Comparative Biochemistry and Physiology Part B 135(2003) 583–591

1096-4959/03/$ - see front matter� 2003 Elsevier Science Inc. All rights reserved.doi:10.1016/S1096-4959(03)00120-9

Phenoloxidases in ascidian hemocytes: characterization of the pro-phenoloxidase activating system

Nicolo Parrinello*, Vincenzo Arizza, Cinzia Chinnici, Daniela Parrinello, Matteo Cammarata`

Laboratory of Marine Immunobiology, Department of Animal Biology, University of Palermo, Via Archirafi, 18, 90123 Palermo, Italy

Received 21 October 2002; received in revised form 15 April 2003; accepted 21 April 2003

Abstract

The phenoloxidase(PO) activity of the hemocytes lysate supernatant from three ascidians species, assayed by meansof 3-methyl-2-benzothiazolinone hydrazone hydrochloride, have been compared. PO-containing hemocytes were identifiedby a cytochemical reaction and the enzymatic activity measured by a spectrophotometric assay of lysate supernatantfrom hemocyte populations separated on a discontinuous Percoll density gradient. InStyela plicata, the enzyme appearedto be contained in morula cells only. InCiona intestinalis, PO activity was shown in univacuolar refractile granulocyteand granular hemocyte. InPhallusia mammillata both compartment cell and granular hemocytes were positive. Enzymaticassay following electrophoretic analysis on polyacrylamide gel electrophoresis(PAGE) or SDS-PAGE indicated thathemocyte lysate presented orthodiphenoloxidase(catecholase) activity. The enzymes from the three species differed inmolecular size, activating substances and trypsin sensitivity.� 2003 Elsevier Science Inc. All rights reserved.

Keywords: Tunicate; Phenoloxidase; Hemocyte;Styela plicata; Phallusia mammillata; Ciona intestinalis; Cell separation; Immunity

1. Introduction

In invertebrates, the cascade reaction called‘prophenoloxidase activating system’(proPO) is amelanogenic pathway that is also involved inimmune reactions of hemocytes(Johansson andSoderhall, 1989; Soderhall and Cerenius, 1998).¨ ¨ ¨ ¨Phenoloxidase(PO) is a bifunctional copper con-taining redox enzyme(o-diphenol:O oxidoreduc-2

tase, EC.14.18.1) that catalyzes the ortho-hydroxylation of monophenol(i.e. tyrosine) form-ing o-diphenol, and then the dehydrogenation ofthe diphenol into ano-quinone. The quinones maypolymerize producing melanin(Nappi and Sey-mour, 1991). The proPO system is activated byb-

*Corresponding author. Tel.:q39-91-6230100; fax:q39-91-6230144.

E-mail address: nicpar@unipa.it(N. Parrinello).

1,3 glucan, lipopolysaccharides(LPS) (Soderhall¨ ¨and Smith, 1983), andyor through limited prote-olysis by a serine protease(Soderhall, 1983). The¨ ¨modulation mechanism resembles the complementcascade and intermediate products can display invitro cytotoxic reactions(reviewed in Nappi andVass, 1993; Sugumaran, 1996; Nappi and Ottavi-ani, 2000).

In tunicates, PO activity has been detected inhemocyte lysates andyor was revealed by cyto-chemical analysis(Cammarata et al., 1996). In theascidiansGoniocarpa rustica, Halocynthia auran-tium (Chaga, 1980), Ciona intestinalis (Jackson etal., 1993), Styela plicata (Arizza et al., 1995),Phallusia mammillata (Cammarata et al., 1999)and the colonialBotryllus schlosseri (Ballarin etal., 1994), proPO is activated by protease action(Arizza et al., 1995) and microbial LPS(reviewed

584 N. Parrinello et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 583–591

in Cammarata et al., 1996). After incubation withblood plasma from incompatible colonies ofBotryllus (Ballarin et al., 1996) in Holothuriaroretzi, PO was released from hemocytes inresponse to stimulation by sheep red blood cellsand yeast cells(Hata et al., 1998). Several hemo-cyte types, described on the basis of their mor-phology (Rowley et al., 1984; Wright, 1981), arecommon to all ascidians: stem(lymphocyte-like)cells, hyaline amoebocytes, granular amoebocytes,signet-ring cells, compartment cells and morulacells. In addition, hemocytes specific to certainspecies have been reported(Parrinello et al.,1996).

