l-ZI.Ul2O Bologna, Italy (lel: o31 Z4.50

Abstract--1. Ribosomes from M. domestica larvae were iso several ribosome-inactivating proteins (RIPs) from plants

2. Ribosome-inactivating proteins inhibited, to differen ribosomes.

3. Analysis of RNA from RIP-treated ribosomes showed fragment resulting from the N-glycosidase activity of the t

4. The release of adenine from saporin 6-treated M. dome analysis.

INTRODUCTION was of M

bosome-inactivating proteins (RIPs) (see reviews Barbieri and Stirpe, 1982; Jimtnez and V~zquez, unexl

RIPs 85; Roberts and Selitrennikoff, 1986; Stirpe and these

the el culiar RNA N-glycosidase activity, through which larva~ ey cleave the N-glycosidic bond of an adenine from ;idue in a precise position of a highly conserved aratk :luence of rRNA (A4324 of 28 S rat liver rRNA) is aft( ndo et al., 1987; Stirpe et al., 1988). This alteration

to ob aders ribosomes unable to bind the elongation activi : tor 2, with the consequent arrest of protein syn: a¢~tlvl

M u s c a domes t ica (Cenini et al., pected, since saporin is one ot

RIPs. To investigate further the acti these ribosomes, experiments were

proteins effects of this RIP on rRNA fr( ~e. These experiments showed

these larvae are easily degra( aration and consequently their syntl~ is affected. Thus, experimental cond

obtain undegraded ribosomes wi activity, and the action of RIPs o

) from the inhibition of activity was reinvestigated, together

L the appearance of an miline at the depurinated :termination of released MATERIALS AND METH

,,le-chain proteins (type 1 larvae were 2 RIPs) consist of two Crespellano, Bologna, Italia.

as the enzymatic activity L-[U-14C]Phenylalanine, spee. act. i galactose-specific lectin from Amersham UK; GTP and pc

)syl-terminated receptors Boehringer Mannheim G.m.b.H., Germ mal cells). Thus, type 2 specific tRNA was from Sigma, St. Lo e potent toxins, the best reagents were of analytical or molecular

when possible, RNase-free. merous structural simi- Chloroacetaldehyde was prepared ace

n of action is apparently [~4C]Phenylalanyl.tRNA, was :her type differ in their Hultin and Nilslund (1978). l 'hus, type 2 RIPs bring Artemia salina S-I05 post-ribosoma :s (Derenzini et al., 1978; prepared according to Sierra et al. (197

et al., 1979), and RIPs Type 2 RIPs were purified as descril~ cantly different activity references: ricin 60 (Nicolson et al., 197 s plants, protozoa and et al., 1978), and volkensin (Stirpe et al ~84; Cenini et al., 1988, was purified from the roots of Bryonia :ects of RIPs on phenyl- by Stirpe et al. (1986). Other type 1 1 ribosomes from various as described by Barbieri et al. (1987):

Dianthus caryophyllus leaves, saporin ) that type 2 RIPs were officinalis seeds, pokeweed antiviral pro

type .1 RIPs; and Phytolacca americana seeds, momordit

by 1985 Barbieri, 1986) are plant peculiar the' residue sec (Endo renders factor thesis. It can be detected (i) protein synthesis; (ii) from rRNA fragment cleaved by aniline site; and (iii) by direct determinal adenine. Most RIPs are sin[ RIPs) and only a few (type chains (an A-chain which has and a B-chain which is a capable of binding to galactosy on the surface of most animal RIPs can enter ceils and are known of which is ricin.

