[CANCER RESEARCH 52. 3622-3628. July 1. I992|

Characterization and Purification of a Guanidinobenzoatase: A Possible Marker ofHuman Renal Carcinoma1

Claudine Poustis-Delpont,2 Roland Descomps, Patrick Auberger, Pascale Delque-Bayer, Pierre Sudaka,

and Bernard RossiLaboratoire de Biochimie, Facultéde Médecine[C. P-D., R. D., P. D-B., P. S.J, and INSERM V 210, Facultéde Médecine[P. A., B. R.J, Avenue de Valombrose 06107Nice Cedex 02, France

ABSTRACT

Guanidinobenzoatases are cell surface-associated proteinases supposed to be involved in cancer metastasis, cell migration, and tissueremodeling. The main features of the guanidinobenzoatase associatedwith human renal carcinoma plasma membrane are weak membraneassociation, continuous cleavage of ;>-nitrophenyl-/?'-guanidinobenzoate

conversely to the site titration effect of this compound when used withtrypsin, and a peculiar sensitivity to serine protease inhibitors, compatiblewith a poorly active form. Plasma membrane preparation followed byagmatine-trisacryl affinity chromatography allows the purification ofguanidinobenzoatase to homogeneity with an apparent enrichment factorof 450. Purified guanidinobenzoatase appears as a single polypeptidechain of M, 80,000, likely stabilized by intrachain disunities bonds. Theproperties of purified guanidinobenzoatase indicate that it is an originalenzyme in spite of some similarities with plasminogen activators. Indeed,in addition to differences in substrate and inhibitor specificity, guanidinobenzoatase is not recognized by specific monoclonal antibodies directedagainst plasminogen activators or their single-chain precursors. Thus,human renal carcinoma guanidinobenzoatase appears to be an originalenzyme whose activity is undetectable in the nontumoral tissue of origin.In this respect, use of purified guanidinobenzoatase would allow us toobtain specific tools to give new insights in cancer cell metastasis.

INTRODUCTION

The regulation of cellular adhesive, migratory and invasiveevents, and the cancer metastasis process involves proteinases(see Refs. 1-3 for review). Among them, we have investigateda cell surface protease referred to as GB' which was originally

described by Steven and Al Ahmad (4). GB cleaves continuouslyunusual substrates such as p-NPGB and methyl-umbelliferyl-guanidinobenzoate, previously known as titrant-active site inhibitors for trypsin-like enzymes (5). GB can be competitivelyinhibited by the fluorescent probe 9-aminoacridine (6). Thesurfaces of tumor cells in tissue sections (6) or of intact ratleukemia cells (7) have been shown to bind 9-aminoacridine,demonstrating that GB is an ectoenzyme. Using this technique,Steven et al. (6) localized GB in numerous human cancer cells.A small number of nontumoral specialized cells or tissues alsopossess GB: colonie mucosa, infiltrating lymphocytes and macrophages, skin basal layer epithelium, hair follicles, embryoniccells, newly proliferative blood vessels, and spermatozoa (6).

Received 9/16/91; accepted 4/24/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported by a grant from the Association pour la Recherche

contre le Cancer (Villejuif, France) and by a grant from la FédérationNationaledes Centres de Lutte contre le Cancer (Paris, France).

2To whom requests for reprints should be addressed.3The abbeviations used are: GB, guanidinobenzoatase; p-NPGB, p-nitro-

phenyl-p'-guanidinobenzoate; t-PA, tissue plasminogen activator; SDS, sodiumdodecyl sulfate; DMSO, dimethyl sulfoxide; u-PA, plasminogen activator ofurokinase type; NPC, nitrophenyl chloroformate; BL, basolateral; BB, brushborder; PBS, sodium phosphate buffer; PMSF, phenylmethylsulfonyl fluoride;PAGE, polyacrylamide gel electrophoresis; p-NA, p-nitroanilide; DFP, diisopro-pyl fluorophosphate; STI, soybean trypsin inhibitor; BBI, Bowman Birk soybeaninhibitor; E64, /rani-epoxy-i.-leucyl-amido-(4-guanidinobutane): octyl-glucoside,octyl-/3-glucopyranoside.

GB protein inhibitors have been localized in the cytoplasm ofboth tumor cells and nontumoral tissues (lung, liver, brain) witha pattern of cross-inhibition specificity suggesting that GBrepresents a family of enzymes rather than a single entity (8).Finally, GB was demonstrated to cleave the adhesiotope peptideGRGD at the COOH side of arginine (9). The presence of GBon the surfaces of cancer cells and migratory cells, or in remodeling tissues, associated with GRGD cleavage supports the ideathat GB might be involved in cell migration and metastasis.

We have assayed GB activity by its lysis of the synthetic esterp-NPGB in homogenates of human renal clear cell carcinomas.Interestingly, this type of activity was undetectable in the nontumoral tissue of origin. In this paper, we describe the characterization and purification of the GB associated with a humancarcinoma (renal carcinoma) using a modification of the ag-matine affinity column method originally reported by Steven etal. (6) for the isolation of GB secreted in mouse Ehrlich ascitesfluid. Since t-PA was recently demonstrated to cleave methyl-umbelliferyl-guanidinobenzoate as a substrate (10), our purposewas to establish that GB was actually an original enzymedistinct from t-PA and to obtain enough purified material toprovide the tools for its further molecular characterization.

