Isolation and characterization of prolyl endopeptidase from eggs of the solitary ascidian Halocynthia roretzi: Comparison with the sperm enzyme

Comp. Biochem. Physiol. Vol. 8611,No. 4, pp. 809-814, 1987 Printed in Great Britain

0305-0491/87 $3.00+ 0.00 © 1987 PergamonJournals Ltd

ISOLATION A N D CHARACTERIZATION OF PROLYL ENDOPEPTIDASE FROM EGGS OF THE SOLITARY ASCIDIAN HALOCYNTHIA RORETZI: COMPARISON WITH THE SPERM ENZYME HIDEYOSHI YOKOSAWA,JUN ITO, MAKOTO NISHIKATA* and SHIN-IcHI ISHII Department of Biochemistry, Faculty of Pharmaceutical Sciences, and *Central Research Division, School of Dentistry, Hokkaido University, Sapporo 060, Japan (Received 16 June 1986)

Abstract--1. A prolyl endopeptidase has been highly purified from eggs of the ascidian Halocynthia roretzi by ammonium sulfate fractionation and five chromatographic operations using DEAE-cellulose, DEAESephacel, hydroxylapatite, Sephadex G-150 and Z-Gly-Pro-Leu-Gly-aminohexyl-Sephrose. 2. The molecular weight and isoelectric point of the enzyme were estimated to 66,000 and 5.0, respectively. The pH optimum of the activity was 7.0-7.5. 3. The enzyme was inactivated with diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, Z-Gly-Pro-chloromethylketone and sulfhydryl-speeific reagents; the susceptibility to these inhibitors was similar to that of the enzyme previously purified from spermatozoa of the same ascidian. 4. The ranking of four prolinal-containing peptides in their inhibitory potencies to the egg enyzme was in good agreement with that to the sperm enzyme.

INTRODUCTION Prolyl endopeptidase (post-proline cleaving enzyme, EC 3.4.21.26) is an enzyme whose function is uniquely limited to the cleavage of peptides at the carboxyl side of proline residues. This enzyme, isolated from kidney and brain of various mammals, has been well characterized and proposed to function in the metabolism of proline-containing peptide hormones (Koida and Walter, 1976; Rupnow et al., 1979; Hersh, 1981; Yoshimoto et al., 1981, 1983). However, the enzyme showing a similar specificity has not yet been isolated in invertebrates except for ascidians (Yokosawa et al., 1983). We have previously shown the presence of a prolyl endopeptidase in spermatozoa of the ascidian Halocynthia roretzi which occupies a phylogenetic position between vertebrates and "true" invertebrates (Yokosawa et al., 1983).This enzyme purified from ascidian sperm has similar properties to those of prolyl endopeptidase from various mammals. Since prolyl endopeptidase has been classified as a scrine protease according to its susceptibility to active-site specific reagents including diisopropylphosphorofluoridate and proline-containing chloromethyl ketones (Yoshimoto et al., 1977) we have synthesized several peptide aldehydes possessing prolinal at the carboxyl termini and found that these were potent inhibitors of the enzyme (Yokosawa et al., 1984; Nishikata et al., 1986). The result that Z-Gly-Prochloromethyl ketone (Yoshimoto et al., 1977) inhibits the fertilization of the ascidian which has been measured on the basis of chorion elevation led us to propose that the prolyl endopeptidase is involved in fertilization of ascidians (Yokosawa et al., 1983). Since the fertilization measured on the basis of cell division was inhibited at the concentrations lower than that required for the inhibition of fertilization C.B.P. 86/41~-L

