Eur. J. Biochem.258, 5402545 (1998) FEBS1998

The deoxyribonuclease activity attributed to ribosome-inactivating proteinsis due to contamination

Philip J. DAY, J. Michael LORD and Lynne M. ROBERTS

Department of Biological Sciences, University of Warwick, Coventry, UK

(Received 24 September1998) 2 EJB 981272/3

The mode of action of ribosome-inactivating proteins (RIPs) has, for many years, been considered tobe depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis.Recently, this view has been challenged by the observation that many RIP preparations have significantDNase activity in addition to theirN-glycosidase activity. In this study, we have investigated the putativeDNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases,the DNase activity is due to the presence of contaminating nucleases. TheN-glycosidase and DNaseactivities of PAP were separately and specifically inactivated by chemical modification and heat. Gelfiltration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAPnor recombinant ricin A-chain purified fromEscherichia colidisplayed DNase activity.

Keywords:deoxyribonuclease; ribosome-inactivating protein; RNAN-glycosidase.

Ribosome-inactivating proteins (RIPs) are a group of struc-turally related proteins which depurinate a specific adenyl resi-due of 28-S ribosomal RNA (A4324 in rat, [1]). Depurinationof this residue prevents binding of elongation factors, therebyinhibiting protein synthesis and resulting in cell death. RIPs canbe divided into two classes: type I, which consist of a singlesubunit possessing RNAN-glycosidase activity, and type II,which have a type I-like catalytic subunit (A chain) linked to acell-binding subunit (B chain). The B chains of plant toxins,e.g. ricin, are typically galactose/N-acetylgalactosamine-specificlectins and are linked to the A chain via a disulphide bond.Bacterial toxins that catalyse the same depurination reaction asRIPs, e.g. Shiga toxin, have a non-covalently bound glycolipid-binding B-chain pentamer.

Most type-II RIPs are potent cytotoxins due to their abilityto bind to and enter mammalian cells. Once inside the cell, thetoxins exploit the cellular retrograde transport system to reachthe endoplasmic reticulum from where they translocate into thecytosol by an as yet undetermined mechanism [2]. As holotoxinsshow no catalytic activity against ribosomes, separation of theA chain and B chain following intoxication is essential in orderto exhibit a toxic effect.

Type-I RIPs are, by comparison, only weakly cytotoxic whenadded exogenously to cells. A number of roles have been pro-posed for type-I RIPs, including defence mechanisms and pro-motion of senescence. Those which are active on con-specificribosomes are typically stored in non-cytosolic compartments oras inactive precursors. Such RIPs are believed to be releasedinto the cytosol following cellular damage, such as that caused

Correspondence toL. M. Roberts, Department of Biological Sci-ences, University of Warwick, Coventry, CV4 7AL, UK

Fax: 144 1203 523568.E-mail : lm@dna.bio.warwick.ac.ukAbbreviations.RIP, ribosome-inactivating protein; PAP, pokeweed

antiviral protein; D30 and D32, dianthins 30 and 32, respectively; RTA,ricin-A chain; RTB, ricin-B chain ; DT, diphtheria toxin ; DEPC diethyl-pyrocarbonate.

Enzyme.RNA N-glycosidase (EC 3.2.2.22).

by viral infection, resulting in local cell suicide around infectedareas. An antifungal role has been proposed for RIPs that areinactive against con-specific ribosomes. Barley RIP is co-ex-pressed with a chitinase, and [1, 3] β glucanase, which, whilehaving intrinsic antifungal activity themselves, also facilitateRIP entry into the cells of invading fungal pathogens [3]. Al-though the mode of entry into the target cell differs for the dif-ferent classes of RIPs, the cause of cell death in each case resultsfrom highly specific depurination of rRNA with consequent in-hibition of protein synthesis.

Recently, a number of groups have proposed additionalactivities for RIPs, including non-specific depurination of a vari-ety of nucleic acids [426], RNase [7] and deoxyribonuclease[8213] activities and superoxide dismutase activity [14]. TheRNase and non-specific depurination activities were found to behighest at low, non-physiological pH, suggesting that they areunlikely to be biologically significant. The DNase activity is,however, high at pH 7.5, and has been proposed as an alterna-tive, physiologically relevant mechanism of cytotoxicity [9],with implications for various antiviral strategies [12, 13]. Here,we present a detailed investigation into the putative DNase ac-tivity of RIPs.

