Eur. J. Biochem.255, 3292335 (1998) FEBS1998

Androgen-sensitive human prostate cancer cells, LNCaP, produce bothN-terminally mature and truncated prostate-specific antigen isoforms

Annakaisa HERRALA1, Riitta KURKELA 1, Mauno VIHINEN2,3, Nisse KALKKINEN4 and Pirkko VIHKO1,2

1 Biocenter Oulu and WHO Collaborating Centre for Research on Reproductive Health, University of Oulu, Oulu, Finland2 Department of Biosciences, Division of Biochemistry, University of Helsinki, Finland3 Institute of Medical Technology, University of Tampere, Tampere, Finland4 Institute of Biotechnology, Protein Chemistry Laboratory, University of Helsinki, Finland

(Received 9 January/27 April1998) 2 EJB 98 0032/3

To characterize prostate-specific antigen (PSA) produced by cancer cells, different isoforms of PSAsecreted by the human prostate cancer cells, LNCaP, were purified. LNCaP-PSA production was inducedby synthetic androgen, R1881. LNCaP-PSA was separated into four pools. The molecular mass ofLNCaP-PSA isoforms in these pools was 34 kDa under reducing conditions and 29 kDa under non-reducing conditions on SDS/PAGE. pI of LNCaP-PSA isoforms varied from 6.8 to 8.2. Pool A had thehighest specific activity, 37 nmol/(min3mg). All the pools formed stable complexes withA1-antichymo-trypsin andA2-macroglobulin. The pools contained10260% of N-terminally correctly processed LNCaP-PSA isoforms. According to the molecular modelling, the addition or deletion of two or four N-terminalamino acids could affect the three-dimensional structure and thereby remarkably reduce the enzymeactivity of LNCaP-PSA.

Keywords:prostate cancer; N-terminal sequencing; molecular modeling; standardization; enzyme ac-tivity.

Human prostate-specific antigen (PSA) is synthesized in the nized as a useful markers for the diagnosis of prostate cancerand for monitoring patients [17219]. Serum PSA concentrationepithelial cells of the prostate and mammary glands and secreted

into the seminal fluid or breast nipple aspirate fluid at concentra- is low, less than 4 ng/ml, in healthy males [17, 20]. PretreatmentPSA values correlate with the pathological stage and grade [21,tions of 0.524 g/l or up to 4 mg/l, respectively [125]. Human

PSA is a single-chain glycoprotein with a molecular mass of 22], tumor volume, and presence or absence of metastases [17].In the serum, PSA forms complexes with serine-protease inhibi-33234 kDa [223, 6]. PSA is a serine protease and it plays an

important role in semen liquefaction, progressively degrading tors, serpins. The major portion of PSA is covalently complexedwith A1-antichymotrypsin, resulting in the inactivation of the en-and fragmenting the seminal vesicle proteins fibronectin and

semenogelin, both of which are essential in the formation of the zyme [23, 24]. A small amount of PSA is free, despite a largemolar excess ofA1-antichymotrypsin andA2-macroglobulin, an-seminal gel [7, 8]. PSA belongs to the human kallikrein gene

family of three different genes: prostate-specific antigen (human other serpin, indicating that this isoform of PSA is not enzymati-cally active. The free forms of PSA and PSA-A1-antichymotryp-KLK3), prostate-specific glandular kallikrein (human KLK2),

and pancreatic/renal kallikrein (human KLK1) [9]. All kallikrein sin complex are immunologically detectable, whileA2-macro-globulin encapsulates the reactive PSA epitopes in such a waygenes are located on chromosome19, and human KLK3 and

human KLK2 are aligned12 kb apart [10212]. Human KLK2 that the complexes are not quantifiable immunologically by con-ventional two-site immunometric assays [25]. There are PSAis most closely related to human KLK3, having 80% coding

sequence identity, and is also expressed in human prostate tissue assays detecting either free, complexed or both PSA forms. Asignificantly higher proportion of serum PSA has been reportedand breast tissue [13215]. PSA possesses the catalytic triad typ-

ical of serine proteases and has a serine residue at position183 to be complexed toA1-antichymotrypsin in patients with prostatecancer than in patients with BPH, the proportion of free serumpredicting chymotrypsin-like activity [6,16].