Recently, we demonstrated(Arizza et al., 1995)that morula cells fromS. plicata, and ‘univacuolarrefractile granulocytes’ fromC. intestinalis containproPO and display cytotoxic activity against tum-our cell line K562 and rabbit erythrocytes via POproducts(Lipari et al., 1995; Cammarata et al.,1997). Quinones could be the cytolytic moleculesfrom the PO-pathway of these solitary ascidians,while ROI were cytotoxic inB. schlosseri nonfusion reaction(Ballarin et al., 2002).

In this article, the PO system ofP. mammillatawas identified and the active enzyme characterized.Moreover, in hemocytes ofC. intestinalis and S.plicata, POs appeared to be distinguishable in theirbiochemical properties, while enzyme localizationstudies in enriched hemocyte populations showeda variety of PO-containing cells.

2. Materials and methods

2.1. Tunicates and collection of hemolymph

Ascidians were collected in the Southern Medi-terranean sea(Mazara del Vallo) kept in filtered,aerated seawater at 158C, and fed with a marineinvertebrate diet(Hawaiian Marine Imports, Inc.,Houston, TX).

Before bleeding, the tunic was cleaned of epi-phytes, and the sample area was sterilized withalcohol. Hemolymph was aseptically withdrawnfrom the heart with a sterile syringe using sterilecalciumymagnesium-free artificial sea water(FSW) containing 10 mM ethylenediamine-tetra-acetic acid (EDTA) as anticoagulant(FSW-EDTA:hemolymph ratio was 1:9). Aftercentrifugation at 400=g for 10 min, the superna-tant was removed and the hemocytes were washedthree times in sterile FSW-EDTA.

2.2. Hemocyte lysate supernatant

Hemocytes were separated from hemolymph-FSW-EDTA by centrifuging(400=g) for 10 minat 4 8C, immediately suspended in ice cold FSW-EDTA, counted in a Neubauer chamber and adjust-ed at 3=10 ml . Hemocyte populations were6 y1

separated by density gradient centrifugation onPercoll, and then adjusted at 3=10 ml . Follow-6 y1

ing a subsequent centrifugation, hemocytes wereresuspended in the same volume of 10 mM cacod-ylate buffer, pH 9,(CAC) and sonicated at 48Cfor 20 s(Branson, model B15, Danbury, CT). Thelysate was then centrifuged at 27 000=g for 20min at 4 8C and the resulting HLS was used inthe experiments.

2.3. Assay of PO activity

PO activity in HLS was measured spectropho-tometrically by recording the formation of theproduct deriving from the reaction of dopa-quinonewith 3-methyl-2-benzothiazolinone hydrazonehydrochloride (MBTH) (Winder and Harris,1991). Briefly, 20 ml of HLS were incubated for20 min at 208C with 150 ml of CAC buffer, pH9, and 150ml of MBTH reaction mixture, contain-ing 0.49 ml of buffer B (CAC buffer, 4%N,N9dimethylformamide), 0.2 ml of 5 mM dihy-droxyphenylalanine(L-dopa) and 0.3 ml of 20.7mM MBTH in buffer B (dopa-MBTH). Afterincubation, the reaction product was detected at505 nm. PO activity was expressed as relativeunits, where 1 Us0.001 DA min mgy1 y1

505

protein.The effect of proteases as a possible activator

was examined by adding 150ml of bovine pancre-as trypsin(type III) (1 mg ml ) solution in CACy1

to the HLS sample(20 ml). After 20 min prein-cubation, the reaction mixture was completed byadding 150ml of the above reaction mixture. Thespectrophotometric measures were compared withcontrols where protease solution was substitutedby 150ml of CAC buffer.

