Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates Miche Á le Brillard-Bourdet 1 , Sophie Re  hault 1 , Luiz Juliano 2 , Miche Á le Ferrer 1 , Thierry Moreau 1 and Francis Gauthier 1 1 Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University FrancË ois Rabelais, Faculty of Medicine, Tours, France; 2 Departamento de Biofõ  sica, Escola Paulista de Medicina, Universidade Federal de Sa Ä o Paulo, Sa Ä o Paulo, Brazil In addition to kallikrein hK3, a serine protease generally reported as PSA (prostate-speci®c antigen), at least two other enzymes in human seminal plasma also cleave syn- thetic peptidyl substrates derived f rom the sequen ce o f human semenogelins. We have identi®ed one of these a s prostatic acid phosphatase (PAP), a major component of prostatic ¯uid whose physiological function is unclear. T he other is a high M r basic protein present at low concentrations in seminal plasma and that remains to be characterized. PAP was puri®ed to homogeneity from freshly ejaculated seminal plasma. Its N-terminal sequence a nd its phosphatase prop- erties (hydrolysis of p ara-nitrophenylphosphate at low p H) were determined, and its inhibition by sodium ¯uoride measured. Both puri®ed and c ommercial PAP also had amidolytic activity on peptide substrates derived from the semenogelin sequence a t neutral and slightly basic pH. The k cat /K m values w ere in t he 10 2 )10 3 M )1 ás )1 range u sing ¯uorogenic semenogelin-derived substrates whose peptidyl moiety included cleavage sites that had been identi®ed ex vivo. PAP cleavage sites d iered fro m those of hK3 and were mainly at P1  Gln residues or b etween residues bearing hydroxyl groups. PAP amidolytic activity was poorly inhibited by all currently used wide spectrum proteinase inhibitors. Only 3±4 dichloroisocoumarin and benzamidine inhibited puri®ed PAP. Puri ®ed human s eme- nogelin was cleaved by puri®ed and commercial PAP at neutral p H; the two ma in cleav age sites were at Tyr292 and Ser170 (semenogelin I sequence), only the former has been identi®ed ex vivo by analysis of seminal plasma. Keywords: amidolytic activity; ¯uorogenic substrates; human kallikrein; phosphatase; semenogelins. The s emenogelins I and II are secreted by the s eminal vesicles into human semen where they immediately form a coagulum that entraps spermatozoa upon ejaculation. This coagulum dissolves within a few minutes after ejaculation as a result of proteolysis involving kallikrein hK3 (prostate- speci®c antigen, PSA) [1,2]. This appears to be the main physiological function of this protease, although it may act on several other biological substrates, such as insulin-like growth factor binding proteins [3±5] and parathyroid hormone-related protein [6].Exvivoanalysis by two- dimensional electrophoresis of fresh ejaculates i denti®ed preferential cleavage sites in semenogelins at Tyr136, Tyr292 and Gln266 of semenogelin I (S. Re  hault & M. Brillard- Bourde t, 3 unpublished data). All three cleavage sites have been identi®ed by in vitro studies [7], the third one only very recently [8]. W e prepared peptidyl ¯uorogenic substrates of hK3 based on these sequences and on others containing previously identi®ed cleavage sites, in an attempt to obtain more sensitive substrates o f hK3 than those that a re available c ommercially. Some of them were cleaved by at least two other proteases obtained by ion exchange chro- matography from crude seminal plasma. This study shows that one of these enzymes is prostatic acid phosphatase (PAP), although this enzyme has never been attributed any proteolytic or amidolytic activity to date. L arge amounts of PAP (1 mgámL )1 ) are present in seminal ¯uid [9], but its physiological function remains unclear even its cellular form has neutral protein-tyrosine kinase phosphatase activity [10]. We have puri®ed PAP to homogeneity and compared its amidolytic properties to those of commercial source using semenogelin-derived ¯uorogenic and puri®ed semenogelins. MATERIALS AND METHODS Materials The following materials were obtained from commercial sources: DEAE±Sephadex A50, Seph acryl S300, Phenyl-Sepharose (Pharmacia), NaF, (Octylphenoxy) poly- ethoxyethanol (IGEPAL 4 CA-630 Sigma), EDTA, dimethyl- formamide, acetonitrile (Merck), CF 3 COOH 5 (Perkin Elmer). All other reagents were of analytical grade. Com- mercial PAP puri®ed from human semen was from Sigma. Puri®cation and characterization of PAP Approximately 50 mL of seminal plasma was obtained from healthy donors, dialysed against 3 ´ 2L25m M Tris/HCl Correspondence to F. Gauthier, Laboratory of Enzymology and Protein Chemistry, INSERM EMI-U 00-10, University FrancË ois Rabelais, Faculty of Medicine, 2 bi, Boulevard Tonnelle  , 37032 Tours cedex France. Fax: + 33 2 47 36 60 46, E-mail: gauthier@univ-tours.fr Abbreviations: PSA, prostate-speci®c antigen; PAP, prostatic acid phosphatase; DCI, 3-4 dichloroisocoumarin 1,2 ;IGEPAL 1,2 , (octylphenoxy) polyethoxyethanol; Abz, O-aminobenzoyl; EDDnp, ethylenediamine 2,4-dinitrophenyl. (Received 23 July 2001, revised 31 October 2001, accepted 9 Nove mber 2001) Eur. J. Biochem. 269, 390±395 (2002) Ó FEBS 2002 pH 7.8, 0.15 M NaCl, and fractionated on DEAE A50. The column (2.6 ´ 40 cm) was washed with 200 mL equilibrium buffer and proteins were eluted at 28 mLáh )1 with a discontinuous gradient of NaCl (25 m M ,80m M ,and 200 m M ). Fractions were assayed for their enzymatic activity towards Abz-ISYQSSSTEEQ-EDDnp as reported under Kinetic measurements. Fractions that were eluted from DEAE A50 w ith 200 m M NaCl were fractionated further on Sephacryl S300. The column (2.6 ´ 100 cm) was equilibrated in t he same buffer a s before and eluted at 60 mLáh )1 . Fractions of the s econd eluted peak were pooled, checked for activity towards the same substrate as before, and run onto a phenyl-Sepharose column (1 ´ 7cm) equilibrated with 25 m M Tris/HCl pH 7.8, 1.5 M (NH4) 2 SO 4 . The column was eluted with a decreasing d iscontin- uous gradient of ammonium sulfate. Enzymatically active fractions were concentrated and checked for purity by four methods. First, HPLC on C4 (C4 uptisphere column, 5 lm, 30 mm ´ 2.1 mm) eluted with a linear gradient of aceto- nitrile (0±60%, v/v) in 0.075% CF 3 COOH for 40 min at a ¯ow rate of 0.2 mLámin )1 ; second MALDI-TOF MS using aBru È cker Re¯ex mass spectrometer; third SDS/PAGE and fourth N-terminal sequencing. The protein concentration was calculated from a BCA assay and the activity is expressed with reference to the phosphatase activity of commercial PAP. Puri®cation of human semenogelins Semenogelins were p uri®ed essentially as described by Malm et al. with some modi®cation s [11]. Ejaculates from healthy volunteers were collected on DEAE cellulose A50 equilibrated in 40 m M Tris/HCl pH 9.7 in the presence of 4 M urea and a cocktail of inhibitors. T here was no signi®cant degradation of semenogelins under these condi- tions, which allow the rapid separation by centrifugation of cellular components and gel-bound proteases. The ®nal step of puri®cation was HPLC fractionation on a C4 column using a 0±60% (v/v) linear gardient of acetonitrile in 0.075% CF 3 COOH for 40 min at a ¯ow rate of 0.2 mLámin )1 . Synthesis of ¯uorogenic substrates The intramolecularly quenched ¯uorogenic peptides were synthesized by solid-phase method; glutamine was the C-terminal residue of all peptides due to a requirement of the synthesis strategy [12]. All syntheses were performed with Fmoc methodology using a multiple automated peptide synthesizer (PSSM-8, Shimadzu Co.). Intramolec- ularly quenched ¯uorogenic substrates were prepared as 2m M stock solutions in dimethylformamide and diluted with activation buffer . Substrate purity w as checked by MALDI-TOF MS (TofSpec-E, Micromass) and by re- versed-phase chromatography on a C18 column eluted with a 10-min linear gradient of 0±60% acetonitrile