PSA is also detected in the serum of patients with prostate PSA is lower in prostate cancer than in BPH [23, 26228]. Thereason for the higher portion of complexed PSA in prostate can-diseases like prostatitis, benign prostate hyperplasia (BPH) or

prostate cancer. For this reason, serum PSA levels are recog- cer is not known. It has been reported that prostate tumor cellsproduce both PSA andA1-antichymotrypsin leading to possible

Correspondence toP. Vihko, Biocenter Oulu and WHO Collaborat- PSA-A1-antichymotrypsin complex formation already in cancering Centre for Research on Reproductive Health, University of Oulu,cells. By constrast, there is noA1-antichymotrypsin productionKajaanintie 50, FIN-90220 Oulu, Finland in BPH nodules containing PSA, which could be the reason for

Fax: 1358 8 3155631. the higher free PSA concentration in patients with BPH [29, 30].E-mail : pvihko@whoccr.oulu.fi

LNCaP is a human prostate cancer cell line derived fromAbbreviations.PSA, prostate-specific antigen;hKLK3, human pros-prostate cancer metastasis in the lymph node [31]. LNCaP cellstate-specific antigen gene;hKLK2, human prostate-specific glandularsynthesize prostate-specific proteins, like PSA and prostate acidkallikrein gene;hKLK1, human pancreatic/renal kallikrein gene; ACT,phosphatase (PAP), that are produced in the epithelial cells ofA1-antichymotrypsin; A2M,A2-macroglobulin ; BPH, benign prostatic

hyperplasia. the prostate glandin vivo [31, 32]. The LNCaP cell line is andro-

330 Herrala et al. (Eur. J. Biochem. 255)

gen sensitive, and in the presence of androgens, the expressionof PSA is clearly up-regulated [33]. A similar regulation is seenwith breast carcinoma cell lines [34].

High serum PSA concentrations of prostate cancer patientsare derived mainly from cancerous and metastastic prostate tis-sue. Presently, the standardization of PSA assays is based widelyon purified PSA fraction from seminal fluid. It is important toknow if it differs from those PSA isoforms produced by prostatecancer cells. In this study we investigated the PSA secreted bythe prostate cancer cells and found out that there existed N-terminally differentially processed LNCaP-PSA isoforms. Withstructural modelling of PSA, we evaluated how the N-terminalamino acid addition or deletion affected the protein folding andenzyme activity, which both obviously have effect on complexFig.1. SDS/PAGE analyses of LNCaP, recombinant and seminalformation. We compared LNCaP-PSA isoforms with the recom-fluid PSA isoforms. (A) Coomassie brilliant blue stained LNCaP-PSA

pools A2D and purified PSA fraction from seminal fluid (*) under non-binant PSA produced in insect cells [35], and the purified frac-reducing conditions. (B) Silver-stained recombinant PSA pools A2Dtion of PSA from seminal plasma. Our results provide informa-and purified PSA fraction from seminal fluid (*) under non-reducingtion about the PSA secreted by prostate cancer cells and showconditions.that the standardization of PSA assays is more complicated than

expected.

