cdna cloning of rat prolyl oligopeptidase and its expression in the ovary during the estrous cycle

656 A. KIMURA AND T. TAKAHASHIJOURNAL OF EXPERIMENTAL ZOOLOGY 286:656–665 (2000)

© 2000 WILEY-LISS, INC.

JEZ 0845

cDNA Cloning of Rat Prolyl Oligopeptidase and ItsExpression in the Ovary During the Estrous Cycle

ATSUSHI KIMURA AND TAKAYUKI TAKAHASHI*Division of Biological Sciences, Graduate School of Science, HokkaidoUniversity, Sapporo 060-0810, Japan

ABSTRACT A cDNA for rat prolyl oligopeptidase was cloned which contained an open readingframe of 2,130 nucleotides encoding a protein of 710 amino acids. The deduced amino acid se-quence is around 95% homologous to other mammalian prolyl oligopeptidases and about 40% tobacterial prolyl oligopeptidases. The recombinant prolyl oligopeptidase generated in E. coli waspurified and its properties were examined. The substrate specificity and the susceptibility to pro-teinase inhibitors were similar to those of the native enzyme. Northern blot analysis showed wideexpression of the prolyl oligopeptidase gene. Using ovaries from hormone-treated rats, it was foundthat both the mRNA expression and enzyme activity increased in the luteal phase. These findingssuggest the involvement of prolyl oligopeptidase in events associated with corpus luteum forma-tion and/or luteal regression. J. Exp. Zool. 286:656–665, 2000. © 2000 Wiley-Liss, Inc.

Prolyl oligopeptidase (EC. 3. 4. 21. 26), alsoknown as prolyl endopeptidase or post-prolinecleaving enzyme, is a peptidase that cleaves thepeptide bonds at the carboxyl side of proline resi-dues. The enzyme is found in various organismsfrom bacteria to mammals (Walter et al., ’71;Koida and Walter, ’76; Rupnow et al., ’79; Yoshi-moto et al., ’80; Moriyama et al., ’88; Ohtsuki etal., ’94). Mammalian prolyl oligopeptidase iswidely distributed in various tissues (Orlowski etal., ’79; Walter et al., ’80; Moriyama and Sasaki,’83; Mizutani et al., ’84; Kalwant and Porter, ’91;Sharma and Ortwerth, ’94). The enzyme has beenpurified from many sources (Welches et al., ’93),and its cDNA has been cloned from several or-ganisms (Rennex et al., ’91; Yoshimoto et al., ’91;Kanatani et al., ’93; Shirasawa et al., ’94; Vanhoofet al., ’94; Ohtsuki et al., ’97; Yoshimoto et al.,’97; Ishino et al., ’98; Kabashima et al., ’98). Werecently discussed the structure of the mouseprolyl oligopeptidase gene and its characteristicsas a housekeeping gene (Kimura et al., ’99). Inaddition, the three-dimensional structure of por-cine muscle prolyl oligopeptidase has been estab-lished by Fülöp et al. (’98).

The physiological function of the enzyme is notyet fully understood, but various roles for the en-zyme have been suggested, such as the matura-tion and degradation of some peptide hormonesand neuropeptides (Wilk, ’83; Mentlein, ’88), DNAsynthesis (Ohtsuki et al., ’97; Ishino et al., ’98),the generation of amyloid β protein (Ishiura et

al., ’90; Fukunari et al., ’94; Shinoda et al., ’97),and cell proliferation and differentiation (Ohtsukiet al., ’94). Furthermore, Williams et al. (’99) havevery recently demonstrated that inhibition of theprolyl oligopeptidase activity caused an increasedconcentration of inositol (1,4,5) triphosphate. Us-ing a Dictyostelium mutant lacking the gene, theysuggested a novel mechanism linking enzyme ac-tivity to intracellular signaling.

We are interested in the role of prolyl oligo-peptidase in the reproductive organs, because itis highly active in the mammalian ovary anduterus as compared with other organs (Ohta etal., ’92). We previously reported that prolyloligopeptidase is specifically expressed in thegranulosa cells of porcine ovaries, and that its ex-pression level is greater in the cells of small fol-licles than in those of large follicles (Kimura etal., ’98). These findings suggest its importance inthe early stage of follicular development. However,it is difficult to conduct further detailed investi-gation on the ovarian role of prolyl oligopeptidaseusing this domestic animal because available ova-ries are almost exclusively in the follicular phase.In this study, we carried out experiments usingrat ovaries to examine the gene expression pat-