MATERIALS AND METHODS

Materials

Sucrose, Tris, SDS, gelatin, sodium acetate, and sodium barbitalwere obtained from Merck (Darmstadt, Germany). P-NPGB, 3-3'-

diaminobenzidine, plasminogen, inhibitors, all purified enzymes commercially available except t-PA, u-PA, and fibronectin-related peptides,octyl-glucoside, DMSO, and anti-mouse IgG antibody were suppliedby Sigma Chemical Co. (St Louis, MO). Biotinylated anti-rabbit antibody and avidin peroxidase complex were purchased from Dako (Copenhagen, Denmark); t-PA and u-PA were obtained from Stago (As-nières, France); acrylamide and bisacrylamide were from Bio-Rad(Richmond, CA); molecular weight proteins markers and Percoli werepurchased from Pharmacia (Uppsala, Sweden) and ampholines werefrom Serva (Heidelberg, Germany). D-Val-Leu-Lys-p-NA (S2251) andpGlu-Gly-Arg-p-NA (S2444) were obtained from KabiVitrum (Stockholm, Sweden); dansyl-Glu-GIy-Arg-CH2-Cl was from Calbiochem(Meudon, France). NPC-trisacryl was a product of Industries Biologiques Françaises(Villeneuve la Garenne, France).

Methods

Preparation of Membrane Vesicles from Human Renal Cortex andRenal Carcinoma. Nephrectomy specimens from 10 patients with untreated renal clear cell carcinomas of various histological grades wereobtained from the urology department. The nephrectomy specimen wasimmediately brought to the laboratory at 0-4°C.Tumor and slices of

nontumoral renal cortex were subjected in parallel to the same membrane purification technique (11). This membrane preparation, foundedon differential centrifugations and MgCl2-CaCl2 precipitation, allowsthe separation of BL and intracellular membranes (pellet) from BBmembranes (supernatant). Thereafter, the crude BL pellet obtained inthe presence of MgCl2 and CaCl2 was subjected to a Percoli density

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RENAL CARCINOMA GUANIDINOBENZOATASE

gradient to obtain purified BL membranes. Except when otherwiseindicated, the results shown were obtained from the same membranepreparation.

Solubilization and Quantitation of Membrane Proteins. Octyl-gluco-

side (1.35%, w/v) was used as previously indicated (12). Protein concentration was estimated during the course of purification by specificabsorbance at 280 nm: A28o1% = 20 units. After concentration dialysisof the eluate or before purification, protein concentration was measuredaccording to the method of Lowry et al. (13) with bovine serum albuminas standard.

Enzyme Activities. GB activity was measured according to a methodused for trypsin-active site titration (14) with a modification: the assaywas carried out in 0. l M sodium barbital/HCl, pH 7.6, without CaCl?.Usually, 200 /ugof octyl-glucoside extract of cancer BL membranes wasused per assay. The sample was first preincubated at 37°Cfor 1 min

and then the reaction was started by the addition of 0.25, 0.8, or 1.5mM p-NPGB, 1% (v/v) DMSO (final incubation volume, 1 ml). Continuous release of /r-nitrophenol was followed at 37°Cat 410 nm for 5

min, except when indicated otherwise. A blank measurement, performed in an identical buffer mixture in the absence of protein extract,was systematically subtracted.

Na*-K*-ATPase activity (EC 3.6.1.3), a BL membrane marker, wasassayed at 37°Cas previously described (15). Before the assay, mem

brane vesicles were permeabilized by incubation with a mixture ofsodium deoxycholate (0.12%) and EDTA (2 mM) for 30 min at roomtemperature.

Aminopeptidase N activity (EC 3.4.11.2), a BB membrane marker,was determined using Ala-p-NA as a substrate (16).

In tracellular membrane markers: cytochrome c oxidase (EC 1.9.3.1),a mitochondrial membrane marker (17), and NADPH-cytochrome creducíase(EC 1.3.99.1), an endoplasmic reticulum marker (18), weremeasured as previously described. /3-GIucuronidase (EC 3.2.1.31), aGolgi marker, was estimated using p-nitrophenyM-glucuronide as a

substrate (19).Trypsin activity (EC 3.4.21.4) was assayed using N-a-benzyl-Arg

ethyl ester as a substrate at pH 7.6 and 25'C (20). Plasmin (EC 3.4.21.7)was measured by following the hydrolysis of a-casein at 37'C at pH

7.5 at 280 nm (21). Plasminogen activator activity was assayed usingD-Val-Leu-Lys-p-NA (3.5 HIM) as a substrate. The sample was incubated at 37°Cin 150 mM of PBS, pH 7.5, in the presence of 3.6 ^M of

plasminogen, and the reaction was quantitated by the release of p-nitroaniline, measured at 410 nm. u-PA activity was measured byfollowing the hydrolysis of its specific chromogenic peptide (3.5 mM)<Glu-Gly-Arg-p-NA over 2 h at 37°Cin PBS, pH 8.5. Cathepsin B

activity (EC 3.4.22.1) was assayed by the hydrolysis of A'-a-benzyl-Lys-p-nitrophenyl ester at 25"C (22).