DNase activity has been proposed for a wide range of RIPs,including type-II holotoxins, which are inactive against ribo-somes unless the A chain is reductively released from the Bchain [8, 9]. In each case, the observed specific activity is verylow and the relative activity of each RIP is seen to vary fromreport to report. Stirpe and co-workers [5] suggested that theDNase activity associated with RIPs could be due to non-spe-cific depurination of DNA and subsequent hydrolysis by a con-taminating nuclease [5]. In contrast, Lee-Huang and othersbelieve this activity to be an intrinsic property of all RIPs [10,13, 15].

DNase activity was once assigned to diphtheria toxin fromCorynebacterium diphtheriae[16, 17]. Again, this activity hadlow specific activity when compared with bone fide DNase I.However, further purification resulted in complete separation ofthe DNase activity from the toxin [18, 19] and, similarly, purifi-

541Day et al. (Eur. J. Biochem. 258)

cation of toxin from a DNase-deficient strain ofC. diphtheriaealso resulted in protein lacking DNase activity. In this paper, wedemonstrate that the DNase activity associated with a type-I RIP,pokeweed antiviral protein (PAP) and a type-II RIP, ricin is alsodue to contamination. TheN-glycosidase and DNase activity ofPAP have been differentially inhibited by chemical treatmentand heating, and those of ricin have been physically separatedby gel filtration.

EXPERIMENTAL PROCEDURES

RIPs were purchased from the following: ricin and recombi-nant RTA (Zeneca Pharmaceuticals) ; native RTA and nativeRTB (Inland Laboratories); Gelonin (Pierce). Trichosanthin, di-anthin 30 (D30) and dianthin 32 (D32) were a kind gift fromProfessor F. Stirpe (University of Bologna, Italy).

DNase assay.DNase assays were performed essentially asdescribed previously [20]. Reaction buffer (13) consisted of50 mM Tris/HCl, pH 8,100 mM NaCl and10 mM MgCl2. Reac-tions (20µl) containing1 µg pUC18 were allowed to proceedfor up to 4 h at 37°C, stopped by addition of 5µl gel loadingbuffer and products analysed by agarose gel electrophoresis.

Preparation of ribosomes.Yeast ribosomes were preparedfrom Saccharomyces cerevisiaestrain ABYS 1 by the methodof Rothblatt and Meyer [21]. Escherichia coliribosomes wereprepared from strain JM101 as described [22]. Purified ribo-somes were stored at270°C in 13Endo buffer (25 mM Tris/HCl, pH 7.6, 25 mM KCl, 5 mM MgCl2).

N-Glycosidase assay.Depurination of ribosomes wasassayed as described [23]. Reactions (containing 20µg ribo-somes in 20µl 13Endo buffer) were incubated at 37°C for30 min and stopped by the addition of100 µl Kirby reagent(made by dissolving 6 g 4-aminosalicylic acid in 25 ml 200 mMTris/HCl, pH 7.6, 40 mM KCl, and adding10 ml 10% tri-isopro-pylnaphthalene sulphonic acid until the solution goes cloudy.Tris-saturated phenol was then added dropwise until the solutionbecame clear again and the volume was finally adjusted to 50 ml[24]). rRNA was prepared from the ribosomes by phenol extrac-tions and subsequent precipitation with ethanol. 325 µg ex-tracted rRNA was treated for 2 min at 60°C with acetic-aniline,pH 4.5 [25]. The rRNA was ethanol precipitated and analysedafter denaturing (formamide) agarose-gel electrophoresis andethidium-bromide staining.

Modification of RIPs with diethylpyrocarbonate. Diethyl-pyrocarbonate (DEPC) (1 µl, 20 mM) was added to RIPs (1 mg/ml) in NaCl/Pi (137 mM NaCl, 2.7 mM KCl,10 mM Na2HPO4,1.8 mM KH2PO4, pH 7.4), in a final volume of10 µl. The reac-tion was allowed to proceed for10 min at room temperature,then terminated by the addition of1 µl 0.1 M imidazole. Themodified enzymes were then tested for DNase (1 µl added di-rectly to a 20-µl reaction) andN-glycosidase (following appro-priate dilution, typically10-fold for assay withE. coli ribosomesand1000-fold for assay with yeast ribosomes) activities.

Heat treatment of PAP. PAP (1 mg/ml in NaCl/Pi) was in-cubated at 70°C for up to10 min and placed on ice for a further10 min. DNase activity was assayed directly andN-glycosidaseactivity assayed following appropriate dilution in NaCl/Pi.

