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Communicated by Sune Bergstrom, October 5, 1992. ABSTRACT. Rat prostatic acid phosphatase (rPAP; ortho- phosphoric-monoester phosphohydrolase (acid ...
Proc. Nad. Acad. Sci. USA

Vol. 90, pp. 799-803, February 1993

Biochemistry

Rat acid phosphatase: Overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization PIRKKO VIHKO*t, RiIrrA KURKELA*, KATJA PORVARI*, ANNAKAISA HERRALA*, AIJA LINDFORS*, YLVA LINDQVISTt, AND GUNTER SCHNEIDER: *Biocenter and Department of Clinical Chemistry, University of Oulu, Kajaanintie 50, SF-90220 Oulu, Finland; and tDepartment of Molecular Biology, Swedish University of Agricultural Sciences, Box 590, Biomedical Center, S-75224 Uppsala, Sweden

Communicated by Sune Bergstrom, October 5, 1992

ABSTRACT Rat prostatic acid phosphatase (rPAP; orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was expressed in the baculovirus expression vector system. Recombinant protein was secreted into the ium at a high yield by infected insect cells, which were cultured at high density in a 30-liter bioreactor allowing high oxygen content for rapidly growing cells. About 20% of the cell protein produced was rPAP. Partial sequence determination of the N terminus of the purified recombinant secreted protein revealed identity to the native secreted protein, showing that the sgnl peptide is recognized and properly cleaved in insect cells. The enzyme was purified by using L-(+)-tartrate affinity chromatography. The purified protein had a high specific activity of 2620 tmolimi~1 Lmg ' with p-nitrophenyl phosphate as the substrate, and it also showed phosphotyrosine phosphatase activity. The molecular mass of the recombinant rPAP was 155 kDa. Two subunits of 46 kDa and 48 kDa could be detected in SDS/PAGE, but only one subunit of 41 kDa was present after digestion with N-glycosidase. The active enzyme is a trimer of subunits differing only in glycosylation. When recombinant rPAP was crystallized with polyethylene glycol 6000 as the precipitant, the crystals were trigonal (space groupP3,21) with cell dimensions a = 89.4 A and c = 152.0 A. The observed diffraction pattern extends to a resolution of at least 3 A.

Activity of an acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2] has been detected in a wide variety of tissues from different species. Conventionally the various types can be differentiated on the basis of sensitivity to inhibition by L-(+)-tartrate, molecular weight, subcellular location, and immunological crossreactivity. Tartrate-inhibitable acid phosphatase has been purified from the human placenta (1), liver (2), and prostate (3), with the majority of the placenta and liver enzymes being expressed in lysosomes, so that they are referred to as lysosomal acid phosphatases (LAP). cDNA for human and rat LAP (hLAP-and rLAP) has been cloned and sequenced (4, 5), and the gene structure for hLAP has been determined (6), showing it to be a housekeeping gene that is expressed in almost any tissue. Prostate acid phosphatase (PAP) is synthesized in the human (hPAP) and rat (rPAP) prostate gland in a precursor form containing a signal peptide sequence of31 amino acids at the N terminus (7, 8). The human enzyme is secreted from the epithelial cells into the spermatic fluid at high concentrations (-1 mg/ml) (9), and the leader sequence is cleaved off. This secretory acid phosphatase is specifically expressed only in the prostate (10). hPAP detected in sera has been used for some 50 yr as a marker for staging and monitoring prostatic cancer (11).