Different RIPs show numerous larities, and their mechanism identical. Still, RIPs of either in vivo and in vitro action. Thus about different lesions in rats Sperti et al., 1979; Barbieri of either type have significantly on ribosomes from various metazoa (Battelli et al., 1984; 1990). In a study on the effects alanine polymerization by metazoa, it was observed (i) generally less inhibitory than (ii) that ribosomes from some sl; inactivated by some RIPs. Among a type 1 RIP from the seeds of Sap~

pp. 223-227, 1991

F PLANT RIBOSOME-Ii~ TEINS ON RIBOSOMES

MUSCA DOMESTICA

:) FERRARI, LUIGI BARBIERI and FIOREN ogia sperimentale dell' Universit~i di Bolo Bolo Italy (Tel: 051 24 30 42); (Fax: (

(Received 16 April 1991)

were isolated and tl was tested.

lifferent extents, 1

)rues showed the appeara RIPs.

domestica ribosc

almost inel

possessing a

rRNA.

nation

Mo domestica

et al. (1983).

,TING

3iacomo 14,

ity to the action of

polymerization by

ae-cleavable rRNA

nstrated by h.p.l.c.

~osomes from the larvae 1990). This was

of the most active action of saporin on

set up to ascertain from M . domes t ica

showed that ribosomes raded during prep-

synthetic performance conditions were set up

with high synthetic on their synthetic

with the effect on

METHODS

obtained from R.G.L.,

19 GBq/mmol, was poly(U) were from

Germany; phenylalanine Louis, MO. All other

biology grade and,

~ared according to McCann

prepared according to

ibosomal supematant was (1974).

described in the respective 1974), viscumin (Ziska

al., 1985). Bryodin-R dioica as described

RIPs were purified diantbin 32, from 6 from Saponaria

)rotein (PAP-S) from Lomordin from Momordica om Trichosanthes kirilowii n multiflorum seeds, and n Hordeum vulgare seeds.

tS-l--l~Sl ouner, p t t 7.3, containing 37.5 mM Mg-acetate, I M KCI and 2 mM dithiotreithol; their concentration was termined by the A260 according to Montanaro et al. (1978) ;uming that 12.5 u/mL was equivalent to 1 mg/mL and Fig. 2 It 1 mg contained 250 pmol of ribosomes. Ribosomes Musct re stored in aliquots under liquid nitrogen, as des Poly(U)-directed phenylalanine polymerization was per- stopl~ ~ e d as described by Montanaro et al. (1978). The ~ction was carried out in 100#L volume containing was ir ,tool ribosomes (unless indicated differently) in 80 mM is-HCI, pH 7.4, 9.9raM Mg-acetate, 135raM KCI, 55:4p] mM dithiotreithol, 100/zg poly(U), 0.2 mM GTP, 10 #L Contr, Artemia salina supernatant (containing 190 #g of pro- adenir as), 18pmol []4C]phenylalanyl-tRNA and RIPs in the propriate concentration. Incubation was for 10min at intern~ 'C (unless indicated differently). A deri ested in ice by the addition of 100/~L of 10% (w/v) (100# :hloroacetic acid. ~.nalysis of rRNA was performed essentially following analyz graph

method of Endo and Tsurugi (1987) as practised by meter rpe et al. (1988). Ribosomes (15 pmol) were incubated for contr¢ min at 24°C in the same conditions as described for Spheri ly(U)-directed phenylalanine polymerization, without

phenylalanyl-tRNA, GTP and buffer, [y(U). RNA was extracted and treated with aniline as 0-16"/, ~eribed previously (Stirpe et aL, 1988). Electrophoresis 1 ml/rr s carried out on a polyacrylamide gel (5%) with a luencing Gel Electrophoresis System Model S2 (Bethesda search Laboratories, USA). Gel size was 0.4 mm (thick-

#L, equivalent to 12% of the reac ,zed with a Kontron high-pressur(

graph equipped with a model SFM 25 meter and a model 450 MT data system f( control and data analysis. The colum

~herisorb Ct8 (I0 #m particle size, 25 c 20 mM Na-tetraborat

', pH 7.7, and eluted with a linear 0-16% methanol in the same buffer. ,

rmin. The spectrophotofluorimeter excitation and 415 nm emission.