Agmatine-Trisacryl Gel. Agmatine (0.1 M) and NPC-activated trisa-cryl (1 g) were coupled in 4.5 ml of 0.1 M sodium carbonate, pH 10,for 6 h at room temperature, with constant gentle stirring. The resultinggel (3 ml) was extensively washed with the coupling buffer and neutralized by incubation in 0.5 M Tris, pH 8, for l h at room temperature.Agmatine-trisacryl gel was then rinsed first with l M NaCl and thenwith 10 mM Tris/HCl, pH 8.3, binding buffer. The gel was thenequilibrated in binding buffer. Usually 6 /tmol of agmatine were coupledper 1 ml of gel.

Guanidinobenzoatase Purification. GB was purified using a modification of the method of F. S. Steven (6). Routinely, 4.5 ml of octyl-glucoside extract of renal cancer BL membranes containing 7.5 mg/mlof proteins was incubated with agmatine-trisacryl (2 ml) equilibrated in10 mM Tris/HCl, pH 8.3, for 2 h at 37°Cwith constant stirring in the

presence of an antiprotease cocktail [aprotinin (200 kilounits/ml),PMSF (0.1 mM), E64 (0.1 HIM) 1,10-phenanthroline (1 mM)]. Thereafter, the mixture was poured into a column and allowed to settle at4°C.The gel was then washed with the binding buffer until the specific

absorbance of the effluent at 280 was zero. Elution was carried outusing 0.1 M sodium acetate/acetic acid buffer, pH 4. (flow rate 3 ml/h). Fraction volumes were 0.8 ml. To neutralize the eluate, 50 ß\of 2M Tris, pH 8.8, was immediately added to each fraction. The activefractions were pooled and concentrated by concentration dialysis (Biob-

lock Scientific, Illkirch, France) using a PM 15 membrane and storedat -80°C.

SDS-Polyacrylamide Gel Electrophoresis. SDS-PAGE (5-12% poly-acrylamide) was run using a LKB 2001 apparatus (Uppsala, Sweden)and the Laemmli buffer system (23). Two-dimensional electrophoresisof purified GB was performed according to the method of O'Farrell

(24). Gels were then silver stained as described previously (25). GBactivity retained in the gel after PAGE was checked as follows. SDSwas removed by incubating gel slabs with 0.15 M PBS, pH 7.5, containing 2% Triton X-100 for 30 min at room temperature. Triton X-100

was then washed away by incubating the gel twice with PBS for 5 mineach time. The gel was then cut into 3-mm-thick slices. Each slice wascrushed and stirred overnight at 4°Cwith 900 n\ of 0.1 M sodium

barbital, pH 7.6. GB activity was determined from the hydrolysis of 1.5mM p-NPGB at 37°Cover 30 min.

Immunodetection on Nitrocellulose. Commercially available t-PA,human u-PA, and purified GB (1 /<g)were subjected to dot blot analysison nitrocellulose sheets (Millipore) in a 96-well dot blot apparatus(Hybridot, Manifold, Bethesda Research Laboratories). Immunodetection of each antigen was assayed using two types of antibodies (1 Mg):(a) a mixture of 5 specific ami t l'.\ monoclonal IgG antibodies (kindlydonated by Professor D. Collen) and (b) a specific anti-u-PA IgGmonoclonal antibody (Biopool, Umea, Sweden). The antigen-antibodycomplexes were bound to l ¿¿gof a rabbit anti-mouse anti-IgG antibody(Sigma) followed by biotinylated anti-rabbit immunoglobulin (Dako).Then, avidin-peroxidase (Dako) was added, and the complexes werestained using 1.4 mM 3,4-diaminobenzidine as a substrate (in 0.1 MTris, pH 7.4) and H2O2 as a cosubstrate.

RESULTS

Guanidinobenzoatase Activity Is Present in Homogenates ofHuman Renal Carcinomas but not in Homogenates of NormalRenal Cortex from the Same Kidney

Si fractions, corresponding to supernatants of homogenatessubjected to centrifugation for 10 min at 1000 x g, were checkedfor their ability to cleave continuously p-NPGB (0.25 HIM) at37°Cand pH 7.6. The average level of GB activity was 1.56 ±

0.3 milliunits/mg as measured from 10 renal carcinomas,whereas no activity was detectable in any of 10 Si fractions ofnormal renal cortex dissected from the same kidneys (Fig. 1).Under these conditions, the activity value significantly differentfrom zero (P > 95%) was 0.145 milliunits/mg. GB activityfrom carcinomas was abolished by a heat treatment of 1 min at100'C or incubation for 24 h at 37°C(not shown).