in 0.075% CF 3 COOH at 0.5 mLámi n )1 . Kinetic measurements All amidolytic assays were carried out at 37 °Cin20m M Tris/HCl pH 9.0. Speci®city constants (k cat /K m )were determined for Abz-peptidyl-EDDnp substrates under pseudo-®rst order conditions, using a substrate concentra- tion far below the K m , as reported elsewhere [13]. Calcula- tions were done usin g ENZFITTER software (Biosoft). Excitation and emission wavelengths were 320 nm and 420 nm for e xperiments with intramolecularly quenched ¯uorogenic substrates. The system was standardized using Abz-FR-OH prepared by total tryptic hydrolysis of Abz- FR-pNA, and its concentration was determined from the absorbance at 410 nm, assuming e 410nm  8800 M )1 ácm )1 for p-nitroanilide. Substrate concentrations of Abz-pept- idyl-EDDnp were determined by measuring the absor- bance at 365 nm, assuming e 365nm  17 300 M )1 ácm )1 for EDDnp. K m s were measured using six substrate concentra- tions (0.2±12 l M ), the ®nal concentration of PAP was in the 0.1 l M range. The phosphatase activity of the puri®ed PAP was measured using p-nitrophenylphosphate as substrate and the method recommended by the manufacturer (Sigma). This activity was compared to that of commercial PAP and the concentration of a ctive enzyme calculated on this basis. The pH activity pro®les were determined by measuring the phosphatase and the peptidase activities of puri®ed PAP over the pH range 3±10 at 0.5 unit intervals at 37 °C. 20 m M citrate/phosphate buffers were used over the pH range 3±7, 20 m M phosphate buffer for p H 7±8, 20 m M Tris/HCl for pH 8±9 and 20 m M carbonate/bicarbonate buffer for pH 9± 10. All other experimental cond itions were as reported above. Inhibition of puri®ed PAP The amidolytic activity of puri®ed PAP (60 n M ®nal) was measured in the presence of a molar excess of inhibitors from all four classes of proteases ) aprotinin, benzamidine, leupeptin, Soya bean t rypsin inhibitor (SBTI) 6,7 ,(1-trans- epoxysuccinyl-leucylamido (4-guanidino) butane (E64 6,7 ), o-phenanthroline, and NaF, w hich inhibits phosphatase activity [14]. Inhibitors were mixed with the enzyme for 15minin20m M Tris/HCl pH 9.0, 0.0 5% IGEPAL at 37 °C, before adding the ¯uorogenic substrate ( 2 l M ). Inhibition is expressed with reference to an inhibitor-free control incubated under the same experimental conditions. Conversely, acid phosphatase activity was measured in the presence of NaF and of those of inhibitors that alter amidolytic activity. Incubation with inhibitors was per- fromed at pH 9 and at pH 4.8, befo re the PAP activity was measured at pH 4.8. The in¯uence of ¯uorogenic substrate binding on phosphatase activity was also assayed by incubating PAP with A bz-SSIYSQTEEQ-EDDnp (2 l M and 20 l M ®nal) under the same experimental conditions and then measuring phosphatase activity. Identi®cation of cleavage sites in peptidyl substrates Fluorogenic peptides (15±30 l M ®nal) were incubated with puri®ed PAP in the 0.1 l M range at 37 °Cin20m M Tris/HCl pH 9.0 containing 0.02% IGEPAL. Aliquots (200 lL) were removed at intervals up to 60 min and the reaction was blocked by adding 4 lLCF 3 COOH. T he samples were fractionated on a C18 cartridge column (5 lm, 30 mm ´ 2.1 mm) using a linear gradient of acetonitrile (0±60%, v/v) in 0.075% CF 3 COOH for 10 min at a ¯ow rate of 0.5 mLámin )1 , with simultaneous recordings at three wavelengths ( 220, 320, and 360 nm). Ó FEBS 2002 Amidolytic activity of prostatic acid phosphatase (Eur. J. Biochem. 