protein concentration of the pools was measured by the methodof Lowry et al. [36]. The S-Sepharose and Superdex 75 columnsMATERIALS AND METHODSwere connected to a BioPilot automated chromatography sys-tem; the Mono-S column was used with the FPLC system (allCell culture. LNCaP-FGC cells (ATCC CRL-1740) ob-

tained from American Type Culture Collection at passage18 from Pharmacia).Characterization of LNCaP-PSA isoforms. The purifiedand passages 20230 were used for PSA production. Cells were

cultured in RPMI1640 medium with Phenol Red supplemented LNCaP-PSA isoforms were characterized by SDS/PAGE andnative PAGE. Both types of electrophoresis were carried out onwith 5% fetal calf serum, 2 mmol/lL-glutamine,100 U/ml peni-

cillin, and 100 µg/ml streptomycin. a PhastSystem (Pharmacia) with PhastGel gradient media10215 for SDS/PAGE and 8225 for native PAGE, and the proteinsLNCaP cells were grown in T-flasks, and two days after in-

oculation the culture medium was changed to RPMI1640 me- were Coomassie brilliant blue or silver stained [37242]. Theproteins were transferred onto nitrocellulose membranes withdium, without serum, containing10 nmol/l of synthetic andro-

gen 17β-hydroxy-17A-methyl-estra-4,9,11-trien-3-one (R1881, PhastTransfer (Pharmacia) for western blotting [43]. Rabbitpolyclonal antibody raised against hPSA purified from seminalNew England Nuclear). R1881 was added to the growth medium

in ethanol. After five days the culture medium was harvested plasma [18] was used with the ProtoBlot AP system (Promega)to detect the LNCaP-PSA isoforms. In both electrophoresis pro-and the amount of PSA secreted into the culture medium was

determined using a time-resolved fluoroimmunoassay kit with cedures, the LNCaP-PSA isoforms were always compared withpurified PSA fraction from seminal plasma (Calbiochem), andtwo monoclonal antibodies (DELFIA, Wallac) measuring the to-

tal PSA of the sample. to the purified recombinant PSA pools produced in insect cells[35]. Isoelectric focusing was performed on a PhastSystem withPurification of LNCaP-PSA isoforms. The harvested me-

dium was concentrated with a Pellicon cassette system (cut-off PhastGel IEF media, pH range 329, and gels were silver-stainedor blotted onto nitrocellulose membrane for western blotting.10 kDa; Millipore), and dialyzed in 50 mM sodium acetate,

pH 5.5. The concentrate was loaded onto a10310-cm cation- The measurement of PSA enzyme activity.The enzymeactivity of LNCaP-PSA isoforms was determined by their abilityexchange column (S-Sepharose HP, BPG100 column; Phar-

macia). After washing, bound protein was eluted with 0.25 mol/l to hydrolyze synthetic peptide substrates, Arg-Pro-Tyr-p-nitro-anilide (NH-Np) for chymotrypsin-like activity, and Pro-Phe-NaCl in equilibration buffer. The eluate was re-dialyzed in

50 mmol/l sodium acetate, pH 5.5, and used for repeated cation- Arg-NH-Np for trypsin-like activity (both from Chromogenix).The reactions, performed at 37°C, were initiated by adding theexchange chromatographies with S-Sepharose HP (3.5310 cm,

10 ml/min; Pharmacia) column and eluted with linear salt gradi- chromogenic substrate (50µl at a final concentration of1mmol/lto 200µl of 50 mmol/l sodium phosphate, pH 7.8, withent from 0 to 0.25 mol/l NaCl. The fractions reacting with rabbit

anti-PSA serum [18] (slot-blot, Schleicher & Schüll) were con- 100 mmol/l NaCl containing LNCaP-PSA (2210 µg). After 1 hthe reaction was stopped by addition of 0.6 mol/l acetic acidcentrated (Amicon) for gel-filtration chromatography with a

Superdex 75 (2.6 cm380 cm, 0.7 ml/min; Pharmacia) column (800µl), and the reaction rate (nmolp-nitroaniline formed/min)was calculated from the standard curve ofp-nitroaniline [44].eluted with 50 mmol/l sodium phosphate, pH 5.5, containing