Purification of recombinant PAP. Recombinant PAP wasexpressed and purified fromE. coli BL21 (DE3) pLysS harbour-ing plasmid pET-PAPstop by cation-exchange chromatography,as described previously [26].

RESULTS AND DISCUSSION

DNase activity of RIPs. A variety of RIP preparations havebeen shown previously to possess DNase activity [8211]. Fig. 1

Fig. 1. Digestion of supercoiled DNA.Reactions were performed asdescribed in Experimental Procedures at 37°C for 4 h. Lanes1 and 9,undigested controls. Lane 2, 2µg DT; lane 3,1 µg pokeweed anti-viralprotein; lane 4,1 µg D32; lane 5,1 µg D30; lane 6,1 µg gelonin ;lane 7,1 µg trichosanthin ; lane 8, 2µg ricin. The upper band is nickedplasmid (N), the middle band linear plasmid (L) and the fastest migratingband supercoiled plasmid (SC).

Fig. 2. Digestion of linearised DNA.pUC18 (0.7µg) linearised withBamHI was digested as described with pokeweed anti-viral protein,lane1 (7.6µg), lane 7 (1 µg); D32, lane 2 (9.6µg), lane 8 (1 µg); D30,lane 3 (9.6µg), lane 9 (1 µg); gelonin, lane 4 (5µg), lane10 (1 µg) ;DT, lane 6 (1 µg) ; trichosanthin, lane11 (1 µg), ricin, lane12 (2µg) orin the absence of RIP, lane 5.

shows the relative activities of several RIPs (five type I and onetype II), which are compared with the activity of a commercialpreparation of diphtheria toxin (DT, Calbiochem) which isknown to be contaminated at a low level with a DNase [18, 19,27]. Of the RIPs tested here, gelonin and trichosanthin had thehighest activity, D30, D32 and PAP had less activity and thetype-II RIP ricin had the lowest activity. However, all had sub-stantially reduced activity compared with that of DT. It has beenshown previously that the DNase contamination of DT yields aspecific activity some four orders of magnitude lower than thatof DNase I [9] and that the contaminant was ~30 kDa in size(the same size as most type-I RIPs). Thus, the DNase activityof RIPs could be due to either a very low intrinsic activity ofthe RIP itself or to very low level contamination with a distinctDNase.

The RIP family of proteins have rather few absolutely con-served residues, and these are clustered near the known glycosi-dase catalytic site, suggesting that any second common activityis most likely to be a product of this same active site. Thus, itmight be expected that all RIPs would possess such a DNaseactivity and that elimination of one activity might have a similareffect on the other activity. Alternatively, if the DNase activityis due to contamination, further purification should separate theRNA N-glycosidase and the DNase activities.

Conformation-specific DNase activity.Previous reports sug-gested that RIP-associated DNase activity was conformationspecific [8, 20]. That is, RIPs only cleave supercoiled DNA ina two-step process, first nicking a single strand before linearisingthe DNA by second-strand cleavage. Fig. 2 shows that no activ-

542 Day et al. (Eur. J. Biochem. 258)

Fig. 3. Effect of Mg2+ on the DNase activity of RIPs.Top panel, diges-tion of supercoiled pUC18 with ricin (2 µg), PAP (1 µg), trichosanthin(1 µg) or in the absence of RIP (U) in the presence of the indicatedmillimolar concentration of Mg21. Middle panel, digestion of pUC18 bygelonin (1 µg) in the presence and absence of Mg21 (mM) and EDTA(mM). Lower panel, effect of Mg21 (mM) and EDTA (mM) on the diges-tion of pUC18 by D30 and D32. U represents incubation in the absenceof RIP.

ity is observed against linear DNA with RIP concentrations thatshow substantial activity against supercoiled DNA. However,when RIP concentrations are increased, digestion of linear DNAcan be readily observed in the case of PAP and gelonin (Fig. 2).The product of this digestion is a smear, suggesting that diges-tion is non-specific. Similarly, digestion of supercoiled plasmidwith high concentrations of active RIPs also produces a smear ofproducts with digestion to small products following prolongedincubation (data not shown).

Mg2+ dependence of DNase activity.Many nucleases requiredivalent metal ions for activity and lack of Mg21 dependence ofthe RIP nuclease activity has been provided as evidence thatpreparations were not contaminated with a conventional DNase[9]. The activity of the six RIPs against supercoiled pUC18 wastested in the absence of added Mg21. The activity associatedwith ricin, PAP and trichosanthin (Fig. 3) was found to be highlydependent on Mg21 as would be expected if the RIP preparationwas contaminated. Omission of Mg21 completely abolished ac-tivity, but full activity was readily restored by the addition of1 mM Mg21 (ricin, PAP) or10 mM Mg21 (trichosanthin).