2.4. Effect of divalent cations, EDTA, soybean-trypsin inhibitor, zymosan, laminarin, LPS, tropo-lone and phenylthiourea on PO activity of P.mammillata

To evaluate the effects of divalent cations, HLS(20 ml) was incubated for 20 min with 150ml

585N. Parrinello et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 583–591

CAC, containing various concentrations of Ca2q

or Mg (5, 10, 100 mM) or EDTA (10 mM).2q

Since it is known that 5–0.5 mg ml soybeany1

inhibitor (STI) inhibits protease activity(Arizzaet al., 1995) experiments were carried out byadding 75ml of STI (1 mg ml ) to the reactiony1

mixture.The effect of zymosan A(Saccharomyces cer-

evisiae) or laminarin(Laminaria digitata), or LPS(Escherichia coli serotype 0111:B4,Pseudomonasaeruginosa serotype 10 Habs, andSerratia mar-cescens) on the activation of proPO system wasevaluated by preparing a reaction mixture where150 ml CAC buffer containing 1 mg ml of eachy1

substance was added.To show the copper-linked PO activity according

to Sugumaran et al.(1988) experiments usingcopper-chelating agents tropolone(Kahn, 1985)and phenylthiourea(PTU), were performed atconcentrations of 100 and 1 mM in CAC,respectively.

Each mixture reaction was incubated with 150ml MBTH mixture for 20 min at 208C, and POactivity estimated at 505 nm. Reagents were omit-ted in controls.

2.5. Protein determination

Protein concentration of HLS was determinedaccording to Bradford(1976), with bovine serumalbumin as the standard. The protein concentra-tions of HLS were usually in the range from 20 to100 mg ml .y1

2.6. Discontinuous density gradient preparation

We used Percoll density-gradients as reportedby Cammarata et al.(1993) with slight modifica-tions. In brief, Percoll solution(Pharmacia, Upp-sala, Sweden) was dialyzed against FSW-EDTA.To avoid clumping ofS. plicata hemocytes, 10mM cysteine was added. Equilibrated Percoll solu-tion adjusted with FSW-EDTA was mixed withappropriate volumes of FSW-EDTA to make stocksolutions at final concentrations of 20, 30, 40, 50,60, 70, 80 and 90%. Discontinuous density-gradi-ents were prepared by sequential overlayering with1 ml of each Percoll stock solutions into 10-mlcentrifuge tubes. Then, 2 ml of washed hemocytesuspension(7–15=10 ml ) were layered onto6 y1

each gradient, and the tubes were centrifuged in aswing-out rotor at 800=g for 30 min at 7 8C.

Bands of cells were gently collected by aspirationwith a micropipette from the top of the tube andwashed three times in sterile FSW-EDTA.

Cell types were identified under a phase-contrastmicroscope(Fluovert Leitz, Wetzlar, F.R.G.) orthrough Nomarski differential interference contrastoptics. The hemocyte types from each ascidianspecies were named according to the literature(Endean, 1960; Wright, 1981; Parrinello et al.,1996). Cell viability was estimated by the eosin-y(0.05% final concentration in FSW) exclusion test.Dead hemocytes in each band never exceeded 5%.

2.7. Cytochemical PO assay and polyphenolreaction

Hemocytes suspended in FSW were layered ona pyrogen-free(heated at 1808C for 4 h) glasscoverslip and incubated for 30 min at 208C. Cellmonolayers were washed three times with FSWand then fixed with 1% glutaraldehyde in FSWcontaining 1% sucrose for 30 min. Finally, afterthree washing in distilled water, cell monolayerswere incubated with dopa-MBTH and observedafter 1–8 h using an inverted microscope. A visiblepink product was shown when MBTH reacted withdopa-quinones(Winder and Harris, 1991).

In controls, substrate was substituted with thesame volume of distilled water. To reveal thespecificity of PO reaction, hemocytes were prein-cubated with the PO inhibitors, tropolone(100mM) or PTU (10 mM) in CAC for 20 min.

To reveal polyphenols, fixed monolayers wereincubated for 5 min in a mixture containingNaNO (10%), urea(20%) and acetic acid(10%);3

finally, cell monolayer were transferred into 2 NNaOH (Reeve, 1951).

2.8. Electrophoresis

Polyacrylamide gel electrophoresis(PAGE)were performed as described by Davis(1964), andSDS-PAGE according to Laemmli(1970), on 7.5%slab gels unless otherwise specified. Proteins werealso stained with Coomassie Blue. Gels werecalibrated with molecular weight standard protein(BIO-RAD); the average of three independentexperiments was evaluated.