0.15 mol/l NaCl. The fractions with the highest LNCaP-PSA The pH optimum was determined for the most active LNCaP-PSA isoform with the same method using Arg-Pro-Tyr-NH-Npcontent were pooled and dialyzed in 50 mmol/l sodium phos-

phate, pH 5.5, for cation-exchange chromatography with Mono- as a substrate at different pH values.N-terminal sequencing.To determine the N-terminal aminoS column (0.535.0 cm, 0.5 ml/min; Pharmacia). The LNCaP

was eluted from the column with linear salt gradient of 0 to acid sequence of different isoforms of LNCaP-PSA, recombi-nant PSA, and purified PSA fraction from seminal plasma, they0.25 mol/l NaCl and a step gradient of1.0 mol/l NaCl, respec-

tively. The1.0-ml fractions reacting with rabbit anti-PSA serum were run by SDS/PAGE and transferred to the Immobilon Pmembrane, cut out of the membrane and analyzed with a gas-(on slot blot) were divided into four different pools : A (fractions

23234), B (fractions 35245), C (fractions 46275) and D (frac- pulsed liquid sequencer [45].The complex formation between LNCaP-PSA isoformstions 76290), as was done with recombinant PSA, respectively

[35]. The pools were concentrated and further characterized. Theand protease inhibitors.10 µg of LNCaP isoforms were incu-

331Herrala et al. (Eur. J. Biochem. 255)

RESULTS

Characterization of the different isoforms of LNCaP-PSA.The LNCaP culture medium containing122 mg/l of PSA washarvested five days after the R1881 induction and used forLNCaP-PSA purification. All LNCaP-PSA in pools A to D andthe purified PSA fraction from seminal plasma had the molecu-lar mass of 29 kDa under non-reducing conditions on SDS/PAGE, while the recombinant PSA in pools A to D had molecu-lar masses of 26228 kDa (Fig.1). LNCaP-PSA proved to haveseveral isoforms. The migration of bands was different from thatof seminal plasma, but rather similar to the recombinant PSA asrevealed by the western blots following native PAGE. LNCaP-PSA was heterogeneous like purified PSA fraction from seminalplasma displaying 425 bands, whereas recombinant PSA dis-played 223 bands (Fig. 2).

The N-terminal sequencing in LNCaP-PSA pools appearedFig. 2. Western blot analyses of LNCaP, recombinant and seminal to have four different N-terminal sequences present. In additionfluid PSA isoforms on native PAGE.Rabbit polyclonal antibody raised to the correctly processed N-terminus of PSA (IVGGWEC-),against purified human PSA fraction from seminal fluid was used to

the sequences WEC- (24 amino acids), ILSRIVGGWEC- (14detect (A) LNCaP-PSA pools A2D and purified PSA fraction from sem-amino acids), and SRIVGGWEC- (12 amino acids) were iden-inal fluid (*) and (B) recombinant PSA pools A2D, and purified PSAtified (Table 1). The same kind of heterogeneity was also de-fraction from seminal fluid (*).tected in four recombinant PSA pools, while the purified com-mercial PSA fraction from seminal plasma had only correctlyprocessed N-terminus (Table1).

bated with 68µg of purified humanA1-antichymotrypsin or LNCaP-PSA pools possessed only chymotrypsin-like en-100 µg A2-macroglobulin (both from Calbiochem). All reactionszyme activity when measured with chromogenic peptide sub-were performed at 37°C for 16 h in 20 mmol/l Tris containing strate 3-carbomethoxypropionyl-Arg-Pro-Tyr-NH-Np. Pool A150 mmol/l NaCl with a pH of 7.4 [35]. The reaction mixtureshad the highest specific activity of 37 nmol · min21 · mg21 andwere analyzed by activity measurements and western blotting ofpool B the lowest, 5 nmol · min21 · mg21. The specific activitySDS/PAGE. was lower when compared with that of PSA from seminal

Structural modelling of LNCaP-PSA isoforms. The PSA plasma,115 nmol · min21 · mg21, or active recombinant PSA,structure has been previously modelled [46] based on crystallo-110 nmol · min21 · mg21, which both had only a correctly pro-graphically determined porcine kallikrein A structure at 2.05-A˚ cessed N-terminus (Table1). The optimal pH for the most activeresolution [47] taken from the Protein Data Bank [48] (entry pool of LNCaP-PSA and recombinant PSA was 8.5, and that for2pka). The model was used to study the consequences of the PSA from seminal plasma was 7.5 (data not shown). The iso-

electric focusing (Fig. 3) of LNCaP-PSA pools A2D when im-differentially processed forms of PSA.