In contrast, D30, D32 and gelonin all had relatively un-changed activities when Mg21 was omitted from the reactionconditions (Fig. 3). However, addition of 2 mM EDTA to thereaction mixture, to chelate any prebound Mg21 largely abol-ished DNase activity in each case. Again, this suggested that adivalent metal ion was required for activity. Addition of Mg21

to EDTA-treated gelonin restored activity (Fig. 3). Thus, the as-sertion that RIPs possess a novel Mg21-independent DNase ac-tivity is wrong. From the data presented here, it is clear that theobserved activity in the absence of added Mg21 is due to carryover of Mg21 with either the RIP or the DNA substrate.

Inhibition of RIP activities with DEPC. The N-glycosidaseactivity of ricin-A chain (RTA) is insensitive to covalent modifi-

Fig. 4. Inhibition of catalytic activities by modification with diethyl-pyrocarbonate. Top: inhibition of the N-glycosidase activity of: (1)Pokeweed antiviral protein, (2) trichosanthin, (3) ricin, (4) gelonin, (5)D30, (6) D32. RIPs were modified with DEPC and theirN-glycosidaseactivity assayed against yeast ribosomes as described in ExperimentalProcedures. The arrowhead indicates the position of the diagnostic rRNAfragment produced by acetic-aniline treatment of depurinated 28-SrRNA. Bottom: inhibition of DNase activity by DEPC treatment of RIPs.Supercoiled plasmid DNA was incubated with RIP that either had orhad not been treated with DEPC.

cation with DEPC [28], whereas many nucleases, which havecatalytic histidyl residues, are inactivated by modification withthis reagent. RIPs that showed nuclease activity were thereforemodified with DEPC and the resulting proteins tested for bothDNase andN-glycosidase activity.N-glycosidase activity ofRIPs was monitored by the production of a characteristic frag-ment of rRNA [23] following treatment of depurinated ribo-somes with acetic-aniline. Hydrolysis of extracted 28-S rRNAby the aniline reagent generates a short (3702390 bp) fragmentfrom the RIP depurination site to the 3′ end of the molecule.Generation of this fragment is diagnostic of RIPN-glycosidaseactivity. Fig. 4 shows such rRNA resolved by electrophoresis ona denaturing formamide gel and visualised by ethidium-bromidestaining. TheN-glycosidase activity of all of the type-I RIPstested was abolished following modification with DEPC. In con-trast, ricin, as previously observed for RTA, was not inactivated.Incubation of PAP and trichosanthin withE. coli rRNA affordedprotection against inactivation by DEPC (data not shown).

Inactivation of D30 was somewhat surprising as this proteinis devoid of histidyl residues [29]. However, DEPC can alsoreact with tyrosyl and lysyl residues at a lower rate than thatwith histidyl residues and all RIPs so far sequenced have twoconserved active-site tyrosyl residues (Tyr 80 and Tyr123 inRTA). Chemical nitration or site-directed mutagenesis of theseresidues reducedN-glycosidase activity by at least an order ofmagnitude [30, 31]. However, as ricin also has active-site tyrosylresidues, it is not easy to rationalise the difference in sensitivitybetween ricin and type-I RIPs based on modification of theseresidues. There is, however, a significant difference in thenumber of lysyl residues in ricin (two) and type-I RIPs (typicallymore than ten), although none is in the active site or obviouslyinvolved in substrate binding.

The effect of DEPC on the DNase activity of RIPs was vari-able. Fig. 4 shows that the DNase activity of ricin, PAP andgelonin on supercoiled DNA was unaffected by modificationwith DEPC, whereas that of trichosanthin, D30 and D32 was

543Day et al. (Eur. J. Biochem. 258)

Fig. 5. Effects of incubating PAP at 70°C on catalytic activity. Uppergel : N-glycosidase activity.E. coli ribosomes (20µg) were incubatedwith 70°C heat-treated PAP (asterisk) or non-heat-treated controls at theconcentrations indicated for1 h at 30°C; Lower gel: DNase activity.Supercoiled pUC18 was incubated with heat-treated PAP (asterisk) ornon-heat-treated controls for 4 h at 37°C. The arrowhead indicates theposition of the diagnostic acetic-aniline cleavage product.

inhibited. Thus, reaction with DEPC resulted in loss of neitheractivity (ricin), both activities (trichosanthin, D30, D32) or lossof only the N-glycosidase activity. Although DEPC treatmentdoes not provide stringent evidence that the two activities arenot in the same protein, our data strongly suggest that, at leastfor gelonin and PAP, the DNase andN-glycosidase activities arelikely to be activities of different active sites if not differentenzymes.