Grant sponsor: Ministry of Education and Culture of Japan.*Correspondence to: Takayuki Takahashi, Division of Biological

Sciences, Graduate School of Science, Hokkaido University, Sapporo060-0810, Japan. E-mail: [email protected]

Received 20 July 1999; Accepted 12 October 1999

RAT OVARY PROLYL OLIGOPEPTIDASE 657

tern and the activity profile of prolyl oligopep-tidase during the whole estrous cycle. Both mRNAand enzyme activity levels were found to be highin the luteal phase, suggesting its involvement inevents occurring during luteinization. We also re-port data on the cDNA cloning of rat prolyloligopeptidase as well as partial characterizationof the recombinant enzyme.

MATERIALS AND METHODSMaterials

Pregnant mares’ serum gonadotrophin (PMSG)and human chorionic gonadotrophin (hCG) wereobtained from Sigma (St. Louis, MO). Peptide 4-methylcoumaryl-7-amide (MCA) substrates, leu-peptin, E-64, pepstatin, and chymostatin werepurchased from the Peptide Institute (Osaka, Ja-pan). DEAE-cellulose (DE-52) was obtained fromWhatman (Clifton, NJ). Poststatin was kindlysupplied from Dr. Takaaki Aoyagi (Institute ofMicrobial Chemistry, Tokyo, Japan). Benzyloxy-carbonyl-prolyl-prolinal (Z-Pro-Prolinal) was a giftfrom Dr. Akikazu Hirashima (Yakult Co., Tokyo).All other chemicals were of analytical grade andreadily available commercially.

Hormonal treatment of ratsImmature female Wistar rats (3 weeks old) were

kept for a few days under controlled conditions(temperature, 25°C; lighting, 14 hr light and 10hr dark). Animals were allowed free access to foodand water. They were injected with 20 IU ofPMSG and 48 hr later with 10 IU of hCG. Ratswere sacrificed at 0, 24, and 48 hr after the PMSGinjection and 6, 12, 24, and 72 hr after hCG injec-tion. The ovaries were obtained and immediatelyfrozen by liquid nitrogen.

Isolation of prolyl oligopeptidase cDNAclones from rat liver cDNA library

Poly(A)RNA was prepared from rat liver by theguanidine isothiocyanate-cesium chloride method(Chirgwin et al., ’79) and oligo(dT)-cellulose col-umn chromatography. The cDNA was synthesizedusing a cDNA synthesis module (Amersham,Buckinghamshire, UK), and was then insertedinto a λgt 10-EcoRI vector arm (Gibco BRL, GrandIsland, NY). A GIGAPACK II GOLD (Stratagene,La Jolla, CA) was used for in vitro packaging. 2.5× 105 plaques were screened by plaque hybridiza-tion using a 1.6 kb PvuII fragment of porcineprolyl oligopeptidase cDNA (Rennex et al., ’91) asa probe. Positive clones were isolated and se-

quenced using an ABI automatic sequencer, model373 (Perkin-Elmer/Applied Biosystems, FosterCity, CA).

Preparation of ovary extract andenzyme activity assay

Tissue extracts were prepared by homogeniza-tion of ovaries using a Polytron homogenizer in500–1,000 µl of phosphate-buffered saline. Thehomogenate was centrifuged at 12,000g for 20 minand the supernatant was used for the enzyme as-say. Prolyl oligopeptidase activity toward varioussynthetic substrates was assayed as described pre-viously (Takahashi et al., ’96).

Expression and purification ofrecombinant rat prolyl oligopeptidase

An open reading frame was amplified by poly-merase chain reaction (PCR) with Pfu DNA poly-merase (Stratagene). The PCR product wassequenced and inserted into a pTrcHis expressionvector (Invitrogen, Carlsbad, CA). The expressionvector construct was transformed to the E. colistrain TOP10. When the transformed cells weregrown at 37°C in 0.8 liter of Luria-Bertani me-dium and the optical density at 600 nm reached0.6, isopropyl β-D(–)-thiogalactopyranoside wasadded at a final concentration of 0.5 mM, followedby incubation at room temperature for an addi-tional 20 hr. The cells were harvested by centrifu-gation, suspended in 60 ml of sodium phosphatebuffer, pH 7.3, containing 2 mM β-mercaptoethnol(PB-ME). An extract was prepared by freeze-thaw-ing and sonication. The crude extract was appliedto a DE-52 column (2.5 × 15 cm) and the columnwas washed extensively with PB-ME. The enzymewas eluted with 50 ml each of PB-ME and thesame buffer containing 0.2 M NaCl. Fractions withenzyme activity were pooled and applied to a Ni-chelate column (1 × 4 cm, Invitrogen). After thecolumn was washed extensively with PB-ME andthen with the buffer (pH 5.5), the enzyme waseluted with 15 ml of PB-ME (pH 4.0).