2.0 ,

O) 1.5 .

îmo

1.0 -

0.5 -

0.0

I

i

cancersn=10

renal cortexn=10

Fig. 1. GB activity in homogenates from human renal clear cell carcinomasand in homogenates from human renal cortex from the same kidneys. GB activity(mean ±SEM) was measured in the presence of 0.25 mM p-NPGB and of 200fig of the supernatant after centrifugation of the homogenate (Si fraction) at 1000x g, pH 7.6, in 0.1 M sodium barbital at 37'C as indicated in "Methods." Oneunit represents 1 fimol of p-nitrophenol released/min.

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RENAL CARCINOMA GUANIDINOBENZOATASE

Subcellular Location of GB Activity

The membrane preparation described by Sheikh et al. (Il)was chosen because it allows the separation of BL membranesfrom BB membranes for which numerous proteinases are associated. In the course of this preparation, plasma membranesfrom renal clear cell carcinomas, although less differentiated,behaved like those of the tissue of origin (Table 1). Thus, thistechnique provided an enriched fraction to study and to purifyGB. Table 1 shows that GB activity was undetectable in anyfraction from nontumoral renal cortex. It has to be mentionedthat only part of the GB activity (25%) was found in BLcarcinoma membranes, whereas 67% of GB activity was recovered in the S2 fraction (not shown). 82 is the supernatantresulting from the centrifugation of the S, fraction at 48,000 xg for 30 min; thus, it corresponds to the cytosolic fraction.Nevertheless, GB activity was enriched only in cancer BLmembranes.

Specificity of p-NPGB as a Substrate

Trypsin, u-PA, cathepsin B, plasmin, and acetylcholinester-ase were unable to hydrolyze 0.25 mivi p-NPGB at a detectablelevel. We verified that these enzymes were active toward theirspecific substrate. Purified t-PA (essentially in two-chain form),however, exhibited a weak esterolytic activity toward p-NPGBof 2.8 milliunits/mg of purified enzyme (not shown).

Characterization of GB Activity in Octyl-Glucoside Extracts ofRenal Carcinoma BL Membranes

We routinely extracted GB activity from BL carcinoma membranes using 1.35% (w/v) octyl-glucoside because it allowed alower blank at 410 nm than did crude membranes. It alsoallowed the use of higher concentrations of p-NPGB thanTriton X-100 extracts, without substrate precipitation. GBactivity could also be completely extracted with l M NaCl orKC1, but, in that case, GB activity was lost and dialysis wasnecessary to partially recover GB activity (30%). In addition,GB activity was not extractable by activated papain (3%, w/v)after l h at 37°C(not shown). GB activity was measurable at

pH 7.2 (3.8 milliunits/mg) and increased at alkaline pH, reaching a maximal value of 8.5 milliunits/mg at pH 9-9.5 (notshown). Despite this increased activity at alkaline pH, wepreferred to routinely measure GB activity at pH 7.6 where thebackground absorbance was significantly lower.

Time Course and Substrate Concentration Dependence of GBActivity. Fig. 2A shows the continuous cleavage of 0.25 mM p-NPGB at 37°Cand pH 7.6 by octyl-glucoside extract of BL

carcinoma membranes (200 ßg).Under the same experimental

0.06-

0.04-

0.02-

g. 0.002468

minutes at 37=C10

10

8

6

4 •¿�

2 .

0.0 0.2 0.4 0.6

p-NPGB.mM

0.8

Fig. 2. Time course and substrate concentration dependence of GB activity inoctyl-glucoside extracts of renal carcinoma BL membranes. Octyl-glucoside extract (200 »jg)was checked for GB activity at 37'C in 0.1 M sodium barbital/HCl,pH 7.6, and 1% (v/v) DMSO. A, time course in the presence of 0.25 mM p-NPGB. B, initial velocity (V) of GB activity as a function of substrate concentration (continuous release of p-nitrophenol was followed at 410 mn for 5 min). Thisresult was typical of the 3 experiments performed, mil, milliunits.

conditions, initial velocity as a function of p-NPGB concentration displayed a curvilinear pattern, suggesting saturation (Fig.2B). Unfortunately, it was not possible to study higher p-NPGBconcentrations than those indicated because of substrateprecipitation.

Inhibition Pattern of GB Activity. As shown in Table 2, GBactivity in the octyl-glucoside extract from renal cancer membranes was not inhibited by metalloprotease inhibitors (EDTA,ethyleneglycol bis(0-aminoethyl ether)-/V,yv,/V",7V"-tetraaceticacid, 1,10-phenanthroline), cysteine protease inhibitor (E64),

or aspartate protease inhibitor (pepstatin). The aminopeptidaseinhibitor bestatin (0.16 mvi) also did not inhibit GB activity(not shown). These results suggest that GB belongs to the serineproteinase family. Among the irreversible inhibitors of serineproteases, only DFP (30 /UM)and dichloroisocoumarin (250(¿M)were able to inhibit GB activity (Table 2). Among thereversible proteinase inhibitors (Table 2), two plasmin inhibitors, aprotinin and «:-macroglobulin (100 ng/m\), and anti-thrombin 3 were not inhibitory for GB. Since amiloride (1 mivi)totally inhibited GB activity and was reported to inhibit the