hPAP and rPAP are subject to androgen regulation (12, 13), but their physiological function and substrate are unknown. It has been suggested that hPAP may hydrolyze phosphocreatine, a high-energy compound present in the seminal fluid (14), and it is known to possess phosphotyrosine phosphatase activity and to dephosphorylate epidermal growth factor receptor in membrane preparations in vitro (15). It has been suggested that the fertilizing capacity of human sperm cells depends on tyrosine phosphorylation of the sperm membrane proteins (16), but whether hPAP present in the spermatic fluid has any implication for the regulation of fertility remains to be elucidated. Protein-tyrosine phosphatases from the rat brain (17) and human placenta (18) nevertheless contain an Arg-Asn-Arg-Tyr-Pro sequence, which is homologous to an Arg-Lys-Arg-Tyr-Arg sequence (residues 54-58) found in hPAP (7, 19), an Arg-Arg-Arg-Tyr-Gly sequence found in rPAP (8), and an Arg-Gln-Arg-Tyr-His sequence subsequently found in hLAP (4). Apart from the tartrate-inhibitable acid phosphatases there also exist tartrate-resistant acid phosphatases (TRAP), which have long been associated with osteoclasts and bone resorption (20), although their role remains to be established. Rat TRAP cDNA has been cloned and sequenced (21), and it has been found to show a 41% overall similarity to rLAP (5) and a 44% similarity to rPAP (8). Comparison of the N-terminal amino acid sequences between residues 3 and 18 in mature rLAP or rPAP with that of residues 7-18 in rat bone TRAP reveals 83% similarity (21). We have been interested for some time in the possible physiological function of hPAP and the regulation of its biosynthesis. To understand the function and mechanism of acid phosphatases, we initiated a three-dimensional (3-D) structure determination using x-ray crystallography, in which hPAP purified from spermatic fluid formed only needleshaped, nondiffracting crystals. Therefore, we decided to mass-produce recombinant rPAP, which shows 75% amino acid sequence identity to and probable similarity with the 3-D structure of hPAP (8), in a secreted form in insect cells, aiming at its molecular characterization and crystallization.

MATERIALS AND METHODS Construction of the Recombinant Plasmid Transfer Vector pVL1392. The coding area of the rPAP-343 cDNA clone (8), including the signal peptide-like sequence (Fig. 1A), was amplified by PCR using two synthetic oligomers. The 5'-

oligomer (5'-GATGACTCGAGAGATCTACAATGAGAGCTGTCCCTCTG-3') contained Xho I and Bgl II restriction Abbreviations: AcNPV, Autographa californica nuclear polyhedrosis virus; LAP, lysosomal acid phosphatase; PAP, prostatic acid phosphatase; hLAP and rLAP, human and rat LAP; hPAP and rPAP, human and rat PAP. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 799

Proc. Natl. Acad. Sci. USA 90 (1993)

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FIG. 1. Strategy for the construction of recombinant plasmid transfer vectors. (A) Representation of rPAP cDNA. The thick, shaded segment indicates the location of the rPAP open reading frame (ORF). Noncoding regions are indicated by solid segments. Arrows P5' and P3' denote the location of synthetic oligonucleotide primers for PCR amplification. The position of the unique Xba I restriction site is indicated. (B) Nucleotide sequences of primers P5' and P3' used for PCR amplification of the rPAP ORF. The artificial restriction sites are highlighted. Random-tailing sequences are shown in lowercase letters. (C) The recombinant plasmid rPAP/ pSP72 consists of the PCR-amplified region of rPAP inserted between the Xho I and Kpn I sites of pSP72 plasmid vector. (D) The recombinant plasmid rPAP/pVL1392 contains the Bgl II fragment of rPAP inserted into the nonfusion transfer vector pVL1392. The direction for transcription and translation of the polyhedrin gene is indicated by an arrow. Open segments indicate the locations of the recombination sequences. bp, Base pairs.