31 cm (width). The gel was RESULTS

t 65 Watts. Samples loaded The condi t ions for poly(U)-direct~ A. Electrophoresis was at ed for 30 min in acetic acid/ polymeriza t ion by r ibosomes from er according to Berry and Ionic condi t ions we

respect to magnes ium and potassil ribosomes was determined and 135 mM, respectively). The rea

~ni et al. (1989) essentially mn et al. (1985). Incubation

j -

I I

1 0 1 ~

I R I P added 16

Fig. 3. Effect of ribosome-inactivating pr saporin 6 on poly(U)-directed phenylalan

iPm°l) by Musca domestica ribosomes. Experi alanine polymerization by were as described in Materials and Methq

/'iLLCLI

the Stir 15 poly(U)-directed Artemia salina S-105, poly(U). described w a s

Sec Research ness) and 38.5 cm (height) and 31 cm pre-electrophoresed for 30 min at contained approx. 3 #g of RNA. 65 Watts for 1 hr. The gel was fixed for 30 methanol and stained with silver Samuel (1982).

Adenine released from treated ribos~ by h.p.l.c, according to Zamboni following the procedure of McCann et

o [ 1 0

.| |

i / | i

-¢ /J I J 2 4 8

R I b o S o m o s

Fig. 1. Poly(U)-directed phenylalanine Musca domestica ribosomes. Reaction all other conditions were as described

Methods.

C. FEIOtAm et al.

addition to ribosomes e enzymatically active ! m the B chain to exert .~

ively with water and of growth media by ~" ~

ere chilled to 0°C and ~ o pparatus fitted with a ~ _" 2 ~se containing 20 mM . - cetate, 100 mM KC1, J 1 L heparin. Ribosomes nini et aL (1988) in o esuspended in 0.4M 37.5 mM M

2. Poly(U)-( Musca domestica

described in 1 )ed at 5, 10,

m the same Each sample cc

~mol of ribo,, ols were r

adenine. The ado an external sta] internal standard

The reaction was A derivatization r

equilibrated in

30 min in acetic been defined.

1 0 0

5O

J I I

15 20 25

3 (rain)

lalanine polymerization by acubation was carried out Jlethods. The reaction was

with 100/zl of 10% TCA.

scribed for rRNA analysis. final volume of 100#L

lppropriate amount of RIP. bosomes and with added time was determined with

enoadenine and with an ted to the reaction mixture. 50% was achieved. Portions

reaction mixture) were -pressure liquid 'chromato-

spectrophotofluori- for chromatography

column, a reverse-phase cm × 0.46 cm), was

aborate/phosphoric acid gradient (20 mL) of The flow rate was was set at 315 nm

)-directed phenyla lanine from M . domes t i ca have

were opt imized in 9otassmm ions ( 1 0 m m

react ion was l inear

I I

l g 104

( n g / m l l

proteins gelonin and enylalanine polymerization

~erimental conditions Methods. Phenylalanine

was 3.15 and I. 15 pmol in nts with gelonin (O) and espectively.

~orted by Cenini et al. (1990), whereas the inhibi- ry effects of type 1 RIPs were generally higher, rticularly so in the case of bryodin-R, Hordeum lgare RIP II and saporin 6. In most, but not all The ex T~ ses, RIPs acted on ribosomes at a less-than-equi- for ~lar concentration (Table 1, Fig. 3). of Incubation of ribosomes with RIPs brought about ala s formation of the so-called "aniline fragment" of IC,. ~IA (Fig. 4). The fragment was present only in mples treated with RIP and aniline and was absent treatr controls and in RIP-treated samples when aniline libera

phor~ mass

1 2 3 ment Th,

more adeni

+ m ml~ m m[~ " meth, M . d ditior with condi could lineai show: The a

l l l q ; 11 ' '~ l ) '~ , l . /~ l l . l~ l .~ O .~UVl ty V l

directly quantitatcd by mea adenine released. In order to do

ad of Zamboni et al. 0989) domestica ribosomes. In our e:

ditions a better separation of adeni a linear gradient of methanol in

conditions. Amounts of adenine lo could be detected, and reaction an linear up to at least 10 pmol of adt shown). H.p.l.c. chromatograms are

amount of adenine liberated by good accordance with the effect on phenylalanine polymerization (Tabl