To further investigate the possibility that the DNase activityof RIPs is due to contamination, we have focussed attention ontwo well-characterised RIPs readily available in our laboratory,ricin and PAP.

PAP. Heat treatment of PAP eliminates DNase activity but notRNA N-glycosidase activity.PAP can be boiled and rapidlycooled without significant loss ofN-glycosidase activity [32]. Incontrast, DNases are often thermolabile. Thus, if PAP hasnuclease activity, it might be expected that this activity wouldalso be stable on heating as is theN-glycosidase activity. If thenuclease activity is due to a contaminant, heat treatment mightbe expected to result in loss of this activity. Incubation of PAPat 70°C for 10 min resulted in only a slight decrease inN-gly-cosidase activity (. 80% activity remaining, Fig. 5) as reportedpreviously, whereas the DNase activity was abolished (,5%activity remaining, Fig. 5). Furthermore, incubation of PAP at70°C had only a small effect on cytotoxicity against HeLa cells(data not shown). Thus, following heat denaturation, PAP eitherrefolds to a conformation which can no longer cleave super-coiled DNA, but can still depurinate ribosomes, or heat treat-ment inactivates a contaminant that does not refold upon cool-ing. Taken together with the differential effect of DEPC on theDNase and RNAN-glycosidase activities, the latter seems morereasonable. Furthermore, the active site of PAP lies in a cleftbetween two domains [33], suggesting that full refolding of bothdomains would be required to restoreN-glycosidase activity. Ittherefore seems unlikely that such refolding would restore onlyone of two intrinsic activities. It should be stressed, however,

Fig. 6. Recombinant PAP lacks DNase activity.Incubation ofE. coliribosomes (upper panel) or pUC18 (lower panel) with native PAP,lane1 ; in the absence of RIP, lane 2; or with recombinant PAP fractions,lane 3 and lane 4. The arrow indicates the position of the diagnosticacetic-aniline cleavage product. Ribosomes were incubated with 50 ngRIP for1 h at 30°C and pUC18 incubated with1 µg RIP for 4 h at 37°C.

that heat treatment does not provide definitive evidence that theactivities are not in the same protein.

Recombinant PAP lacks DNase activity.The PAP gene hasbeen cloned and the recombinant protein expressed at low levelsin E. coli [34, 35]. Recombinant PAP is identical in sequence tothe mature form extracted from pokeweed leaves, except for theaddition of a methionine residue at the N-terminus. The purifiedrecombinant protein hasN-glycosidase activity identical to thatof PAP purified from plant extracts (Fig. 6). Thus, if PAP hasnuclease activity, the recombinant protein might also be ex-pected to possess the same activity. Recombinant PAP, however,cannot cleave supercoiled pUC18 (Fig. 6), strongly suggestingthat the protein purified from the plant extract contains a con-taminating nuclease.

Ricin. Activity of individual subunits.Ricin holotoxin has noN-glycosidase activity, but reductive separation of the A chain andB chain reveals the catalytic activity of the A chain. In contrast,DNase activity was reported to be a function of both holotoxinand free A chain [20]. We tested holotoxin, reduced holotoxin,highly purified native A chain and B chain and recombinant Achain for DNase activity (Fig. 7a). Holotoxin displayed DNaseactivity that was unaffected by reduction of the interchain disul-phide (lane 8 and lane 9), whereas separated subunits (lanes 4and 5) and equimolar mixtures of purified A chain and B chaindid not (lane 2). Similarly, recombinant RTA had no DNase ac-tivity (lane 6). Thus, only holotoxin purified from bean extractshad DNase activity, suggesting that purification of native sub-units had removed a contaminant or an essential co-factor. If thisis the case, then removal of the DNase activity from ricin holo-toxin should be possible by further purification steps.