2.9. Cresolate and catecholase activities of proteincomponents separate by PAGE

Just after PAGE, gels were washed twice withCAC buffer, then incubated with MBTH. When

586 N. Parrinello et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 583–591

Fig. 1. PO activity of HLS(1.0=10 cells ml ) j with or6 y1

h without incubation with trypsin(1 mg ml ) from Percolly1

separated hemocyte bands fromP. mammillata (A); C. intes-tinalis (B) andS. plicata (C) hemolymph. *P-0.001.

the appropriate bands were visualized, the gelswere rinsed with water and the reaction wasstopped by adding 7% acetic acid.

Cresolate and catecholase activities were detect-ed on polyacrylamide gels as described by Nel-laiappan and Vinayagem(1986). Briefly, afterelectrophoresis, the SDS-PAGE gel was washedtwice for 10 min each in phosphate buffer, pH 8,containing 2.5% Triton X-100 and once for 10min in phosphate buffer without detergent. Thegel was then incubated in phosphate buffer con-taining 10 mM substrate solution(4-methyl cate-chol for catecholase activity or tyrosine methylester for cresolase activity) and 0.3% MBTH.Reactions were stopped by adding 7% acetic acid.To inhibit PO activity, the gel was treated withdithiocarbamic acid(DETC) (10 mM).

2.10. Elution of PO from SDS-PAGE

The PO was recovered from SDS-PAGE gel bycentrifugation–elution. After migration, the PO-containing gels were cut out with a razor, slicedinto small pieces and placed in centrifugation–elution concentrators with 10 kDa cut off(Micro-con, Amicon), centrifuged at 14 000=g, at 4 8Cfor 30 min. Finally, the recovered samples weredialysed overnight in phosphate buffered saline(KH PO 6 mM, Na HPO 0.11 mM, NaCl 302 4 2 4

mM, pH 7.4).

2.11. Statistical analysis

All experiments were repeated three times. Thevalues are the means of three assays performed intriplicate"S.D. Significance was determined withStudent’s t-test and differences were consideredsignificant atP-0.05.

2.12. Chemicals

Unless otherwise reported, all chemicals werefrom Sigma(USA).

3. Results

3.1. PO activity in Percoll density separated hemo-cytes from the three ascidians species

PO activity of lysate supernatant from theunfractionated hemocytes HLS, examined by usingL-dopa as substrate and MBTH as a specific

reagent, reveals that quinones are produced by thePO-pathway of the three species.

In P. mammillata seven bands of hemocyteswere separated(Fig. 1a), the spontaneous POactivity was low in HLS from the bands 1(fromthe top), 2, 3, 4, 6, 7 while it reached the highestvalue in relative units(50"7 DA min mgy1 y1

505

protein) in the sample from band 5(85% com-partment cells, 10% morula cells). The enhancereffect of trypsin was ten times higher(P-0.001)in the HLS from band 3(mainly composed of22–42% granular hemocytes, 10–12% hyaline

587N. Parrinello et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 583–591

Fig. 2. P. mammillata hemocytes after cytochemical stainingwith MBTH (a, c) or in the presence of inhibitors(b, d): com-partment cell(a, b); granular hemocyte(c, d), C. intestinalishemocytes after cytochemical staining with MBTH(e) or inpresence of inhibitors(f): Granular amebocytes(Ga); Univa-cuolar refractile granulocyte(Urg). S. plicata morula cell aftercytochemical staining with MBTH(g) or in the presence ofDITC (h). Bar (g, h)s3 mm.

Table 1Properties of POs fromP. mammillata, C. intestinalis andS. plicata hemocytes

Species SDS-PAGE SDS Trypsin Hemocyte(kDa) sensitivity sensitivity types

P. mammillata 70 q qq Granular hemocyte150 y " Compartment cell

C. intestinalis 74 y q Granular amebocyte188 y q Granular amebocyte175 y q URG

S. plicata 122 y q Morula cell

amebocytes, 20–30% pigment cells) (Fig. 1a).Trypsin also activated PO activity of band 5(P-0.05).

In C. intestinalis six bands of hemocytes wereseparated(Fig. 2b). The spontaneous PO activitywas low in HLS from bands 1, 3, 4, 6: it washighest (50"7 DA min mg protein) iny1 y1

905

band 2(50% hyaline amebocytes, and 50% gran-ular hemocytes) or 5 (40% in univacuolar refrac-tile granulocytes(URGs) and 58% morula cells)(Fig. 1b). Trypsin significantly (P-0.001)enhanced PO activity in hemocytes from band 2and 5, respectively(Fig. 1b).