Table 1 Marker enzymes activities in fractions of membrane preparations from renal carcinoma and renal cortex from the same kidneyS,, supernatant of the homogenate centrifuged at 1000 x g 10 min. Enzyme activities were measured as indicated in "Methods." One unit of enzyme activity, 1

mmol of substrate hydrolyzed/min. Numbers in parentheses, enrichment factor versus homogenate. Yield of GB activity in the course of the preparation from cancerhomogenate was 2630 milliunits in homogenates (100%), 3025 milliunits in S (115%), 90 milliunits in BB fraction (3.5%), and 655 milliunits in BL fractions (25%),whereas the protein yield was 7.1% in BL fraction.

Normal renalcortexHomogenateS,BBBLRenal

cellcarcinomaHomogenateS,BBBLNa-K-ATPase

(milliunits/mg)21(1)21(1)38(1.8)205

(10)10.6(1)12(1.1)29

(2.6)104(9.8)Aminopeptidase

N(units/mg)715(1)768(1.1)5303

(7.4)1287(1.8)115(1)279

(2.4)741(6.4)202

(0.7)/j-glucuronidase

(milliunits/mg)0.17

(1)0.20(0.5)0.13(0.8)0.13(0.8)0.10

(1)0.09(0.9)0.125(1.2)0.07

(0.7)Cytochrome

c oxidase(units/mg)2.3

(1)7(1.2)1(0.45)0.8(0.35)5.3

(1)5.75(1.1)1.4

(0.3)1.2(0.2)Cytochrome

c reducíase(units/mg)5.9

(1)7(1.2)4.3(0.8)4.3(0.8)4.9

(1)6.75(1.4)3.5

(0.7)3.2(0.7)GBase

(milliunits/mg)0000KD1.2(1.2)0.8

(0.8)3.5(3.5)

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RENAL CARCINOMA GUAN1DINOBENZOATASE

catalytic activity of urokinase (26), other inhibitors of plasmin-ogen activators were checked. Neither benzamidine nor t-ami-nocaproic acid were inhibitory toward renal carcinoma GB(Table 2). In addition, although they did not inhibit plasmino-gen activators, STI, BBI, and a,-antitrypsin were able to inhibitrenal carcinoma GB in a dose-dependent manner (Table 2).

Purification of GB

After incubation of octyl-glucoside extract of BL carcinomamembranes with agmatine-trisacryl as indicated in "Methods,"

no GB activity was detectable in the supernatant of the gel. GBstarted to elute below pH 5. The elution profile is shown onFig. 3, and the main features of GB purification are shown onTable 3.

SDS-PAGE Analysis of Agmatine-Trisacryl Eluates. Whenanalyzed by SDS-PAGE, concentrated agmatine-trisacryleluates contained a protein having an apparent M, of 55,000 or80,000 under nonreducing or reducing conditions, respectively(Fig. 4A). This feature is typical of a single-chain protein witha high degree of disulfide-linked secondary structure. After two-dimensional electrophoresis, purified GB appeared as a singlehomogeneous protein (Fig. 4B).

Identification of Isolated GB Activity

GB Activity in the Crushed Aery lamido Gel. After 5-12%SDS-PAGE, the gel was treated to regenerate GB activity, andthen the lane where GB activity had been loaded (9 milliunitsmeasured in the presence of 1.5 m\i p-NPGB) was sliced, andGB activity was measured as indicated in "Methods." Three

independent experiments were performed using various concentrations of p-NPGB. In all three experiments, there was significant GB activity in the slice containing the M, 55,000 band (P> 99.7%). Fig. 5 shows a typical experiment in which theliberation of p-nitrophenol in the slice containing the singleprotein band (0.172) was significantly higher compared to thatof the background (0.116 ±0.009, n = 39). A smaller peak of

5 10 15

fraction number

20

Fig. 3. Elution pattern of agmatine-trisacryl gel affinity column. Agmatine wascoupled to NPC-trisacryl gel as indicated in "Methods" and equilibrated in 10niM Tris, pH 8.3. Agmatine-trisacryl gel (2 ml) was incubated 2 h at 37'C with

4.5 ml of octyl-glucoside extract of renal carcinoma BL membranes in the presenceof an antiproteinase cocktail (see "Methods"). Thereafter, the gel was poured ina column and allowed to settle at 4°C.After the column was washed with the

binding buffer, elution was carried out with 0.1 M sodium acetate buffer, pH 4 (3ml/h). Fractions (0.8 ml) were immediately neutralized with 50 »ilof 2 M Tris,pH 8.8. GB activity was measured with 100 n\ of eluate in the presence of 0.8mM p-NPGB in 0.1 M sodium barbital, pH 7.6, for 10 min at 37'C. This

experiment was typical of the 6 purifications carried out under the same conditionswith aliquots from the same membrane preparation, nil milliunits.

presumed activity at the top of the gel was also constantlyobserved and significant in each of the three experiments (P >95%), whereas the intermediary peak was only significant inone. Since we have observed that GB was able to aggregate intotetrameric forms (not shown), the occurrence of tetramer, underthe limit of detection of silver staining, is a likely hypothesis toaccount for the activity in the upper band. This peak corresponds to Mr 220,000-240,000. No gelatinase activity wasobserved under the M, 55,000 band in 1% gelatin, 9% acryl-amide gel after 60 h of incubation at 37°Cin PBS with or

without 2 mM CaCl2 and MgCl? (three experiments, data notshown).