DISCUSSION

Present experiments were under study the effects of RIPs on i observed in a previous screening (C¢ To evaluate RIP activity in more de ary to prepare a ribosome system of efficiency. The M. domestica ribosc indicated in Materials and Methods more active in phenylalanine pol) previously described (Cenini et al. contamination is very difficult to

Table 2. Effects of saporin 6 on protein syntbe,, from Musca domestica ribos(

Adenine released Saporin 6/ribosome (pmol/pmol of

(mol/mol) ribosomes) br N-glycosidase activity. 1 0.82 )nditions were as described 0.1 0.18 lne 1, saporin 6 (6 pmol); me 3, con t ro l w i thou t RIP. Adenine released was measured by h.p.l

Materials and Methods and in the leget d with a minus were not .... ,h.o;. ;nh;h;. . . . . . . dJt~; . ,~ h.,,h~

Fig. 4. Ana lys i s o f r R N A for Reaction and electrophoresis conditions in Materials and Methods. Lane lane 2, momordin (0.6 pmol); lane Samples in the lane designated treated with aniline while those designate treated with aniline. The RNA fragm

aniline treatment is indicated by

Ribosome-inactivating proteins

(Fig. 1), indicating Table 1. Effect ol R. Poly(U)-directed type 2 on poly(U)-d atinued for at least a if 2 pmol of ribo- Fig. 2). The activity Ribosome-inactivati rues was evaluated U)-directed phenyl- Type I Dianthinary°din'lq an of the "aniline Hordeum depurinated RNA G-clonin

mine from treated Momordil PAP-S

eported in Table 1. Saporin 6 re similar to those Trichokiri

Type 2 Ricin 60 Viscumin Volkensin

The experimental pl ype 2 RIPs we

1 hr prior to RIPs, riboso

alanine in 10 mJ IC~ is the cone

treatment was liberated RN.A )horetic migral

of the R1 ~ of mamr~

The N-glyco

4-

Livating proteins type l and mine polymerization by Musca ,osomes

Ribosome/RIP ICs0 at the ICso

(riM) (pmol/pmol)

53.52 0.19 0.41 24.39

79.19 0.13 1267 0.01

8.32 1.20 13.77 0.73 5.63 1.78 1.04 9.58 2.33 4.29

503 0.02 75.44 0.13

bed in Materials and Methods. 1% 2-mercaptoethanol at 37°C • eaction mixture. In the absence lymerized 2-3 pmol of phenyl- ts arc means of duplicates. The ) giving 50% inhibition.

: molecular mass of the as judged by electro-

3arently identical to the produced by RIP treat- nes (Stirpe et al., 1988). y of RIPs is better and

measurement of the so, the h.p.l.c.

was adapted to experimental con-

adenine was obtained tool instead of isocratic

lower than 1 pmol and detection were adenine (results not are shown in Fig. 5.

saporin 6 was in poly(U)-directed

(Table 2).

undertaken to further msect ribosomes

Cenini et al., 1988). detail it was necess- of great purity and

ribosomes prepared as is were 20-25-times

rmerization than aL, 1990). RNases

avoid preparing

,nthesis and adenine release ribosomes

Inhibition of protein synthesis (% of controls)

88% 27%

a.l.c, as described in legend to Fig. 5. Protein

ted by the inhibition of poly(U)- ration as described in Materials of RIPs, ribosomes (2 pmol)

lalanine in 10rain at 24°C.