269) 391 This allowed direct identi®cation of EDDnp-containing peptides prior to N-terminal sequencing of all signi®cant peaks. Amino acid sequence analysis of peptide products The amino-acid sequen ces were determined using an Applied Biosystems 477A pulsed liquid sequencer with the chemicals and program recommended by the manufacturer. Phenylthiohydantoin derivatives were identi®ed using an on line model 120A PTH a nalyser. RESULTS AND DISCUSSION Sperm lique®es very rapidly after ejaculation due to the proteolytic cleavage of the coagulum-forming proteins semenogelins I and II. This results in the progressive release of motile spermatozoa and also in the generation of biologically active semenogelin fragments, the function of which is not yet fully understood [7,15]. Kallikrein hK3 has long been described as t he protease responsible for this proteolysis, based on its high concentration in seminal plasma and o n its chymotrypsin-like s peci®city. It thus cleaves puri®ed semenogelins mainly at tyrosine residues, although cleavage may also occur at histidine and glutamine residues after prolonged incubation of puri®ed semenogelins with hK3 [7,8]. However, total ex vivo hydrolysis of semenogelins is completed within minutes following ejacu- lation, suggesting that some other proteases participate to this process. We have prepared an intramolecularly quenched ¯uorescent substrate (Abz-ISYQSSSTEEQ EDDnp) that encompasses a sequence containing a major ex vivo cleavage site, the Gln266±Ser267 bond of semenog- elin I (S. Re  hault & M. Brillard-Bourdet, unpublished data) 8 . This cleavage site has been also recently identi®ed in puri®ed semenogelins after extensive cleavage by hK3 [8], although it was initially reported to be one residue upstream in this sequence, at the Tyr265±Gln266 bond [7]. Hydrolysis of this substrate by samples from the fractionation of seminal plasma on an anion exchanger revealed two fractions that c leaved this peptide, one eluting with t he ionic strength used for PSA/hK3, and the other, with a much lower pHi, with the buffer containing 200 m M NaCl (Fig. 1A). This s econd peak was fractionated further by gel chromatography (Fig. 1 B), to give two peaks with amido- lytic activity, one was eluted in the void volume and the other in the 50±100 kDa fractions. Semen thus contains at least three proteases that have amidolytic activity on this semenogelin-derived substrate. They had different substrate speci®cities; hK3 cleaved at the tyrosine±glutamine bond, whereas the Sephacryl S300-excluded fraction cleaved at the glutamine±serine bond and the ®ltered fraction at the serine±tyrosine bond, as shown by HPLC fractionation and N-terminal sequencing. Part of the last ®ltered fraction was puri®ed further by hydropho bic chromatography on phe- nyl-Sepharose. Fractions with enzymatic activity were analysed by MALDI TOF MS and reversed-phase HPLC on a C4 cartridge. A molecular mass of 47 798 Da was determined by MS and a single, symetrical peak was eluted from the C4 column (Fig. 1C). The collected fraction was immediately equilibrated in n eutral buffer to d etermine residual peptidase activity. Its N-terminal sequence (20 residues) was 100% identical to PAP and showed no trace of contamination. This fraction still had enzymatic activity on Abz-ISYQSSSTEEQ-EDDnp when buffered at neutral pH immediately after elution from the C4 cartridge, demonstrating that the sequenced product and that with peptidase activity were the same. The puri®ed enzyme was checked for its phosphatase activity on p-nitrophenyl phosphate and this activity w as compared to that of commercial PAP. Based on a molar concentration of about 9 l M estimated from a BCA protein assay for the PAP monomer, a value of 1.67 p hosphatase unitsánmol )1 was calculated for puri®ed PAP, which is in the same range as that reported for commercial PAP. Con®rmation that PAP had amidolytic activity was obtained by showing that commercial PAP hydrolysed the Abz-ISYQSSSTEEQ- EDDnp substrate at neutral pH, as does the puri®ed enzyme. The ratio of the amidolytic activity of puri®ed to that of commercial PAP (2.5) was also in agreement with that obtained by measuring phosphatase activity (1.75). PH-dependence of PAP amidolytic activity The pH-dependence of PAP amidolytic activity was studied over the pH range 3±9. The Abz-ISYQSSSTEEQ-EDDnp substrate