sites in addition to the rPAP sequences, whereas the 3'oligomer (5'-GCTGAGGTACCAGATCTTTACAGCGACGCTTGGTGGTTG-3') contained Bgl II and Kpn I sites (Fig. 1B). The amplified rPAP cDNA fragment was subcloned into the pSP72 vector (Promega) by using Xho I/Kpn I restriction enzymes (New England Biolabs) and checked by dideoxy sequencing (22) (Fig. 1C). A 1.15-kilobase (kb) Bgl II rPAP fragment was isolated from the pSP72 vector and subcloned into the pVL1392 nonfusion transfer vector (Invitrogen) (Fig. 1D). The recombinant transfer vectors were screened for the correct orientation of the rPAP cDNA fragment under control of the polyhedrin promoter by using restriction enzyme digestions (23). Generation of the Recombinant Baculovirus rPAP Autographa californica Nuclear Polyhedrosis Virus (rPAPAcNPV). The transfer vector pVL1392, containing the rPAP DNA fragment (6 ,g), was cotransfected with wild-type AcMNPV DNA (1 ,g, Invitrogen) into Spodoptera frugiperda (Sf9) cells (Invitrogen) by using a modification of the calcium phosphate precipitation technique as described by Summers and Smith (24). After 6 days of incubation of the cotransfection mixture at 27°C, the viral supernatant was harvested, and the titer was determined (24). The recombinant viruses were screened by DNA slot-blot hybridization (25). After three plaque assay screening rounds, the purity of the recombinant virus and the correct position of the rPAP DNA was confirmed by PCR with recombinant baculovirus primers (Invitrogen). Infection of Sf9 Suspension Cultures and Overexpression of rPAP on a Mass Scale. Sf9 cells were grown at a density of 1.5-2.0 x 106 cells per ml in 1000-ml spinner flasks (Techne, Cambridge, U.K.) containing 10o (vol/vol) fetal calf serum (FCS) in TNM-FH insect medium (Sigma). Exponentially growing cultures were pelleted and infected with recombinant or wild-type AcNPV [multiplicity of infection (moi) = 1-101 (24). This was maintained at room temperature with occasional rocking for 1 hr. The cells were resuspended in 1000 ml of 4% FCS in TNM-FH medium. Infections were

monitored for the expression of rPAP in the cell culture medium by using the rPAP activity assay (see below). Optimal levels of expression were normally reached 4 days after infection. rPAP production was then scaled up, first to a 2-liter bioreactor, Biostat MD (B. Braun Biotech International), and subsequently to a 30-liter bioreactor, Biostat UD 30. The infection in Biostat UD 30 was produced by adding the rPAP-AcNPV at a moi of 5 directly to the final working volume once a cell concentration of 1.5-2.0 x 106 per ml had been reached. For harvesting, the cells were centrifuged at 2000 x g for 10 min, and the supernatant was collected and stored at 40C for further purification of the recombinant rPAP. Recombinant rPAP Activity Assays. The activity of recombinant rPAP was assayed as described (3), with the exception that 50 mM citrate (pH 5.4) was used as the substrate buffer. The enzyme activity was expressed in micromoles of p-nitrophenyl phosphate split by 1 ml of enzyme solution at 37TC within 1 min (Amol mind1ml-1), and the specific activity is expressed as ,umol-min-' (mg of protein)-'. Sensitivity to L-(+)-tartrate was measured by using various concentrations of sodium L-(+)-tartrate (0-100 mM). The phosphotyrosine phosphatase activity of rPAP was also measured by using o-phospho-L-tyrosine as the substrate (26, 27). Enzymatic activity was related to the inorganic phosphate liberated, which was quantified by measuring the absorbance at 820 nm. L-[mS]Methionine Labeling. Cells 66 hr after infection were cultured for 1 hr in Grace's medium deficient in methionine (GIBCO) and then for 4 hr in the same medium containing 50 mCi (1 Ci = 37 GBq) of L-[35S]methionine (Amersham) (24, 28). The medium was collected, centrifuged at 5500 x g for 5 min, and concentrated with Centriprep 10 (Amicon). The cell monolayer was washed with 50 mM sodium acetate buffer (pH 6.0) and then lysed on ice with vortexing in a lysis buffer (1% Nonidet P40/50 mM sodium acetate, pH 6.0/6.6 jLM aprotinin/1.2 mM phenylmethylsulfonyl fluoride). Any insoluble material was removed by centrifugation as before. The reduced samples were analyzed by SDS/10% PAGE (29) followed by autoradiography. Purification of Recombinant rPAP. The harvested medium from the recombinant virus infection was concentrated with a Pellicon cassette system (cut-off, 10 kDa; Millipore) and dialyzed in 25 mM Bis-Tris buffer (pH 6.5). The concentrate was then loaded onto a Q-Sepharose HP anion-exchange chromatography column (2.6 x 10 cm, 10 ml/min) connected to a BioPilot (Pharmacia). The anion-exchange chromatography was performed twice, the column being eluted with a linear salt gradient from 30 to 75 mM NaCl in the first step and with a linear pH gradient from 6.5 to 5.0 in the second step. The catalytically active fractions were pooled and dialyzed in 50 mM sodium acetate (pH 6.0). The dialyzed sample was loaded onto an L-(+)-tartrate affinity chromatography column (1.0 x 12 cm, 1 ml/min) connected to a fast protein liquid chromatograph (Pharmacia). The L-(+)-tartrate was coupled to aminohexyl-agarose (AH-Sepharose 4B, Pharmacia) as described (3). Recombinant rPAP was eluted from the affinity column with a linear L-(+)-tartrate gradient from 0 to 50 mM in 50 mM sodium acetate buffer (pH 6.0). The fractions with the highest activities were pooled for gel filtration chromatography. A Sephacryl S-200 column was connected to the BioPilot, and rPAP was eluted with 50 mM sodium acetate buffer (pH 5.5) containing 0.15 M NaCI. The pure rPAP peak was collected and dialyzed in 50 mM sodium acetate (pH 5.5). Protein concentrations were measured by the method of Lowry et al. (30) with bovine gamma globulin (Bio-Rad) as the standard. Antibodies. rPAP antisera were generated against the synthetic peptide fragments rPAP-(41-55) and rPAP-(365-381), the first corresponding to the N-terminal region of rPAP and the second corresponding to the C-terminal region (8). The