Substrate Concentration Dependence of Purified GB Activity.The initial velocity of purified GB activity plotted as a functionof the substrate concentration (Fig. 6) was approximately linear,suggesting that GB displayed a higher Kmtoward p-NPGB than

Table 2 Inhibition of GB activity by various proteinase inhibitorsOctyl-glucoside extract of renal carcinoma BL membranes (200 ng) was incubated with the indicated inhibitors for 30 min at room temperature and then GB

activity was measured in the presence of 0.25 mM p-NPGB at pH 7.6 and 37'C. Residual activity was compared to control activity in the absence of inhibitor. Results

are means from two experiments. The variation was <10%.

InhibitorNoneEDTAEthyleneglycol

bis(/3-aminoethylether)-AVV,/V'^'-tetraaceticacid1,10-PhenanthrolinePepstatinE64DFPDichloroisocoumarinPMSF4-Amidino-PMSFTosyl-i

-lysine-chloromethyl-ketoneTosyl-i-phenylalanine-chloromethyl-ketoneSTIBBIChymostatinBenzamidineAprotininAmiloride(-Aminocaproic

acidAntithrombinIIIa2Macroglobulin«,-AntitrypsinConcentrationI

mM1mM1

mM1

mM4.2

mM30

MM250>iM8mM8mM1mM1

mM14>1M37>iM100(jg/ml2

mMlOUMg/mlI

mM1m.M100fig/ml100»ig/ml100

Mg/mlResidual

activity(%)10010297100981030299897101103642910210498510397963050%inhibitoryconcentration25

JIM10»iM0.25

mM0.8

MM

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RENAL CARCINOMA GUANID1NOBENZOATASE

Table 3 Purification of guanidinobenzoatase from renal clear cell carcinomaGB was purified from 3 human clear cell renal carcinomas (wet weight of 50, 35, and 48 g) as indicated in "Methods". GB activity was assayed with 0.80 mM p-

NPGB. Results are means ±SEM of 3 membrane preparations and 3 purifications.

HomogenateBL fractionOctyl-glucoside extract of BL fractionAgmatine-trisacryl eluateSpecific

activity(milliunits/mg)2

±0.27 ±0.3

10 ±0.35900 ±100Protein

yield(%)100

7.1 ±0.83.8 ±0.3

0.01 5 ±0.001Activity

yield(%)100

24.9 ±6.419 ±2.9

6.8 ±1.9Enrichment

factor1

3.5 ±0.55 ±0.75

450 ±90

KDa

KDa

220-

94 -

67-60-43- "

30-20-

r

-94

_67

-43

-30

-20

Fig. 4. Purified GB on one- and two-dimentional SDS-PAGE analysis. In A,purified GB (1 j/g) was subjected to 5-12% SDS-PAGE under non reducingconditions (lane I) and then under reducing conditions [10% (v/v) 0-mercapto-ethanol, 3% (v/v) SDS; 3 min at 100'C; lane 2]. In B, purified GB (10 /»g)waselectrophoresed under reducing conditions on cylindral pH 4-9 gradient gelscontaining 6% polyacrylamide. Then, the gel was removed from the tube andreduced for 30 min at room temperature in 3 mM Tris/HCl, pH 6.8, containing10% (v/v) glycerol, 2.5% (w/v) SDS, and 5% /3-mercaptoethanol. Thereafter, thegel was placed on top of a 10% polyacrylamide gel and electrophoresed. Molecularweight markers (in thousands; far left and far right lanes) were run in parallel inthe second dimension. Gels were silver stained. This result was confirmed in 2experiments.

did the crude solubili/ed enzyme. Therefore, the determinationof the Kmvalue from a Lineweaver-Burk plot has been inferred,since precipitation occurred at p-NPGB concentrations >3 mM.It is noteworthy that the kinetic features of GB after purificationare quite different from those observed with octyl-glucosideextracts of renal carcinoma BL membranes (Fig. 2B ) and arecompatible with a partial loss of activity during the purification.Therefore, the enrichment factor of 450 is likely anunderestimation.