Polyacrylamide gel electrophoresisThe purified recombinant enzyme was electro-

phoresed in the presence of sodium dodecyl sulfate(SDS) according to the method of Laemmli (’70).

Northern blot analysisTotal RNAs were isolated from various tissues

by the guanidine isothiocyanate-cesium chloridemethod (Chirgwin et al., ’79). Twenty microgramsof total RNAs were electrophoresed on a formal-

658 A. KIMURA AND T. TAKAHASHI

Figure 1.


Fig. 2. SDS-PAGE analysis of the purified recombinantenzyme. About 0.3 µg of the purified enzyme was electro-phoresed in the presence of SDS under nonreducing condi-tions using a 10% SDS-PAGE gel. The gel was silver-stained.The positions of the molecular size markers are shown tothe left and calculated molecular mass of the purified en-zyme to the right.

was washed at 50°C in 0.1% SDS/0.1× SSC andexposed to either Kodak X(OMAT)AR or Biomaxfilm. To normalize the prolyl oligopeptidase mRNAsignal, rat 18S ribosomal RNA (18S rRNA) oligo-nucleotide probe, 5′-CGGCATGTATTAGCTCT-AGAATTACCACAG-3′, was used as an internalstandard (Chan et al., ’84). A DNA fragment ofrat β-actin amplified by reverse transcriptase-poly-merase chain reaction (RT-PCR) was also used asa control probe (Nudel et al., ’83).

Genomic southern blot analysisRat lung was homogenized and treated with 20

µg/ml RNase A and then with 100 µg/ml protein-ase K (Boehringer-Mannheim). After repeated ex-traction with phenol, genomic DNA was isolatedby ethanol precipitation. Five micrograms of thegenomic DNA was completely digested with EcoRI,HindIII, BamHI, and XbaI. The DNA was frac-tionated on a 0.7% agarose gel and alkaline-trans-ferred to a Nytran membrane (Schleicher &Schuell). The blot was hybridized at 60°C for 16hr in 6 × SSPE, 5× Denhardt’s solution, 1% SDS,10% dextran sulfate, and 100 µg/ml denaturedherring sperm DNA with [32P]-labeled probes, e5and e9. The probes were prepared by PCR usingthe following primer pairs: for e5, 5′-GTTATGC-CTTCAGCGAAGAT-3′ (identical to 386-405) and5′-TCGCTCTTGCCGTCCTGCT (complementaryto 576–595); and for e9, 5′-AGTGGGTGGCTT-GCGTCAG-3′ (identical to 1016–1035) and 5′-CTGGAGATAAGAAGGATGTG-3′ (complemen-tary to 1194–1213). Although the exon-intronorganization of the rat prolyl oligopeptidase geneis not known, these two probes correspond to ex-ons 5 and 9 in the mouse gene structure (Kimuraet al., ’99). The hybridized membranes werewashed at 60°C in 1× SSC/0.1% SDS and exposedto Kodak Biomax film.

RESULTS AND DISCUSSIONIsolation of rat prolyl oligopeptidase cDNA

We isolated two phage clones by screening 2.5× 105 clones of the rat liver cDNA library. Oneclone contained a ca. 2.5 kb insert and the othera 3.0 kb insert. Nucleotide sequencing analysisrevealed that the two clones share the same se-quence and that the longer clone has a completeopen reading frame. This clone was 2,754 bp longand included 2,130 bp of open reading frame, en-coding a protein of 710 amino acids (Fig. 1).1 The

1The nucleotide sequence reported in this paper has been submit-ted to the DDBJ/EMBL/GenBank™ Data Bank with the accessionnumber AB012759.