Separation of N-glycosidase activity from DNase activity bygel filtration of ricin. The nuclease contaminating DT could beseparated from DT by a number of chromatographic procedures[18, 19]. Gel filtration of ricin over a Superose 6 FPLC columnresulted in elution of a single protein peak, corresonding to ricinholotoxin, as visualised by SDS/PAGE (not shown). The absor-bance profile of the eluted protein showed a shoulder, presum-ably due to the different glycosylation states of RTA, but corre-sponded well with the profile observed by SDS/PAGE, with the

544 Day et al. (Eur. J. Biochem. 258)

Fig. 7. Separation of theN-glycosidase activity from the DNase activ-ity of a ricin preparation. (a) Digestion of supercoiled pUC18 for 4 hat 37°C with the following: lane 2, an equimolar mixture of purifiednative RTA and RTB subunits (1 µg each) ; lane 3 and lane 8, ricin holo-toxin (2µg) purified from bean extracts, lane 4, purified native RTB(1 µg); lane 5, purified native RTA (1 µg); lane 6, recombinant RTA(1 µg); lane 9, reduced native holotoxin (2µg). Lane1 and lane 7 arebuffer controls; (b) Gel filtration of ricin in the absence of galactose.The solid lines represent absorbance (280 nm) and broken lines representDNase activity (ratio of nicked to supercoiled plasmid following a 4-hdigestion). The gel shown is a Northern blot showing the distribution ofaniline cleavage products following treatment of yeast ribosomes withthe eluted fractions. The fractions reduced with 50 mM dithiothreitol for1 h at 37°C prior to assay. (c) Gel filtration of ricin in the presence of100 mM galactose.

fully glycosylated form eluting at19.2 ml and the mono-glyco-sylated form at 20.4 ml (Fig. 7b). Elution ofN-glycosidase ac-tivity corresponded well with the absorbance profile (Fig. 7b).In contrast, the peak of DNase activity preceded that of ricin byseveral fractions (elution volume5 18.6 ml). Non-correspon-dence of the DNase andN-glycosidase activities strongly sug-gests that the two activities are the result of action by differentproteins, rather than the dual activities of a single protein.

Gel filtration in the absence of galactose resulted in retarda-tion of ricin on the column, presumably due to the lectin activityof ricin-B chain (RTB). When gel filtration was repeated in thepresence of galactose (100 mM), as expected, the peak of DNaseactivity eluted at the same volume (18.6 ml) as in the absence

of galactose (Fig. 7c). Ricin, however, eluted from the columnmuch earlier (18.3 ml) in the presence of galactose. As in theabsence of galactose, gel filtration in the presence of galactoseresulted in separation of the peak of DNase activity from that ofthe bulk protein, although separation of the activity was some-what better when galactose is omitted. Thus, gel filtration overSuperose 6 separates ricin from the DNase activity, confirmingthat the observed DNase activity cannot be due to ricin itself,but is due to low level contamination with an as yet unidentifiednuclease.

That ricin lacking DNase activity remains cytotoxic has beenamply demonstrated by the use of recombinant toxin subunitsproduced in and purified fromE. coli [36237]. When theDNase-free recombinant RTA used in Fig. 7a was reassociatedwith recombinant RTB, the expected IC50 value (1 ng/ml) wasobtained (data not shown). Thus, theN-glycosidase activity isclearly associated with cell killing.

The findings described in this paper are in good agreementwith previous observations. Elimination of theN-glycosidase ac-tivity of PAP (by site-directed mutagenesis) abolishes antiviralactivity [38]. Furthermore, the ability to prevent viral infectionof pokeweed leaves by addition of exogenous RIP is dependenton the ability of the added RIP to depurinate pokeweed ribo-somes [39]. Equally, the cytotoxicity of ricin to mammalian cellsis directly related to theN-glycosidase activity, with a decreasein the N-glycosidase activity, induced by mutagenesis, resultingin a corresponding decrease in cytotoxicity ([28, 40], Svinth andRobertus, personal communication).

For many years, the single mode of action of RIPs has beenconsidered to be highly specific depurination of 28-S rRNA,with subsequent inhibition of protein synthesis and cell death.More recently, this view has been challenged by the observationthat a number of RIP preparations have both non-specific depur-ination and DNase activity. In this paper, we have demonstratedthat such DNase activity is due to contamination. Gel filtrationof ricin allows facile separation of DNase andN-glycosidaseactivities. Furthermore, the two activities associated with PAPpreparations can be separately and specifically inhibited. Thus,the N-glycosidase activity is abolished by DEPC treatment, butnot by high temperature, whereas the DNase activity is inhibitedonly by heat. The separate nature of the two activities was fur-ther demonstrated by purification of a recombinant form of PAPwhich possessed onlyN-glycosidase activity.

This work was supported by grant BO8000 from the United King-dom Biotechnology and Biological Sciences Research Council.

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