Plasminogen Activator and Trypsin-like Activity. Purified GB(25 up,)was unable to cleave plasminogen (3.6 ¿¿M)into plasminafter 2 h at 37°Cas demonstrated by the absence of release of

p-nitroaniline from the plasmin substrate D-Val-Leu-Lys-p-NA(3.5 mM). In addition, purified GB (25 ¿ig)exhibited no significant u-PA activity using the u-PA-specific chromogenic substrate <Glu-Gly-Arg-p-NA (3.5 mM) (not shown). Similarly,the benzoyl-Arg-p-NA (1 HIM)substrate of miscellaneous tryp-sin-like enzymes was not cleaved by purified GB under the sameconditions (not shown). In addition, in contrast to t-PA, purified GB activity was not significantly enhanced in the presenceof 10-100 Mg/ml of fibrin (not shown).

Inhibition Pattern of Purified GB Activity. The data presentedin Table 4 globally confirmed those obtained with octyl-gluco

side extracts of renal carcinoma BL membranes. In addition,dansyl-Glu-Gly-Arg-CH2Cl (0.4 HIM), a reversible inhibitor ofsingle-chain u-PA and irreversible inhibitor of two-chain u-PA(27), had no effect on renal carcinoma GB activity. Interest

ingly, the adhesiotope sequence GRGDS (3 HIM) was able toinhibit 90% of purified GB activity.

Immunodetection by a Mixture of Monoclonal AntibodiesDirected against t-PA or u-PA. Dot blot analysis revealed thatGB did not cross-react with either a mixture of 5 monoclonalantibodies directed against t-PA and its single-chain precursoror a monoclonal antibody specific for u-PA and its single-chainprecursor (Fig. 7).

DISCUSSION

This is the first report of a detailed characterization andpurification of a guanidinobenzoatase associated with theplasma membrane of a human carcinoma. GB activity is enriched only in BL fractions from renal carcinoma, demonstrating that GB activity is associated with the plasma membrane.This result is in agreement with the previous observations ofSteven et al. (6) made via GB active site labeling with fluorescentprobes. GB activity can be extracted by nonionic detergents orby ionic strength but not by the action of papain. This observation suggests a membrane-bound enzyme rather than anintegral membrane protein. This feature is shared by severalproteases, including u-PA (1).

Since GB activity is not diminished by inhibitors known tolower the activity of metalloproteases, aspartate proteases, orcysteine proteases, it appears to be a serine protease, althoughit displays a peculiar inhibition pattern. Indeed, like other serineproteases, GB activity is inhibited by DFP, dichloroisocou-marin, BBI, STI, and a,-antitrypsin. However, GB is not sensitive to other serine proteinase inhibitors, including aprotinin,PMSF, tosyl-L-lysine-chloromethyl-ketone and tosyl-L-phenyl-alanine-chloromethyl-ketone. Curiously, GB activity appears tobe inhibited by amiloride. This feature has to be analyzed inview of a recent report stating that u-PA is also sensitive toamiloride (26). Plasminogen activators (u-PA and t-PA) areserine proteases that share sufficient features with GB that ledus to consider them as possible candidates displaying GB activity. However, the possibility that GB belongs to the plasminogen activator family can be excluded on the basis of the following arguments: (a) in contrast to plasminogen activators, GBactivity was inhibited by BBI, STI, and a,-antitrypsin and wasinhibited by neither e-aminocaproic acid nor benzamidine, (¿>)purified GB was unable to activate plasminogen, (c) the lack ofdetection of GB by the monoclonal anti-t-PA and anti-u-PAantibodies rules out the possibility of an identity with u-PA, t-PA, and their single-chain forms.

Since a member of the GB family has been demonstrated tocleave GRGD (9), GB was designed as a protease. However,the absence of cleavage of benzyl-Arg-p-NA and the lack ofgelatinase activity by GB after purification does not favor thisidea. Although it must be noted that during the purificationprocedure, a significant variation of GB activity kinetic properties was observed (variation of the apparent affinity for p-NPGB), this might be due to the loss of a regulatory component.

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RENAL CARCINOMA GUANIDINOBENZOATASE

B10 20 30

slice number40

Fig. 5. GB activity in polyacrylamide gel slices after SDS-PAGE of agmatine-trisacryl eluate. In A, ISO ¡Aof eluate containing 6 Mgof proteins and about 9milliuniis of GB activity measured in the presence of 1.5 mM p-NPGB weresubjected to 5-12% SDS-PAGE. Then, the gel was treated as to regenerate GBactivity as indicated in "Methods." Thereafter, the lane was cut into 3-mm-thickslices. Each slice was crushed and stirred overnight at 4"C in 0.1 M sodiumbarbital, pH 7.6. GB activity was measured in the presence of 1.5 mM p-NPGBover 30 min at 37*C. These results were confirmed in 3 experiments. In B, ÌMgof proteins of the same aliquot was subjected to 5-12% SDS-PAGE and silverstained.

elucidating the role of this enzyme but cannot be conducteduntil further experiments obtain a fully active GB.

Interestingly, GB activity was only observed in renal carcinomas (10 of 10), whereas it was not detectable in the normaltissue of origin (0 of 10). Our observation is in agreement witha previous report by Steven et al. (30) who also failed to detectany GB-active center in normal rat kidney using a very sensitivemethod of detection based on the labeling of GB-active centerby a fluorescent probe (30). In this respect, GB activity couldbe used as a marker of malignancy in renal tumors and possiblyin other human tumors as well.