was not hydrolysed at acidic pH, at which phosphatase activity is greatest (Fig. 2). There was amido- lytic a ctivity at n eutral pH, w ith a maximum at about pH 9.0. Inhibition of PAP amidolytic activity A series of inhibitors of all classes of enzymes were tested for their ability t o inhibit PAP. Only 3±4 dichloroisocoumarin (DCI; 9 200 l M ®nal) and benzamidine (0.5 m M ®nal) partially inhibited PAP amidolytic activity under the experimental conditions used. Pepstatin, leupeptin, PhCH 2 SO 2 F, SBTI, E64 and EDTA did not signi®cantly inhibit PAP amidolytic activity. However, NaF (10 m M ), which strongly inhibits phosphatase activity at acidic pH, inhibited PAP amidolytic activity at neutral pH. Converse- ly, DCI and benzamidine partially inhibited phosphatase activity (data not shown). Incubating the substrate A bz-SSIYSQTEEQ-EDDnp with puri®ed or commercial PAP at basic or acidic pH also resulted in the inhibition of phosphatase activity, demon- strating that the ¯uorogenic substrate, although not cleaved at pH 4.6, binds to th e e nzyme active site under t hese conditions. PAP amidolytic activity on semenogelin-derived substrates The amidolytic activity of PAP was assayed u sing four ¯uorogenic substrates based on the ex vivo cleavage sites of semenogelin I (S. Re  hault & M. Brillard-Bourdet, unpub- lished data) 10 . Those sites are at Tyr136, Tyr292 and Gln326 and all three have been identi®ed as putative hK3/PSA cleavage sites [ 8]. T wo of these substrates included the Gln326±Ser327 site, but within two different peptides corresponding to residues 321±329 (substrate 3) or 323± 332 (substrate 4) of the semenogelin I sequence. The peptide containing Tyr136 (peptide 1) was cleaved at the glutamine± tyrosine bond by puri®ed PAP and the peptide containing Tyr292 (peptide 2) was cleaved at the glutamine±threonine 392 M. Brillard-Bourdet et al. (Eur. J. Biochem. 269) Ó FEBS 2002 bond, with a s econd cleavage after tyrosine. PAP also preferentially cleaved after a glutamine residue, at the glutamine±serine bond in Abz-NKISYQSSSQ-EDDnp, and secondarily at tyrosine±glutamine (Table 1). Speci®city constants (k cat /K m ) were measured under ®rst-order condi- tions (Table 1). They indicated a rather low c leaving ef®ciency, even though there was a tight interaction between PAP and Abz-peptidyl-EDDnp substrates, as deduced from the low values of K m in the micromolar range for substrate 1 (0.23 l M ) and substrate 4 (4 l M ) which are the only two having a single cleavage site. A shift in PAP speci®city w as observed depending on the length and/or the nature o f the pep tidyl moiety in the substrate. This occurred with substrate 4, whose cleavage site is shifted towards the serine±tyrosine bond when the N-terminal asparagine±lysine dipeptide was removed from substrate 3. This phenomenon, which demonstrates the in¯uence of surrounding subsites on P1 occupancy, was also reported by Coombs et al. [16] for hK3/PSA hydrolysing Fig. 1. Puri®cation of prostatic acid phosphatase from human seminal plasma. (A) SDS/PAGE of the 200 m M NaCl elution from the DEAE A50 column equilibrated in 25 m M Tris/HCl pH 7.8. Lane 2 shows a sample from the 80 m M NaCl elution containing hK3/PSA (M r(app)  33 kDa). (B) The 200 m M NaCl-eluted fractions were pooled, fractionated on Sephacryl S300, checked for their amidolytic activity (dashed line) and those corresponding to the shaded area (lanes 1±3 on SDS/PAGE) wer e puri®ed further by h ydrophobic chromatography on phenyl-Sepharose . (C) Phenyl-Sepharose fractions were pooled and checked for purit y by SDS/PAGE and C4 reversed-phase chromatography using a gradien t of 0±60% acetonitrile in 0.075% CF 3 COOH. Fig. 2. pH activity pro®le of PAP activity (full line) and PAP amidolytic activity (dashed line). Resul ts are norm aliz ed rates and ar e the means of two experiments at each pH . Ó FEBS 2002 Amidolytic activity of prostatic acid phosphatase (Eur. J. Biochem. 