Fig. 1. Nucleotide and deduced amino acid sequence ofrat prolyl oligopeptidase. The amino acids essential for en-zyme activity (Ser554, Asp642, and His680) are marked withasterisks. The conventional polyadenylation signal sequence,AATAAA, is underlined.

dehyde/agarose gel and transferred to a Nytranmembrane (Schleicher & Schuell, Dassel, Ger-many). The blot was hybridized for 16 hr with a[32P]-labeled 2.2 kb HindIII fragment of rat prolyloligopeptidase cDNA at 42°C in 50% formamide,5× Denhardt’s solution, 5× SSPE, 1% SDS, and100 µg/ml salmon sperm DNA. The membrane


nucleotide sequence of the open reading frame was86.7%, 86.9%, 92.1%, 46.9%, 50.8%, and 49.1% ho-mologous to that of the porcine (Rennex et al.,’91), human (Shirasawa et al., ’94; Vanhoof et al.,’94), mouse (Ishino et al., ’98), Flavobacteriummeningosepticum (Yoshimoto et al., ’91), Aero-monas hydrophila (Kanatani et al., ’93), andShingomonas capsulata (Kabashima et al., ’98)prolyl oligopeptidase; amino acid sequences were95.1%, 94.9%, 96.9%, 37.5%, 39.7%, and 37.3% ho-mologous, respectively. To further confirm thatthis clone is the rat prolyl oligopeptidase cDNA,a recombinant protein was produced by expres-sion in E. coli. The recombinant protein showedactivity toward succinyl(Suc)-Gly-Pro-MCA, thesubstrate of prolyl oligopeptidase, indicating thatwe cloned the rat prolyl oligopeptidase cDNA.

Characterization of the recombinantprolyl oligopeptidase

The recombinant enzyme was purified by two-step column chromatography, as described in Ma-

terials and Methods. SDS-PAGE analysis of thepurified protein showed one major band, the mo-lecular mass of which was calculated to be 75,000Da (Fig. 2). Because the recombinant enzyme wassynthesized as a fusion protein having 30 extraamino acid residues at its NH2-terminus, the netmolecular mass of prolyl oligopeptidase should be72,000 Da. This value is in good agreement withthose previously reported for the native rat prolyloligopeptidase (Rupnow et al., ’79; Hersh, ’81;Kusuhara et al., ’93).

The substrate specificity of recombinant prolyloligopeptidase was examined using various MCAsubstrates. The results are shown in Table 1,where the specificity of the porcine enzyme(Takahashi et al., ’96) is included for comparison.The recombinant enzyme was most active on Suc-Gly-Pro-Leu-Gly-Pro-MCA. In addition, Suc-Gly-Pro-MCA and Suc-Ala-Ala-Ala-MCA were goodsubstrates for the enzyme. It is also evident fromthe data in Table 1 that the recombinant enzymeexhibited a few times as great activity on the pep-

TABLE 1. Enzyme activity on various substrates1

Enzyme activity (%)Substrates Rat recombinant Pig liver2 Pig ovary2 (follicular fluid)

Suc-Gly-Pro-MCA 100 100 100Suc-Gly-Pro-Leu-Gly-Pro-MCA 642 699 781Suc-Ala-Pro-Ala-MCA 19 8.1 8.4Suc-Ala-Ala-Ala-MCA 137 45.8 52.1Boc-Gln-Arg-Arg-MCA 1 0 0Suc-Leu-Leu-Val-Tyr-MCA 0 0 01Enzyme activities were determined at pH 8.0 in the presence of 10 mM beta-mercaptoethanol with 0.1 mM substrate and expressed aspercentages of activity for Suc-Gly-Pro-MCA. Suc, succinyl; Boc, butyloxycarbonyl.2Data cited from Takahashi et al. (’96).

TABLE 2. Effects of proteinase inhibitors on enzyme activity1

Relative activity (%)Inhibitors Concentration (mM) Recombinant enzyme Brain enzyme2

None 100 100DFP 1 1 0.1 (0.267 mM)PMSF 1 98 66 (0.45 mM)SBTI 0.1 mg/ml 122 100 (0.09 mM)EDTA 1 121 100 (0.90 mM)Z-Pro-prolinal 0.02 1 NTPoststatin 0.02 2 NTo-Phenanthroline 1 52 79 (1.10 mM)Leupeptin 0.02 99 95 (0.93 mM)E-64 0.02 91 NTChymostatin 0.02 52 NTPepstatin 0.02 89 12 (0.67 mM)1Prolyl oligopeptidase purified from various tissues incubated at room temperature for 5 min in 0.1 M Tris-HCl (pH 8.0) containing 10 mMbeta-mercaptoethanol with proteinase inhibitors at concentrations indicated. The activities were assayed with Suc-Gly-Pro-MCA as sub-strate. NT indicates not tested. DFP, diisopropyl fluorophosphate; PMSF, phenylmethanesulfonyl fluoride; SBTI, soybean trypsin inhibitor.2Data cited from Andrews et al. (’80).