Table 4 Inhibition pattern of purified GB activityAgmatine-trisacryl eluate containing 595 milliunits/ml of GB activity was used

as a GB source. After 60 min of preincubation with the inhibitor at roomtemperature, except as otherwise indicated, GB activity was measured in thepresence of 0.8 mM p-NPGB and 100 M!of eluate at 37°C for 5 min and comparedto the activity without inhibitors (results with 10', of variation were obtained

from two experiments).

InhibitorNoneHgCl2E64I

.onpoplinDFPAmilorideBenzamidinea,-AntitrypsinBBIGRGDSDansyl-Glu-Gly-Arg-CH2-ClConcentration1

1T1M34

MM1m.M1

m\i°30MM*1

mM10mM100Mg/ml33

MM3HIM"0.4

mM°Residual

activity(%)10009990104301896202210103

' Without preincubation.*After 15 min of preincubation.

2.0-,

1.5.

1.0

E 0.5 .m"a

0.00.0 0.5 1.0 1.5 2.0 2.5 3.0

p-NPGO,mM

Fig. 6. Purified GB activity as a function of substrate concentration. Agmatine-trisacryl eluate (200 uI containing 10 Mgof proteins) was incubated at 37*C at pH7.6 in the presence of various concentrations of p-NPGB and 1% (v/v) DMSO.Initial velocity of GB activity was plotted as a function of substrate concentration.Data was obtained from 2 experiments with <10% variation. mU, milIlimits.

The occurrence of a tetrameric fully active enzyme, as observedfor tryptase (28), is another possibility. However, in the octyl-glucoside extract, the unusually high 50% inhibitory concentration values found for STI, BBI, and a,-antitrypsin (Table 2)suggest that GB could be in a poorly active form (precursor orunbound form) even before purification.

Inhibition of the renal carcinoma GB activity by the GRGDSsequence may be of interest owing to the involvement of thistype of sequence in cell adhesion to several extracellular matrixcomponents (29) and considering the fact that GB purified fromEhrlich ascites fluid was reported to cleave this peptide (6).However, it remains to be clarified whether GRGDS acts as aninhibitor or as a substrate for the renal carcinoma GB activity.A search for the natural substrate(s) for GB would help in

1.

2.

3-

4-

5.

6.

iLabcFig. 7. Lack of immunodetection of GB by specific monoclonal antibodies

raised against either t-PA or u-PA. Immunoblotting was performed as indicatedin "Methods." One Mgof GB (a), t-PA(e). or u-PA (c) was checked. Lane I, eachantigen was checked versus the rabbit anti-mouse anti-IgG antibody (1 Mg):/<""'2, each antigen was checked versus an irrelevant monoclonal IgG antibody ( 1 Mg),and then the second anti-mouse anti-lgG antibody was added; lanes 3 and 4, eachantigen was subjected to immunodetection by 1 Mgof 5 monoclonal anti-t-PAantibody mixtures (lane 3) or an anti-u-PA monoclonal antibody (lane 4) in theabsence of second anti-mouse anti-IgG antibody; lanes 5 and 6, each antigen wasimmunodetected by 1 Mgof anti-t-PA monoclonal antibody mixtures (lane 5) orby the anti-u-PA antibody (lane 6), and then the second anti-mouse anti-IgGantibody was added. Immuncomplexes were revealed by a biotinylated anti-rabbitantibody and avidin-peroxidase staining as indicated in "Methods." This obser

vation was confirmed in 2 experiments.

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RENAL CARCINOMA GUANIDINOBENZOATASE

In conclusion, the guanidinobenzoatase associated with human renal carcinoma plasma membrane can be purified in onestep using a agmatine-trisacryl affinity column. Although thisenzyme shares some properties with the single-chain form ofplasminogen activators, it is clearly distinct from them. Sinceone member of the GB family has been demonstrated to be aprotease likely involved in cell migration and cancer metastasis(9), it could be used as an index of putative metastatic dissemination. In this respect, its purification to homogeneity wouldallow us to obtain specific tools, such as antibodies (in courseof production), which could be helpful in detecting and quantifying this type of activity. Furthermore, the design of veryspecific inhibitors of activity of this kind may provide pharmacological means of metastasis limitation.

ACKNOWLEDGMENTS

We thank C. Milesi-Fluet for the optimization of the indirect avidin-peroxidase-labeling method and Dr. S. Heydrick for the revision of themanuscript. We thank Dr. P. Burtin for his stimulating discussions andhelpful comments on the manuscript. Professor R. Loubiere and hiscollaborators from the Laboratory of Pathology as well as Professor J.Toubol and the surgical team of Urology (Hôpital Pasteur, Nice,France) are acknowleged for their active collaboration.

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1992;52:3622-3628. Cancer Res   Claudine Poustis-Delpont, Roland Descomps, Patrick Auberger, et al.   Possible Marker of Human Renal CarcinomaCharacterization and Purification of a Guanidinobenzoatase: A

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