In S. plicata five bands of hemocytes wereobtained after separation. PO activity wasenhanced by trypsin treatment especially in band2 (mainly enriched in morula cells, 55%) (Fig.2c).

3.2. Inhibition and activation experiments on POactivity of P. mammillata HLS

Although chymotrypsin preincubation was inef-fective, the enhancing effect of trypsin was signif-icant (P-0.01) when values of PO activity ofsamples with(66.6"11.2) or without (42.5"9.8)the enzyme were compared(Tables 1 and 2).Accordingly, soybean trypsin inhibitor added tothe reaction mixture reduced the PO activity, whichreached control levels at 1 mg ml STIy1

concentration.A significant (P-0.01) increase in PO activity

was observed when HLS was preincubated withLPS of E. coli, S. marcescens, P. aeruginosa andlaminarin. No effect was found with zymosan.Preincubation with tropolone and PTU completelyinhibited PO activity(Table 2), establishing thatcopper-dependent PO activity was involved.

588 N. Parrinello et al. / Comparative Biochemistry and Physiology Part B 135 (2003) 583–591

Table 2PO activity ofP. mammillata HLS after 20 min of incubationwith proteases(1 mg ml ), LPS, carbohydrates and POy1

inhibitors

Treatment PO activity(DA min mg protein)y1 y1

505

Buffer control 42.5"9.8Trypsin (1 mg ml )y1 66.6"11.2*

TrypsinqSTI (1 mg ml )y1 39.4"7.2Chymotrypsin(1 mg ml )y1 41.8"6.7

LPS (S. marcescens) 52.3"6.0LPS (E. coli) 94.3"12.4**

LPS (P. aeruginosa) 60.6"11.6*

Zymosan 50.0"7.4Laminarin 70.5"8.3*

PTU (1 mM) 17.2"5.3**

Tropolone(100 mM) 12.1"10.0**

Values are the mean of three experiments performed intriplicate"S.D.

P-0.01.*

P-0.001.**

Fig. 3. Detection of PO activity in SDS-PAGE. The HLS(100mg) were electrophoresed through a polyacrylamide gel, sub-sequently the gel was treated with a MBTH reaction mixture.(A) P. mammillata, lanes:(1) B5 hemocytes lysate supernatant(B5HLS), SDS-PAGE 7.5%;(2) B3 Hemocytes lysate super-natant(B3HLS); (3) B3HLS after 10 min trypsin pretreatment;(4) B3HLS after 30 min trypsin pretreatment. The HLS(100mg) were electrophoresed through a 7.5% polyacrylamide gel(Lanes 2, 3, 4). (B) C. intestinalis, lanes:(1) B2 hemocyteslysate supernatant(B2HLS), SDS-PAGE 7.5%;(2) B5 hemo-cytes lysate supernatant(B5HLS), SDS-PAGE 7.5%.(C) S.plicata. lanes:(1) B4 hemocytes lysate supernatant(B4HLS),SDS-PAGE 7.5%.

3.3. Cytochemical PO activity with MBTH

In P. mammillata hemocyte smears of separatedbands, PO was found in ‘compartment cells’,mainly abundant in B5(Fig. 2a). This cell typepresents a spherical shape(8–10mm in diameter),with a variable number of large round or angularvacuoles enclosed by a peripheral rim of cyto-plasm. Granular hemocyte abundant in B3(Fig.2c), spherical in shape(6–12 mm in diameter)with a cytoplasm containing distinct granules, werealso PO positive. The use of inhibitors and controlssupported the specificity of the cytochemical reac-tion: no staining was observed by substitutingMBTH mixture with distilled water, while, PTUinhibit the reaction(Fig. 2b). The effect of inhib-itors was also evident after preincubation of thehemocytes with these substances before fixation.

In C. intestinalis PO localized to URGs mainlyabundant in B5, and in granular hemocytes abun-dant in B2(Fig. 2e). URGs, varying in size(4–12 mm) (Fig. 2e and f) present a single prominentrefractile inclusion in a large vacuole that nearlyfilled the whole cells. Granular hemocyte withpositive granules(Fig. 2e) appeared as ameboidcells, when examined in absence of EDTA(notshown); they vary in size(6–12mm), and presenta coarsely granular endoplasm.