Table 1. The comparison between LNCaP-PSA, Rec-PSA and PSA from seminal plasma.The salt concentrations are those used for theseparation of PSA pools in the final purification step.

PSA forms [NaCl] Molecular mass N-terminal sequence Specific activity

mM kDa nmol · min21 · mg21

LNCaP-PSAA 802115 34 IVGGWEC (50%) 37

WEC (50%)B 1152150 34 ILSRIVGGWEC (40%) 5

SRIVGGWEC (50%)IVGGWEC (10%)

C 1502250 34 ILSRIVGGWEC (30%) 10IVGGWEC (50%)

SRIVGGWEC (20%)D 1000 34 IVGGWEC (60%) 12

SRIVGGWEC (40%)

Rec-PSAA 902110 31 IVGGWEC(100%) 110B 1102120 31 n.d. 49C 1202140 31 APLILSRIVGGWEC (25%) 8

LSRIVGGWEC (25%)SRIVGGWEC (25%)

RIVGGWEC (25%)

PSAa from seminal plasma 34 IVGGWEC(100%) 115

a Commercial PSA.

332 Herrala et al. (Eur. J. Biochem. 255)

Fig. 3. Western blot analyses of isoelectric focusing of LNCaP, re-combinant and seminal fluid PSA isoforms.Rabbit polyclonal anti-body raised against purified human PSA fraction from seminal fluid wasused to detect LNCaP-PSA pools A2D (lanes124), active recombinantPSA, pool A (lane 5), inactive recombinant PSA, pool D (lane 6), andpurified PSA fraction from seminal fluid (lane *).

munostained with polyclonal anti-PSA antibodies showed thatLNCaP-PSA isoforms have several isoelectric points within apH range of 6.828.2. Active recombinant PSA, pool A, showedtwo bands with a pH of 7.7 and a pH of 7.4, and inactive recom-binant PSA, pool D, had a major band with a pH of 8.2 and asmaller band with a pH of 7.7, while PSA from seminal plasmaexists in several isoforms with isoelectric points within a pHrange of 6.027.7.

Complex formation between LNCaP-PSA isoforms and pro-tease inhibitors.Active isoforms of all LNCaP-PSA pools (A2D) formed stable complexes with humanA1-antichymotrypsinandA2-macroglobulin. An approximately 90 kDa band could beseen in the western blot analyses of reduced SDS/PAGE whenLNCaP-PSA isoforms were complexed with twofold molar ex-cess ofA1-antichymotrypsin (Fig. 4A). The loss of enzyme ac-tivity was seen in all LNCaP-PSA/A1-antichymotrypsin com-plexes when measured with 3-carbomethoxypropionyl-Arg-Pro-Tyr-NH-Np (Fig. 4B). WhenA2-macroglobulin was added toLNCaP-PSA, a complex of approximately110 kDa could beseen in the western blots of reduced SDS/PAGE (Fig. 4A). Theaddition ofA2-macroglobulin increased the activity of LNCaP-PSA pool A twofold, pool B fourfold, and for pools C and Dthe activity increased slightly from the original level (Fig. 4B).