Biochemistry: Vihko et al. synthetic peptides were conjugated to keyhole limpet hemocyanin (31) and used to immunize Finnish domestic rabbits according to the standard procedure (32). Gel Electrophoresis and Immunoblotting. The purity and characterization of the recombinant rPAP were evaluated by SDS/PAGE and native PAGE. Both electrophoreses were carried out on a PhastSystem (Pharmacia) with PhastGel gradient media 10-15 for SDS/PAGE (33, 34) and 8-25 for native PAGE (35, 36) and were silver-stained (37). The rPAP activity in the native PAGE gels was located by acid phosphatase activity staining, the gel being incubated with a-naphthyl phosphate (0.5 mg/ml) and fast garnet GBC salt (0.6-0.75 mg/ml) dissolved in 0.1 M sodium acetate (pH 5.0). Western blotting of recombinant rPAP separated by native or SDS/PAGE was performed with a ProtoBlot Western blot AP system (Promega). The proteins were transferred from the PhastGels to nitrocellulose membranes with PhastTransfer (Pharmacia) (38). Rabbit polyclonal antibodies raised against the synthetic peptides (see above) recognized our recombinant protein in the first step of the ProtoBlot system, and an affinity-purified goat antirabbit IgG antiserum conjugated to alkaline phosphatase in the second step (Promega). Enzymatic Deglycosylation of rPAP. Ten micrograms of protein (6.5 gl) was mixed with 1.5 jul of 0.5 M sodium acetate (pH 6.0) and 0.5 1.l of 10%0 SDS, the total volume was adjusted to 15 al with distilled water, and the mixture was boiled for 3 min. After the pretreatment, 5 /.l of 10% (vol/vol) Triton X-100, 2 1LI of 0.5 M sodium acetate (pH 6.0), and 10 ul of neuraminidase solution (10 milliunits) or 2.5/4l of O-glycosidase solution (1 milliunit) or 3/4l of N-glycosidase F solution (0.6 unit) were added to the protein solution (all enzyme solutions were purchased from Boehringer Mannheim). The final volume was adjusted to 35 ,4 with distilled water. The mixtures were incubated at 370C overnight and analyzed by SDS/PAGE under reducing conditions. Preparation of the Rat Prostate Extract. Prostate tissue from adult Sprague-Dawley rats was homogenized as described (3). Native rPAP extract was obtained by 50-70%o ammonium sulfate precipitation. Crystallization and Data Collection. Recombinant enzyme samples were concentrated to 8-10 mg/ml, and crystallization was achieved by vapor diffusion using the hanging drop method. A 7.5-/l aliquot of protein solution was mixed with the same amount of the reservoir solution, placed on coverslips, and allowed to equilibrate over 1 ml of the well solution at 40C. The reservoir solution contained 0.1 M sodium acetate (pH 5.4) and 12-15% polyethylene glycol 6000 as the precipitant. The crystals were mounted in the usual way and analyzed on a Rigaku rotating anode operating at 50 kV and 90 mA. An x-ray data set was collected in a Nicolet area detector system (39) by using the software described by Blum et al. (40). Further data processing was carried out with the PROTEIN program (41).