In S. plicata PO was only present in morulacells mainly abundant in B4(Fig. 2g). This cell

type appeared as a globular granulocyte(5–11mm) with large, membrane bound vacuoles and aberry-like shape.

3.4. Cytochemical polyphenol reaction

As already shown forS. plicata (Cammarata etal., 1997) morula cells, URGs fromC. intestinaliswere positive for the polyphenol reaction.

3.5. Activity and size of PO from the three ascidianspecies

SDS-PAGE ofP. mammillata THLS and B5HLS(enriched in compartment cells) showed a proteinband of approximately 150 kDa with the specificenzymatic activity (Fig. 3, lane 1). Such, onactivity was not found after electrophoretic sepa-ration in SDS of the B3HLS(enriched in POpositive granular hemocyte), even when a trypsinpretreatment was carried out to activate the pro-enzyme. In contrast, a PO positive band wasevident in the electrophoretic pattern obtained inthe absence of SDS(Fig. 3, lanes 2–5). In thiscondition, pretreatment of B3HLS with trypsinincreased the mobility of a single fraction thatshowed the highest activity(more intensely stained

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Fig. 4. SDS-PAGE of HLS on 10% polyacrylamide:(A) gelincubated with 4-methyl catechol for catecholase activity(B)gel incubated with tyrosine methyl ester for cresolase activity.Lane 1: purified mushroom tyrosinase(Sigma); Lane 2: HLSfrom C. intestinalis; Lane 3: HLS fromS. plicata; Lane 4: HLSfrom P. mammillata.

band in Fig. 3). The enhancing effect was sup-ported by spectrophotometric measures of POactivity in HLS (data not shown). The PO ofB3HLS band, separated in PAGE in the absenceof trypsin was eluted by centrifugation and sub-jected to SDS-PAGE in reducing or non-reducingcondition. In both cases, the enzyme showed amolecular mass of approximately 70 kDa(datanot shown).

The PO associated with the B5HLS(mainlyenriched in URGs) was restricted to a 175 kDaband(Fig. 3, lane 5). SDS-PAGE ofC. intestinalisB2HLS (enriched in granular hemocytes) showeda protein band of 188 kDa and a smeared band ofapproximately 74 kDa(Fig. 3, lane 6).

Both S. plicata THLS and B2HLS(enriched inmorula cells) presented a protein band of approx-imately 121 kDa(Fig. 3, lane 7). Since, PO canshow catecholase(oxidation ofo-diphenols) andyor cresolase(o-hydroxylation of monophenols)activities, specific substrates were assayed. Afterelectrophoretic separation of the THLS from thethree ascidians, a band of activity was developedonly with 4-methyl-catechol suggesting catechola-se activity. To rule out the possibility that copper-independent peroxidases oxidized catecholsubstrates(Okun et al., 1972), gel strips werepreincubated with the copper-chelator DETCbefore reaction with the specific substrate. Thistreatment was effective in abolishing any detecta-ble activity(data not shown). To test the specificityof the enzymatic assay, purified mushroom tyrosi-nase was included in the SDS-PAGE as positivecontrol (Fig. 4).

4. Discussion

Melanogenic processes are widespread in allliving organisms, from fungi to plants, from inver-tebrates to vertebrates, and melanin-containingcells take part in the vertebrate pigmentary systemand may be involved in natural immunity(Robb,1984; Cicero et al., 1982). In arthropods, thispathway may also have a multifunctional role. Ithas been claimed to take part in several immunereactions(Johansson and Soderhall, 1989), and to¨ ¨be involved in the sclerotization of cuticle ininsects(reviewed in Sugumaran, 1996). The cen-tral enzyme, PO, is responsible for the pathwaythat produces intermediate and end products ful-filling these functions.