Structural modelling of LNCaP-PSA isoforms. The structureFig.4. The complex formation between LNCaP-PSA isoforms andof PSA has been modelled [46] by using porcine kallikrein [47]protease inhibitors,A1-antichymotrypsin (ACT) and B2-macroglobu-as a template. The sequences have an overall sequence identitylin (A2 M). Formation of complexes with LNCaP-PSA pools A2D were

of 60%. The model has been useful in interpreting mutant andfollowed by western blotting of reduced SDS/PAGE stained with rabbitepitope mapping data and in modifying properties of the prote-polyclonal antibody raised against purified human PSA fraction fromase or developing diagnostic detection methods for prostate can-seminal fluid. All samples were reduced with 5% 2-mercaptoethanolcer. Here the model was used to delineate properties of the dif-before analysis. The purified PSA fraction from seminal fluid is marked

with * (Fig. 4A). Chromogenic activities of uncomplexed and com-ferently processed forms of PSA. PSA has also been modelledplexed LNCaP-PSA pools A2D were measured after complex formationfor other purposes [49250].(Fig. 4B).The inactive precursors of serine proteases are activated by

proteolytic cleavage. One of these cleavages removes the resi-dues14 and15 and generates another N-terminus at residue16.The serine proteases are highly conserved in this region, and in was inspected by introducing all the observed alterations to the

modeled structure (Fig. 5). The insertions and deletions at theall known three-dimensional structures residue16, either isoleu-cine or valine, is bound in a pocket in the second subdomain. N-terminus affect the hydrogen bond between residues I16 and

D194 in the mutations. The insertions and deletions further af-This cleft is close to the catalytic site and residue16 forms oneor more hydrogen bonds with D194 from the oxyanion hole. fect the conformation and activity of the catalytic site due to

increased flexibility.This residue is next to catalytic S195. The effect of mutations

333Herrala et al. (Eur. J. Biochem. 255)

The mature PSA consists of a single-chain polypeptide of237 amino acids with a molecular mass of 26079 for the peptidemoiety of the molecule, and contains a single N-linked carbohy-drate side chain attached to Asn45 [16, 51]. All the LNCaP-PSAisoforms had a molecular mass of 29 kDa when measured bynon-reduced SDS/PAGE, like purified PSA fraction from semi-nal plasma, while recombinant PSA isoforms produced in insectcells had molecular masses of 26228 kDa. The reduction of themolecular mass in the recombinant PSA is caused by the im-proper glycosylation in insect cells [35]. In isoelectric focusing,LNCaP-PSA and recombinant PSA isoforms had a similar pIrange of 7.028.2, which was higher than in seminal PSA (6.027.7). The pI range of LNCaP-PSA corresponds to earlier re-ported pI ranges (7.028.3) of free PSA in sera of prostate cancerpatients or PSA from LNCaP cells [19]. It was interesting tonote that the free PSA in sera of patients with BPH had a pIrange of 6.627.3 [19], which was lower than that of LNCaP-PSA, but similar to purified PSA fraction from seminal fluid inour experiments.

The N-terminal sequencing of LNCaP-PSA pools showedthat there were several N-terminally differently processed PSAisoforms. The recombinant PSA had the same kind of N-termi-nal heterogeneity, while the purified fraction of PSA from semi-nal plasma had only correctly processed N-terminus. The N-terminal sequencing was done through17 residues to confirmthat the all LNCaP-PSA pools (A2D) did not contain hK2,which has been recently detected in LNCaP culture media [52].The first 16 amino acids in N-terminus of PSA and hK2 areidentical [13]. Several purification procedures of PSA from sem-inal fluid have been reported resulting in pure PSA fractions,which have correctly processed N-termini [2,16, 53, 54]. Incontrast, internal peptide bond cleavage (K145-K146) causinginactivation of PSA has been reported [6, 24, 53]. The internallycleaved PSA remained connected with internal disulfide bridgesand migrated with the intact PSA under non-reducing conditionson SDS/PAGE, while under reducing conditions several smallersized fragments from the clipped form of PSA could be seen inaddition to intact protein [6]. The internal cleavage was alsodetected with LNCaP-PSA pools and purified PSA fraction fromseminal fluid (Fig. 4A). The reduction of five internal disulfidebridges of PSA [6,16] on SDS/PAGE by 2-mercaptoethanolincreased the molecular masses of LNCaP-PSA isoforms andpurified PSA fraction from seminal fluid to 34 kDa (Fig. 4A).