Proc. Natl. Acad. Sci. USA 90 (1993)

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inside the cells, constituted 20% of the total synthesized soluble protein from the rPAP-AcNPV-infected Sf9 cells, as determined by densitometric scanning of the autoradiographed film of the SDS/PAGE gel (Fig. 2). The estimated molecular masses of the secreted and cleaved recombinant rPAP subunits were 46 kDa and 48 kDa, respectively (Fig. 2). Properties of Recombinant rPAP. The recombinant rPAP was purified from the recombinant virus-infected Sf9 cell culture medium, the specific activity of the pure protein being 2620 tumol mind mg-1 with p-nitrophenyl phosphate as the substrate. rPAP also displayed phosphotyrosine phosphatase activity (specific activity, 12 ,umol mind mg-1 with o-phospho-L-tyrosine as the substrate) and was sensitive to L-(+)tartrate, its catalytic activity being 22% of the noninhibited enzyme when 10 mM L-(+)-tartrate was added. Human PAP behaves in a similar manner (6), having 17% of its catalytic activity left under the same reaction conditions. Purified recombinant rPAP was present in silver-stained native PAGE in two forms, with molecular weights of 100 kDa and 155 kDa, respectively (Fig. 3), but only the latter band had catalytic activity (Fig. 4). The rat prostate extract analyzed with native PAGE (Fig. 4) showed a catalytically active protein band of 150 kDa. Subunits of 46 and 48 kDa were detected in

RESULTS Expression of rPAP in Sf9 Cells. When a baculovirus expression vector system was used to overproduce rPAP in Sf9 cells, catalytically active recombinant rPAP could be detected in the medium 2 days after infection and reached a maximum 4 days after infection. Ninety percent of the recombinant rPAP was secreted into the culture medium. Media from noninfected and wild-type virus-infected Sf9 cells did not have any acid phosphatase activity. To evaluate the expression of rPAP in infected cells and secretion into the culture medium, L-[35S]methionine-labeling experiments were carried out 66 hr after infection. Recombinant rPAP was detected in the medium 4 hr after labeling. The 47-kDa and 51-kDa bands, representing the recombinant rPAP protein

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Proc. Natl. Acad. Sci. USA 90 (1993)

Biochemistry: Vihko et al.

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FIG. 4. Acid phosphatase activity staining of native PAGE. The gel was stained with monosodium 1-naphtyl phosphate and fast garnet GBC disodium salt. Lanes: 1, hPAP purified from seminal fluid; 2, recombinant rPAP; 3, partially purified rat prostate extract. Sizes are shown in kDa.