® 15o Cenin '~ inac

75 of

i ~ Cenin: 5 10 15 plat

vari Time (min)

;. 5. H.p.l.c. chromatograms for the identification and Deren Iermination of adenine released by saporin 6 from (19~ ~ca domestica ribosomes. Experimental procedures are of 1 ~cribed in Materials and Methods. The volume analyzed s 100/z L, corresponding to 7.9 pmol of control ribosomes Endo p) and to 7.9 pmol of saporin 6-treated (1:1 molar ratio) The osomes (bottom). The retention time of the adenine peak on

was 15.5 min. of t] the

Endo ~osomes from a source like whole larvae, but the of r ntamination was sufficiently low to allow for the Hulti~ ;ntification of the "aniline fragment" of RNA. enz~ The effects of type 1 RIPs on this more purified by ~tem were higher than those obtained previously ribc

Jimrm enini et al., 1990) especially regarding the activity tein saporin 6. The pattern of inhibition by the various synt ~e 1 RIPs resembles that obtained with other McCa imal r ibosome sources (Cenini et al., 1990). Pret

U l t l l U I l I U U l I I q , . i l L I U I I 111 t l l U ,~O O I I I J U ~ i U I I

toxins. J. biol. Chem. 262, 5908-5 Endo Y. and Tsurugi K. (1987) RNA N-

of ricin A-chain. J. biol. Chem. 262, Hultin T. and N/islund P. H. (197

ymatic phe-tRNA binding to ma~ thallium ions at concentration

ribosomal functions. Eur. J. Biochem Jimrnez A. and V~izquez D. (1985) Hal

and glycoprotein toxins inhibitin~ rnthesis. A. Rev. Microbiol. 39, 649-

McCann W. P., Hall L. M. and NonJ 3aration, titration, and storage oft

saponn 6 showed the for fluorometric determination of idase activity of the RIP, derivatives. Analyt. Chem. 55, 1455-1 of R N A after treatment McCann W. P., Hall L. M., Siler W

~enine. These effectswere Whitley R. J. (1985) High-pressure s of saporin 6 less-than- graphic methods for determining ar," • rming that the release monophosphate, arabinosyl-adenine

hypoxanthine in plasma and urine enzymatic activity. The Chemother. 28, 265-273.

)f RIPs in this system Montanaro L., Sperti S., Zamboni ,er min) can be explained Testoni G., Gasperi-Campani A. am • ature in the assay. This Effect of modeccin on the steps

near physiological for elongation. Biochem. J. 176, 371-379. ames and may not be Nicolson G. L., Blaustein J. and Etzler M dase activity, ization of two plant lectins from Ric

their quantitative interaction with Biochemistry 13, 196-204.

Dr A. Mattioli for the Roberts W. K. and Selitrennikoff P. Lyde and Dr M. Brigotti proteins that inactivate foreign riboso| p.l.c, determinations. The 19-29.

Ministero dell'Universital me, and by the Consiglio Sierra J. M., Meier D. and Ochoa S in the Progetto finalizzato development on the translation of n

Artemia salina embryos. Proc. natn. .~ 2693-2697•

Sperti S., Montanaro L., Derenzini M., A. and Stirpe F. (1979) Effect of mo ribosomes in vivo. Biochim. biophys.

., Derenzini M., Betts C.M., Stirpe, F., Bailey S., Miller S. P. and B lesions of acinar pancreatic Modification of ribosome-inactivatil

ribosom~ contamination identifi

The system (Cenini of sa tyl animal

Ribosomes treated with typical effect of the N-glycosidase act i.e. production of a fragment of with aniline and release of adenine. obtained with concentrations equimolar to ribosomes, confirmin of adenine was the result of e n r apparently low efficiency of (very little adenine released per min) c by the relatively low temperature temperature was chosen as M . domestica larvae ribosom, optimal for R N A N-glycosidase

Acknowledgements--We thank preparation of chloroacetaldeh for excellent advice on the h. research was supported by the e della Ricerca scientifica, Rome Nazionale delle Ricerche, within "Bioteenologie e Biostrumentazione".

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