Ascidian PO is ao-diphenoloxidase(Barringtonand Thorpe, 1968; Chaga, 1980; present work)that can be generally revealed in hemocyte lysatesupernatants(Jackson et al., 1993; Arizza et al.,1995; Ballarin et al., 1994). In P. mammillata HLStwo enzymes are contained, one of them wasspontaneously activated, probably depending onthe hemocyte manipulation during the hemolymphandyor cell lysate preparation. In this respect, it isknown thatP. mammillata hemocytes contain ser-ine proteases(Guerrieri et al., 2000) that mayactivate the proPO system. It is significant that thehighest level of PO activity from separated gran-ular hemocytes(B3), was reached after incubationwith trypsin, suggesting that the pro-PO system ismodulated by a serine protease-dependent mecha-nism as supported by the inhibitory effect ofsoybean trypsin inhibitor(Arizza et al., 1995;Cammarata et al., 1996, 1999). The activationexperiments suggest that the proPO system ofP.mammillata presents some differences with theB.schlosseri (Ballarin et al., 1994) andC. intestinalisproenzyme(Jackson et al., 1993), being insensitiveto zymosan, laminarin(respectively, a and b

glycans) and polygalacturonic acid.Enzymatic assay of the hemocyte lysate and

cytochemical staining of the separated hemocytesrevealed heterogeneity in the ascidian PO-contain-ing cells. In P. mammillata, ‘granular hemocytes’and granulocytes named ‘compartment cells’ con-tain the enzyme. However, differences were foundbetween the B3HLS and B5HLS hemocyte frac-tions enriched in granular hemocytes and com-

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partment cells, respectively. Granular hemocytespresented a proPO sensitive to trypsin activation,whereas compartment cells, abundant in the B5band, were less sensitive to proteolysis by trypsin,being largely spontaneously activated. The bio-chemical and electrophoretic analyses providedevidence of distinctive properties of the two LPS-sensitive enzymes that catalyze the same reaction.The PO of B3HLS can also be inactivated by SDStreatment that usually affects a polymeric proteinstructure. In addition, trypsin treatment signifi-cantly enhanced both the PO activity and theelectrophoretic(PAGE) mobility of the B3HLSactive fraction suggesting cleavage of a peptidefragment. Conversely, SDS-PAGE analysis of theB5HLS indicated that the single 150 kDa compo-nent, insensitive to trypsin and SDS treatments,may be the PO of the ‘compartment cells’ whereasthe 70 kDa PO component characterizes the HLSfrom granular hemocytes.

In C. intestinalis the granular hemocytes(ame-bocytes) and the URGs are the PO containing-cells. The PO activity of granular hemocyte(amoebocyte) HLS is represented by two bands of188 and 74 kDa, whereas PO of the URG lysateis contained in a band of 175 kDa. The possibilityexists that the smeared band of approximately 74kDa represents a monomeric form. InS. plicatamorula cells, PO activity appears to be related toa 121 kDa band.

In accordance with these results, reducing poly-phenols, that are substrates of PO activity havebeen identified in morula cells and URG.

We recently reported thatS. plicata morula cellsreveal a PO-dependent cytotoxic activity againstrabbit erythrocytes and tumour cell lines(Cam-marata et al., 1997). Moreover, it is known thatthe PO-positive hemocytes ofC. intestinalis namedURGs are cytotoxic against erythrocytes(Parrinel-lo et al., 1996) and tumour cell lines(Parrinelloet al., 2001). Therefore, preliminary assays wereperformed on the cytotoxic activity of the unfrac-tionated and density gradient separatedP. mam-millata hemocytes against K562 tumour cell linesand rabbit erythrocytes, but we were unable toshow any cytotoxic reaction(unpublished).

In contrast with arthropod monophenoloxidase,an orthodiphenoloxidase activity is expressed bythe enzymes from ascidian hemocytes. This activ-ity was found in various hemocytes types includingmorula cells, granular hemocytes, and URGs. Wedo not know if these hemocyte types belong to

the same developmental cell line as suggested byBallarin et al.(1993, 1994) for B. schlosseri andParrinello et al.(1996, 2001) for C. intestinalis.

Based on biochemical properties, several ascid-ian POs can be distinguished, presumably relatedto various functional roles. Although they showcatecholase activity, differences in size, trypsinsensitivity, activating substances, and chain SDSsensitivity were found between the POs of theexamined species. A specific PO was also foundin the colonial ascidianB. schlosseri (Frizzo etal., 1999). This enzyme, contained inside thevacuoles of morula cells, has a molecular mass of160 kDa, is composed of two subunits and itsactivity is enhanced by calcium ions. Finally,differences can be found within a species: differentsizes and trypsin sensitivity characterize theenzymes from granular hemocytes and morulacells of C. intestinalis. Cloning and expressionstudies are in progress to reveal the phylogeneticrelationships of these enzymes.

Acknowledgments

This work was supported by MURST grants.

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