SDS/PAGE (Fig. 3) from both native proteins of 100 kDa and 155 kDa. Both native forms of rPAP were recognized in Western blotting with the rabbit polyclonal antibodies raised against peptides from the N and C termini of rPAP, and when these antibodies were used in SDS/PAGE, 46-kDa and 48-kDa subunits were detected (Fig. 5). After deglycosylation, rPAP was seen only as one subunit of 41 kDa in SDS/PAGE (Fig. 6), the reduction in molecular weight being mainly due to the cleavage of N-linked carbohydrates. O-Glycosidase did not have any detectable effect on the molecular mass. Crystallization. Crystals of acid phosphatase were obtained with polyethylene glycol 6000 as the precipitant (Fig. 7). The largest crystals grew to a size of 0.8 x 0.6 x 0.2 mm. The cell dimensions and space group symmetry were determined from precession photographs and an x-ray data set collected on a Nicolet area detector. The crystals are trigonal, with a = 89.4 A and c = 152.0 A. The space group was found to be P3121 (or its enantiomorph, P3221). The crystals are stable in the x-ray beam and diffract to a resolution better than 3 A. Estimates of the content of the asymmetric unit and the packing density of the crystal can be obtained from the Vm value, as described by Matthews (42). Assuming a molecular mass of 48 kDa for the acid phosphatase subunit, only the values for n = 2 (two subunits in the asymmetric unit) (1.8 A3/Da) and n = 1 (3.6 A3/Da) are within the empirical range of packing densities, 1.7-3.7 A3/Da. An x-ray data set to 3-A resolution (R-merge§ = 5.4%) was collected on a Nicolet area detector. Based on these intensity data, rotation-function calculations were carried out (43). None of these calculations revealed the presence of an axis of symmetry. A noncrystallographic symmetry axis parallel to a twofold or threefold crystallographic axis will not be detected by these calculations, however. A native Patterson

FIG. 5. Western blotting of the purified rPAP. Lanes: 1, native PAGE separation; 2, SDS/PAGE. The blot was immunostained with polyclonal rabbit antibody raised against peptide from the N terminus of rPAP. Sizes are shown in kDa.

tion because of the proposed functional involvement of the enzyme in the spermatic fluid (14, 16), its phosphotyrosine phosphatase activity (15), its role in the formation of bone metastases originating from prostatic cancer (44), and the down-regulation of the hPAP gene via androgens (12). Until now, only the structure of the cytoplasmic tail of hLAP has been analyzed by two-dimensional nuclear magnetic resonance spectroscopy (45). We have previously cloned and sequenced hPAP (7) and rPAP (8) cDNAs and further classified the structure of the hPAP gene (46) to study the regulation of the biosynthesis of PAP in both normal and malignant mammalian prostatic tissue, and we describe here the use of a baculovirus system for the mass production of rPAP required for large-scale purification of this protein. This baculovirus expression system in insect cells (Sf9) cultured in a bioreactor gave high expression levels of recombinant protein, about 20%o of the cell protein produced. Use of the bioreactor ensured a high oxygen supply, which is vital for increased cell density accompanied by a high production rate. The resulting enzyme was secreted, and the signal sequence was properly cleaved, as confirmed by peptide sequencing. Pure rPAP has not been described previously, but the specific activity of the enzyme when p-nitrophenyl phosphate was used as the substrate was comparable to the previously described specific activity of pure hPAP (3). rPAP also has Neuraminidase

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FIG. 6. Silver-stained SDS/PAGE analysis of the deglycosylation of recombinant rPAP. rPAP was digested with neuraminidase (lane 2), O-glycosidase (lane 3), N-glycosidase F (lane 4), neuraminidase and O-glycosidase (lane 5), neuraminidase and N-glygosidase F (lane 6). Nondigested rPAP is shown in lane 1. Sizes are shown in kDa.

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Biochemistry: Vihko et al.

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6. Geier, C., von Figura, K. & Pohlmann, R. (1989) Eur. J. Biochem. 183, 611-616. 7. Vihko, P., Virkkunen, P., Henttu, P., Roiko, K., Solin, T. & Huhtala, M.-L. (1988) FEES Lett. 236, 275-281. 8. Roiko, K., Janne, 0. A. & Vihko, P. (1990) Gene 89, 223-229. 9. Rdnnberg, L., Vihko, P., Sajanti, E. & Vihko, R. (1981) Int. J. Androl. 4, 372-378. 10. Solin, T., Kontturi, M., Pohlmann, R. & Vihko, P. (1990) Biochim. Biophys. Acta 1048, 72-77. 11. Gutman, A. B. & Gutman, E. N. (1938) J. Clin. Invest. 17,473-478.