The LNCaP-PSA isoforms formed stable complexes withhuman A1-antichymotrypsin (90 kDa) andA2-macroglobulin(110 kDa) in the same way as active recombinant PSA in poolA and purified PSA fraction from seminal fluid [35].A1-antichy-

Fig. 5. Model of the three-dimensional structure of the PSA.(A) Rib- motrypsin inactivates PSA by binding to the active site of thebon model of the structure indicating the normal N-terminus (I16 in

protein [23, 26], which was seen also with LNCaP-PSA poolsyellow) and the truncation position at W20 (green). The ribbon runsA-D (Fig. 4B). It is known thatA2-macroglobulin encapsulatesthrough the backbone of the protein and only the interesting side chainsthe antigenic epitopes of PSA in such a way that the proteinhave been shown. The catalytic site residue H57, D102, and S195 areescapes immunodetection in native conditions [30].A2-macro-shown in magenta, orange and blue, respectively. The truncated form is

clearly unable to form the hydrogen bond with D194 (red), preceding globulin is a tetrameric molecule of 720 kDa composed of fourthe catalytic residue S195. Therefore, insertions and deletions are likelyidentical subunits disulfide-bridged in pairs, which for their partto affect at least the local conformation of the catalytic center. (B) Space-are held together by non-covalent forces [55]. WhenA2-macro-filling presentation indicating the tight binding of the normal N-terminusglobulin is subjected to conditions which favour distribution ofto the PSA structure. Color coding as in (A). non-covalent linkage, it dissosiates in half-molecules. In the

presence of reagents that reduce disulfide bonds, half-moleculesfurther produce subunits with molecular mass of180 kDa [55].The protease attack to the bait region of theA2-macroglobulinDISCUSSIONsubunits splits it nearly in halves (85 kDa) and initiates the shiftin A2-macroglobulin’s molecular structure. Only one half of theIt was shown that prostate cancer cells, LNCaP, isolated

from a lymph node metastasis, secrete multiple isoforms of PSA,A2-macroglobulin subunit links covalently the attacking prote-ase. Reducing SDS/PAGE ofA2-macroglobulin-protease com-some of them are active and some inactive. In the last purifica-

tion step, LNCaP-PSA isoforms were divided into four different plexes producesA2-macroglobulin fragments which have a mo-lecular mass of approximately 85 kDa and fragments complexedpools.

334 Herrala et al. (Eur. J. Biochem. 255)

to these half-size subunits [55]. When the LNCaP-PSAs heterogeneous PSA isoforms are detected in the commerciallyavailable PSA assays.(34 kDa) were covalently bound to these fragments ofA2-macro-

globulin, bands of approximately110 kDa could be detected inimmunoblots (Fig. 4A).A2-macroglobulin did not affect the We thank Ms Marja-Riitta Hurnasti, Ms Marja-Liisa Norrena andactive site of PSA and when a small synthetic peptide substrateMs Pirkko Ruokojärvi for expert technical assistance. This work waswas used, the chromogenic activities of the LNCaP-PSAssupported by the Research Council for Health of the Academy of Finland

(Project no. 3314), and the Technology Development Centre of Finlandincreased immediately afterA2-macroglobulin addition(TEKES). World Health Organization Collaborating Centre for Research(Fig. 4B). The specific activity of the LNCaP-PSA in the poolson Reproductive Health is supported by the Ministries of Education,A2D was lower when compared with the recombinant PSASocial Affairs and Health, and Foreign Affairs, Finland.pool A and the PSA fraction purified from seminal fluid, which

both had only correctly processed N-terminus (Table1). TheLNCaP-PSA pool B had only10% N-terminally correctly pro-cessed PSA, causing the low specific activity. Although 50%REFERENCESand 60% of the LNCaP-PSA isoforms in pools C and D, respec-

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