. Wiky. New York. 174. Bar-Nun, N, Mayer. A. M. and Sharon, N. (1981) PhyrochmLtrry 20,407. 175. Vaitkcvki~ R, Vctu~c. V. Carps, N, Barus. R. and Kulys. J. (1984) Bldrhimiyo, Moscow 49. IOOO. 176. Mims, W. B, Avis, J. L and Pckach, J. (1984) Biophys. 1. 43. 755. 177. Briving. C.. Gandvk, E.-K. and Nyrnan. P. 0. (1980) Btiphys. Bidwm. Rrs. Commm 93,454. 178. Alkndorf, M. D.. Spira D. 1. and Sobmoq E I. ( 1985) Proc. Nmn. Acai. .%I. L/‘S.A. 8l. 3063. 179. Penner-Hahn J. E. HaIrnan, B.. Hodgson. K. 0, Spua D. J. and Solomon, E I. (1984) B&km. Biophys. Res. cmun. 119. 567. 180. GoLdberg M.. F~IWX. 0. and Pccht. I. (1980) 1. Biol. Chm. 2%. 7353. 181. Hansen. F. B.. Koudclka, G. B.. Nobk. R. W. and Ettinger. M. J. (1984) Biochcmisrry 23. 2057. 182. Frank. P., Farvcr. 0. ami PcchL 1. (1984) Imrg. Chrm. Acre 91. 81. 183. KoudeLa G. B, H~sen, F. B. and Etdnger. M. 1. (1985) J. Biol. Chm. 260. 155661. 184. Hunmchvright, R. S, Eickruanq N. C.. LuBin C. D.. Lerch. K. lad Sobmoa, 1. (1980) J. Am. Chm. Sot. 102 7339. 185. Lerch, K. (1981) in Inwrrebmre Oxygen-Binding Proreuu: Prcc. Workshop (kmy. J. and Lpmy. J., cds) pp. 259-265. Dekka. New York. 186. PfilT~. E. IDd Lcrch, K. (l9fIl) Biakmirrry 21.6029. 187. Lerch. K., Longooi, C. and Jordi, E. (1982) 1. Biol. Chem. 257.6408. 188. Lcrch. K. (1982) 1. Biol. Chm. 2!!7, 6414. 189. Rueeg C.. Ammcr. D. and Lerch K. (1982) 1. Biol. Ckm. 2~7.6420. 190. Kahn, V, Golan-Goldhirsh, A. and Whrtaka. J. R. (1983) Phyrorhemisrry 22, 1875. 191. Andrawis. A. and Kahn. V. (1985) Phyro&misrry 24, 397. 192. Strothkamp, K. G.. Jolky. R. L and Mason, H. S. (1976) Biachmt. Biophys. Res. Commun. 70. 519. IY3. Robb. D. A. (1979) Bicdwm. Sot. Trans. 7. 131. 194. Robb. D A. (1981) Phyrochemurry 20, 1481.