FIG. 7. A crystal of rPAP. The largest dimension of the crystal is 0.8 mm.

phosphotyrosine phosphatase activity. The molecular mass of the secreted enzyme is 155 kDa, corresponding to that of the enzymatically active acid phosphatase protein (150 kDa) detected with rPAP-specific antibodies in a rat prostatic tissue extract. The molecular mass (470 kDa) of partly purified rPAP published previously (13) does not agree with our results. Human PAP has a molecular weight of 110 kDa and is a homodimer (3). We detected two subunits of 46 kDa and 48 kDa in SDS/PAGE of rPAP that were reduced to one band of 41 kDa after N-glycosidase digestion. The calculated molecular mass of the mature rPAP polypeptide of 350 amino acids is 40,599 Da (8). We also found one band of 100 kDa in the purified recombinant preparations that was immunoreactive towards rPAP antibodies but enzymatically nonactive. Both of the 100- and 155-kDa forms of the enzyme contain the same subunits with the same N-terminal peptide sequence, and we conclude that the 100-kDa band is a nonactive dimer and that the active form of rPAP is a trimer of 155 kDa. A trimeric quaternary structure would conform with the crystals obtained, which contain a threefold crystallographic axis. The crystals of rPAP diffract to high resolution and are suitable for a structural determination by protein crystallographic methods. We thank Dr. Ake Engstrom of Uppsala University for performing the N-terminal amino acid sequencing. This work was supported in part by a grant to Y.L. from the Tryggers Foundation and grants to P.V. from the Sigrid Juselius Foundation, the Finnish Cancer Foundation, the Research Council for Medicine of the Academy of Finland, and the Technology Development Centre of Finland (TEKES). The Department of Clinical Chemistry, University of Oulu, is a World Health Organization Collaborating Center for Research in Human Reproduction supported by the Finnish Ministries of Education, of Health and Social Affairs, and of Foreign Affairs. 1. Gieselmann, V., Hasilik, A. & von Figura, K. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 651-660. 2. Saini, M. S. & Van Etten, R. L. (1987) Arch. Biochem. Biophys. 191, 613-624. 3. Vihko, P., Kontturi, M. & Korhonen, L. K. (1978) Clin. Chem. 24, 466-470. 4. Pohlmann, R., Krentler, C., Schmidt, B., Schroeder, W.,

Lorkowski, G., Cully, J., Mersmann, G., Geier, C., Waheed, A., Gottschalk, S., Grzeschik, K., Hasilik, A. & von Figura, K. (1988) EMBO J. 7, 2343-2350. 5. Himeno, M., Fujita, H., Noguchi, Y., Kono, A. & Kato, K. (1989) Biochem. Biophys. Res. Commun. 162, 1044-1053.

12. Henttu, P., Liao, S. & Vihko, P. (1992) Endocrinology 130,766-772. 13. Vanha-Perttula, T., Niemi, R. & Helminen, H. J. (1972) Invest. Urol. 9, 345-352. 14. Lee, H. J., Fillers, W. S. & Iyengar, M. R. (1988) Proc. Nati. Acad. Sci. USA 85, 7265-7269. 15. Lin, M. F. & Clinton, G. M. (1986) Biochem. J. 234, 351-357. 16. Naz, R. K., Ahmad, K. & Kumar, R. (1991) J. Cell Sci. 99, 157-165. 17. Guan, K., Haun, R. S., Watson, S. J., Geahler, R. L. & Dixon, J. E. (1990) Proc. NatI. Acad. Sci. USA 87, 1501-1505. 18. Chemoff, J., Schievella, A. R., Jost, C. A., Erikson, R. L. & Neel, B. G. (1990) Proc. Natl. Acad. Sci. USA 87, 2735-2739. 19. Van Etten, R. L., Davidson, R., Stevis, P. E., MacArthur, H. & Moore, D. L. (1991) J. Biol. Chem. 266, 2313-2319. 20. Susi, F. R., Goldhaber, P. & Jennings, J. M. (1986) Am. J. Physiol. 211, 959-962. 21. Ek-Rylander, B., Bill, P., Norgird, M., Nilsson, S. & Andersson, G. (1991) J. Biol. Chem. 266, 24684-24689. 22. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 23. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold

Spring Harbor, NY).

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