269) 393 peptide substrates. Nevertheless the preferential cleavage sites for PAP in the p eptides used here were with glutamine at P1 and hydroxyl-bearing (Ser, Thr, Tyr) residues at P1¢. We do not know why PAP has amidolytic activity at neutral pH. But its speci®city differs from that of hK3/PSA, which also cleaves these substrates, but at different sites (S. Re  hault & M. Brillard-Bourdet, unpublished data) 11 .Wefurther con®rmed that enzymatic activity of the PAP preparation is not due to the presence of trace amounts of hK3 by showing that: (a) the hK3 substrate Suc-AAPF-pNa is not cleaved by PAP at a 1 l M ®nal concentration; (b) a polyclonal antibody against PSA does not reveal any band after immunoblotting; (c) the amidolytic activity is strictly associated with the peak of PAP after reversed-phase HPLC, and no trace of contaminant at the elution time of PSA was observed; (d) commercial PAP has amidolytic activity similar to that of puri®ed PAP. The fact that there was a shift in the cleavage of synthetic peptides by PAP raised the question of whether PAP cleaves a protein substrate such as semenogelin, which also questions the biological relevance of PAP amidolytic activity. We therefore measured the ability of pu ri®ed and of commercial PAP to cleave freshly prepared semeno- gelin I, and we identi®ed the main cleavage sites in the protein. PAP proteolytic activity on puri®ed semenogelins Semenogelins were puri®ed from seminal plasma in such a way that no proteolysis could occur upon sample collection. Puri®ed semenogelin I was checked for purity by reversed- phase HPLC, and used as a substrate for puri®ed and for commercial PAP. PAP ( 1 l M ®nal) was incubated with puri®ed semenogelin 1 (16 l M ®nal) for 18 h at 37 °Cin 20 m M Tris/HCl pH 9.0, 0.05% IGEPAL. The mixture was then fractionated b y H PLC o n a C4 column, a nd the cleavage sites identi®ed by N-terminal sequencing (Fig. 3). Two cleavage sites were identi®ed, one at Ser170 and the other at Tyr292. The former has not been described before, whereas the latter is a m ajor cleavage site for hK3 [8]. Tyr292 is also present in substrate 2 used in this study (Table 1). It is cleaved by PAP at the Tyr292±Ser bond and at the Gln294±Thr bond, suggesting that accessibility to t he sensitive bond is different in the peptide substrate and in the protein. Semenogelin was hydrolysed by a PAP concentration lowerthanthatinthe10 )5 M range found in semen [9]. Only a few sites were identi®ed however, and the exte nt of proteolysis was lower than that obtained with hK3 under similar conditions (data not shown). N evertheless, pros- tatic P AP could b e i nvo lved i n s emenogelin processing and d egradation because of its high concentration in seminal plasma, its rather wide substrate speci®city, with cleavage after glutamine or hydroxylated residues, and its relatively slow inactivation in semen. The rapid hydrolysis of semenogelin by hK3/PSA gives large fragments that could be further processed by PAP, which is stable as a protease at neutral pH, and whose phosphatase activity requires an acid environment. This condition occurs physiologically only when the enzyme reaches the female genital tract [17]. ACKNOWLEDGEMENTS The authors are indebted to C. Barthe  le  myandM.H.Saussereau (Department of Human Reproduction CHRU Bretonneau, Tours) for their collaboration and helpful advice, and in Brazil to the FundacË a Ä ode Amparo a Pesquisa do Estado de Sa Ä o Paulo (FAPESP). The English text was checked by Amanu ensis Scienti®ca. REFERENCES 1. Lilja, H. (1985) A kallikrein-like serine protease in prostatic ¯uid cleaves the predominant seminal vesicle protein. J. Clin. Invest. 76, 1899±1903. 2. Lilja, H., Oldbring, J., Rannevik, G. & Laurell, C.B. (1987) Seminal vesicle-secreted proteins and their reactions during gelation and liquefaction of human semen. J. Clin. Invest. 80,281± 285. 