tide bond of Ala-X as the native porcine enzyme.Since the substrate specificity for these MCA sub-strates of rat prolyl oligopeptidase has not beenreported, it is not known at present whether thedifference in the enzyme’s actions toward the twoMCA substrates containing an Ala-X bond is dueto species difference or due to the fusion proteinnature of the recombinant enzyme. The effects ofproteinase inhibitors on the activity of this en-zyme are summarized in Table 2. Enzyme activ-ity was strongly inhibited by DFP, but not byPMSF, SBTI, EDTA, leupeptin, E-64, and pep-statin. The recombinant enzyme was also stronglyinhibited by Z-Pro-Prolinal and poststatin, bothof which are the specific inhibitors of prolyloligopeptidase (Wilk and Orlowski, ’83; Aoyagi et

al., ’91). These inhibition profiles were similar tothose of native porcine prolyl oligopeptidase(Takahashi et al., ’96) and rat prolyl oligopeptidase(Andrews et al., ’80).

Tissue distribution of the ratprolyl oligopeptidase

Northern blot analysis was conducted using to-tal RNAs from various tissues (Fig. 3). A majorband was detected at 3.0 kb in all tissues exam-ined. The size corresponded to the longer prolyloligopeptidase cDNA insert (3.0 kb) that containedthe entire coding and noncoding regions. There-fore, this major band probably represents themRNA encoding the enzyme. Prolyl oligopeptidasemRNAs in other mammalian species have alsobeen detected at about 3.0 kb (Rennex et al., ’91;Shirasawa et al., ’94).

In addition to the 3.0 kb band, a minor bandwas reproducibly detected in a position corre-sponding to approximately 4.0 kb. Such a bandhas not previously been observed in any other spe-cies. Since existing evidence points to the pres-ence of other types of prolyl oligopeptidase inmammalian species (Matsubara et al., ’98; O’Learyand O’Connor, ’95; O’Leary et al., ’96; Cunninghamand O’Connor, ’97), we conducted a genomic South-ern blot analysis in order to see if this minor bandcould be a mRNA encoding a homologue of prolyloligopeptidase. Two hybridization probes wereused, namely, e5 and e9; these probes correspondto exons 5 and 9, respectively, in the mouse prolyloligopeptidase gene (Kimura et al., ’99). For bothprobes, only a single hybridization signal was de-tected when rat genomic DNA was digested withfour independent restriction enzymes (Fig. 4). Thedata clearly show that a single-copy gene encodesrat prolyl oligopeptidase, indicating that no ho-mologue of prolyl oligopeptidase exists in the ratgenome. Based on these results, we surmise thata portion of the probe used in the Northern blotanalysis was similar enough to affect hybridiza-tion with an unknown gene(s). It is also con-ceivable that the 4.0 kb band could be mRNAproduced as a result of alternative splicing of theprolyl oligopeptidase primary transcripts.

Prolyl oligopeptidase expressionduring the estrous cycle

We previously reported that prolyl oligopep-tidase expression was more noticeable in the earlystage of follicular development in the porcineovary (Kimura et al., ’98). However, using ovariesobtained from a local slaughterhouse, we were

Fig. 3. Northern blot analysis of prolyl oligopeptidase inrat tissue. Twenty micrograms of total RNA from the indi-cated rat tissues were applied to each lane. The probe was a2.2 kb-HindIII fragment of rat prolyl oligopeptidase cDNA. A3.0 kb of prolyl oligopeptidase signal is indicated as rPOP,and a 4.0 kb band is also shown. Rat β-actin cDNA was usedas a control.


only able to analyze its expression in the follicu-lar phase of the whole estrous cycle. To furtherinvestigate the role of prolyl oligopeptidase in thisorgan, rat ovaries are obviously more suitablethan porcine ovaries, since the hormonal condi-tions can be controlled in the rat.

Using RT-PCR on mRNA isolated from ovariesof superovulated rats, we first amplified the ratovarian prolyl oligopeptidase cDNA. It was con-firmed that the prolyl oligopeptidase expressed inthe ovary had the same nucleotide sequence asthat in the liver (data not shown).

Northern blot analysis of prolyl oligopeptidasewas conducted using total RNAs isolated from theovaries of hormone-treated rats. As shown in Fig-

ure 5, prolyl oligopeptidase mRNA expressionraised slightly after PMSG injection. Furthertreatment with hCG led to a steady increase inprolyl oligopeptidase mRNA expression. At 72 hrafter hCG injection, the mRNA expression levelreached about 2.7 times that observed at the timeof injecting PMSG (0 hr after PMSG injection).These results are inconsistent with our previousobservation that the amount of the prolyl oligo-peptidase mRNA in the granulosa cells of smallfollicles was 40% greater than that in large preo-vulatory follicles in porcine ovaries (Kimura etal., ’98), indicating some differences in the geneexpression between the two species.

Prolyl oligopeptidase activity was measured at

Fig. 4. Genomic southern blot analysis of the rat prolyloligopeptidase gene. Five micrograms of rat genomic DNAwere used in each digestion. The blot was hybridized with[32P]-labeled probes, e5 (a) or e9 (b). The membrane was

washed with a final stringency of 1× SSC/0.1% SDS at 60°Cbefore autoradiography. Molecular size markers in kilobases(kb) are indicated to the left.


the same time points as was the mRNA. Ovariesisolated from the hormone-treated rats were ho-mogenized and the extracts were analyzed for en-zyme activity. The activity of prolyl oligopeptidasewas almost constant after PMSG injection (Fig.6). In contrast, the hCG treatment initially led toa reduction in enzyme activity: the activity reachedits lowest level at the time of ovulation (12 hr af-ter hCG injection). Here we must bear in mindthat at this time the follicles are ovulating. Theloss of some ovarian tissues at ovulation could haveaffected the prolyl oligopeptidase activity. After thisinitial reduction, the activity was elevated. The ac-tivities determined at 12 and 72 hr after hCG in-jection were both significantly different from that

at the time of PMSG injection. These results areconsistent with our previous observation that inthe porcine ovary, prolyl oligopeptidase activity de-creased during follicular development. However,the present data are apparently in conflict withthe finding of Ohta et al. (’92) that the prolyloligopeptidase activity is high at estrous and lowat diestrous in the SHN strain of mice. It is diffi-cult to sufficiently explain these conflicting results,as they are from different experimental systems.It should be noted, however, that the enzyme ac-tivity profile is, as a whole, in parallel with themRNA expression pattern (Figs. 5 and 6).

We previously suggested the importance of prolyloligopeptidase in the early stage of follicular develop-

Fig. 5. Messenger RNA expression of rat prolyl oligo-peptidase during an estrous cycle. Northern blot analysis oftotal RNA from hormone-treated rat ovaries was conductedusing 2.2 kb-HindIII fragment of rat prolyl oligopeptidasecDNA as a probe. The total RNA was prepared at 0, 24, and48 hr after PMSG injection and 6, 12, 24, and 72 hr after

hCG injection. The signals (rPOP) were quantified and nor-malized by 18S rRNA signals. The value at 0 hr after PMSGinjection was adjusted to 1.0. The means ± S.E. obtained fromthree separate experiments are given. A typical result isshown at the top of the figure. The time points of PMSG andhCG injection are indicated at the bottom.


ment (Kimura et al., ’98). In this study, we have con-ducted similar experiments using rats to gain furtherinsight into the role of this enzyme in the whole es-trous cycle. The present study clearly shows that theprolyl oligopeptidase mRNA level, as well as the en-zyme activity, were significantly elevated after hCGtreatment, suggesting the involvement in the ovarianevents that take place during the luteal phase. In thiscontext, it is of particular interest to note that lutealcells synthesize and secrete peptide hormones suchas oxytocin, vasopressin, and substance P (Braden etal., ’94), all of which could be physiological substratesof the enzyme (Moriyama et al., ’88). However, it re-mains to be determined whether or not this may beoperative in the corpus luteum of the ovary. In addi-tion, an elevation of the enzyme activity in the lutealphase tempts us to speculate about its involvementin luteolysis. Drastic morphological changes associ-ated with luteal regression are well known (Bradenet al., ’94). Such changes require the enhanced activ-ity of intra- and extracellular enzymes capable of de-grading proteins and peptides. Prolyl oligopeptidaseis possibly one of these enzymes.

ACKNOWLEDGMENTSA.K. is supported by a Research Fellowship of

the Japan Society for the Promotion of Science.

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Fig. 6. Prolyl oligopeptidase activity in rat ovaries dur-ing an estrous cycle. The ovaries from hormone-treated ratswere prepared at 0, 24, and 48 hr after PMSG injection and6, 12, 24, and 72 hr after hCG injection, and the extractswere assayed for enzyme activity with Suc-Gly-Pro-MCA. Themeans ± S.E. obtained from four separate experiments aregiven. The time points of PMSG and hCG injection are indi-cated at the bottom of the figure.


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