3. Rehault, S., Monget, P., Mazerbourg, S., Tremblay, R., Gutman, N., Gauthier, F. & Moreau, T. (2001) Insulin-like growth factor binding proteins (IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3. Eur. J. Biochem. 268, 2960± 2968. 4. Cohen, P., Graves, H.C., Peehl, D.M., Kamarei, M., Giudice, L.C. & Rosenfeld, R.G. (1992) Prostate-speci®c antigen (PSA) is an insulin-like growth factor binding pro tein-3 protease fo und in seminal plasma. J. Clin. Endocrinol. Metab. 75, 1046±1053. Fig. 3. Hydrolysis of puri®ed semenogelin I by PAP. HPLC fraction - ation on C4 of semenogelin incubates with (solid line) or without (dashed line) puri®ed PAP. All m ajor fractions were sequenced N-terminally to identify two cleavage sites, one at Ser170 (peak 1) and one at Tyr292 (peak 2). The main peak on each chromatogram was the IGEPAL and the last peak eluted was PAP. Table 1 . Second order rate constants (k cat /K m ) for the hydrolysis of semenogelin-derived synthetic peptidyl substrates by puri®ed prostatic acid phosphatase. k cat /K m values were determined under pseudo-®rst order conditions. The results are the mean of two recordings for each substrate. Cleavage sites are identi®ed after numbered residues i n peptide sequences. Numbers refer to the positioning of the residue in the human smenogelin I s equenc e. Substrate k cat /K m ( M )1 ás )1 ) 1 Abz-SSQ 135 YSNTEEQ- EDDnp 303 2 Abz-SSIY 292 SQ 294 TEEQ-EDDnp 600 3 Abz-NKISY 325 Q 326 SSSQ-EDDnp 1028 4 Abz-IS 324 YQSSSTEEQ-EDDnp 250 394 M. Brillard-Bourdet et al. (Eur. J. Biochem. 269) Ó FEBS 2002 5. Sutkowski, D.M., Goode, R.L., Baniel, J., Teater, C., Cohen, P., McNulty, A.M., Hsiung, H.M., Becker, G.W. & Neubauer, B.L. (1999) Growth regu lation of prostatic stromal cells by prostate- speci®c antigen [in process citation]. J. Natl Cancer Inst. 91, 1663± 1669. 6. Iwamura, M., Hellman, J., Cockett, A.T., Lilja, H. & Gershagen, S. (1996) Alteration of the ho rmonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-speci®c antigen. Urology 48, 317±325. 7. Robert, M., Gibbs, B.F., Jacobson, E. & Gagnon, C. 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(1996) Isolation and charac terization of the major gel proteins in human semen, semenogelin I and semenogelin II. Eur. J. Biochem. 238, 48±53. 12. Hirata, I.Y., Cezari, M.H.S., Nakaie, C.R., Boschcov, P., Ito, A.S. & Juliano, M.A. (1994) Internally quenched ¯uorogenic protease substrates: solid phase synthesis and ¯uorescence spectroscopy of peptides containing ortho-aminobenzoyl/ dinitro- phenyl groups as d onor-accepto r pairs. L ett. Pept. Sci. 1, 299± 308. 13. Bourgeois, L., Brillard-Bourdet, M., Deperthes, D., Ju liano, L., Tremblay, R., Dube  , J .Y. & Gauthier, F. (1997) Serpin-derived substrates for investigating the substrate speci®city of human tissue kallikreins hK 1 and hK2. J. Biol. Chem. 272, 29590± 29595. 14. NagDas, S.K. & Bhattacharyya, A.K. (1984) The kinetics of inhibition of human seminal plasma acid phosphatase b y sodium ¯uoride. Biochem. Int. 9, 659±668. 15. Robert, M. & Gagnon, C. (1999) Semenogelin I: a coagulum forming, multifunction al seminal vesicle protein. Cell Mol. Life Sci. 55, 944±960. 16.Coombs,G.S.,Bergstrom,R.C.,Pellequer,J.L.,Baker,S.I., Navre, M., Smith, M.M., Tainer, J.A., Madison, E.L. & Corey, D.R. (1998) Substrate speci®city of prostate-speci®c antigen (PSA). Chem. Biol. 5, 475±488. 17. Ramsey, P.S., Ogburn, P.L., Harris, D.Y., Heise, R.H. & Ramin, K.D. (2000) Eect of vaginal pH on ecacy of misoprostol for cervical ripening and labor induction. Am. J. Obstet. Gynecol. 182, 1616±1619. Ó FEBS 2002 Amidolytic activity of prostatic acid phosphatase (Eur. J. Biochem. 269) 395 . Amidolytic activity of prostatic acid phosphatase on human semenogelins and semenogelin-derived synthetic substrates Miche Á le Brillard-Bourdet 1 ,. e e nzyme active site under t hese conditions. PAP amidolytic activity on semenogelin-derived substrates The amidolytic activity of PAP was assayed u sing