809

measured on the basis of chorion elevation, it may be considered that a prolyl endopeptidase is also present in eggs, and that this enzyme is responsible for the process of division of egg. The present paper describes the isolation and characterization of this enzyme from eggs and gives the comparison of its properties including the susceptibility to various prolinal-containing peptides with those of the sperm enzyme. MATERIALSAND METHODS Biologicals The procedure of collecting eggs and spermatozoa from a pair of gonads with gonaducts of the solitary ascidian Halocynthia roretzi, type A, was described previously (Sawada et al., 1982). The eggs from each individual were washed with sea water, pooled, and stored at -40°C until use, while spermatozoa were suspended in 3 vol of 50mMTris--HC1, pH 8.0, containing 0.5MNaCI and stored at -40°C until use. Chemicals Sueeinyl-Gly-Pro-MCA, GIy-Pro-MCA, pyroglutamic aeid-MCA, neurotensin, luteinizing hormone-releasing hormone, angiotensin II and Z-Gly-Pro-Leu-Glywere purchased from the Peptide Institute, Inc., Osaka. Phenylmethylsulfonyl fluoride and dithiothreitol were obtained from Sigma Chemical Co., St. Louis. Diisopropylphosphorofluoridate and fluorescamine were products of Kishida Kagaku Kogyo Co., Osaka and F. Hoffman-La Roche Inc., Diagnostia, respectively. Z-Gly-Pro-chloromethyl ketone was a gift from Drs T. Yoshimoto and D. Tsuru of Nagasaki University. Prolinal-containing peptides including Z-Pro-prolinal were synthesized as described previously Orokosa~va et aL, 1984; Nishikata et al., 1986). Z-Gly-Pro-Leu-Gly-aminohexyl-Sepharose was prepared by coupling Z-Gly-Pro-Leu-Gly to aminohexyl-Sepharose in the presence of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide as described previously (Yokosawa et al., 1983).

HIDEYOSHI YOKOSAWAet al.

810 'l-

z

(a)

,%

0.06

ta.I , ~ O30

N

1

O

:).1

6

,~ o.o4

/

:

L,,J Z

q

0.02 0 O3

30

40

50

60

Z ~ 3.0

(b)

o

1

ii

.~ 2.0

!i

L,J U '7

;~ 1.o 0 O3

10

20

30

40

FRACTION NUMBER Fig. 1. (a) Sephadex G-150 gel filtration of prolyl endopeptidase fractions from the hydroxylapatite column. The column (2.7 x 53 cm) was equilibrated and developed with 10mMTris-HC1, pH7.0, containing 1 mM EDTA and 0.5raM dithiothreitol. The effluent was collected with 4ml-fractions. (b) Z-Gly-Pro-Leu-Gly-aminohexylSepharose chromatography of prolyl endopeptidase fractions from the Sephadex G-150 column. The column (I0 ml in volume) was equilibrated with 10 mM Tris--HC1, pH 7.0, containing 1 mM EDTA and 0.5 mM dithiothreitol. The arrow indicates the replacement of the intial buffer with the buffer containing 0.1 M NaC1. The effluent was collected with 2ml-fractions. Absorbance at 225 nm (--); prolyl endopeptidase activity ( - - O - - ) .

Enzyme assay Proly endopeptidase activity was assayed at 25°C in 25 mM sodium phosphate, pH 6.8, containing 0.1 M NaCI and 0.5 mM dithiothreitol by using succinylGly-Pro-MCA (50/~M) as substrate. The appearance of fluorescence due to 7-amino-4-methylcourmarin was monitored with excitation at 380 nm and emission at 460 nm. Activities toward GIy-Pro-MCA and pyroglutamic acidMCA were similarly assayed. Enyzme purification Prolyl endopeptidase from spermatozoa of the aseidian H. roretzi was prepared as described previously (Yokosawa et al., 1983) and the enzyme preparation of the DEAESephacel chromatography was used in this communication. The enzyme from eggs of the same ascidian was purified according to the modification of the procedure employed for the purification of the sperm enzyme. All the procedures were carried out at 4°C, except that the Z-Gly-Pro-LeuGly-aminohexyl-Sepharose chromatography was performed at 0°C. Frozen eggs (83 ml) were thawed and homogenized in an equal volume of artificial seawater (50 mM Tris-HCl, pH 8.0, containing 460 mM NaCI, 10 mM CaCI 2, 10 mM MgC12 and 10mM KCI) using a Potter-Elvehjem Teflonglass homogenizer (1000 rpm, 5 strokes) at 0°C, and then the supernatant was separated by centrifugation (10,000g, 30 rain). The pellet was extracted again by stirring for 2 hr with 83 ml of artificial seawater, and the pellet was discarded after centrifugation. The supernatants obtained by the two

extraction procedures were pooled and dialyzed against 50 mM Tris-HC1, pH 8.0, containing l0 mM CaCI 2. After removing insoluble materials by centrifugation, the extract was applied to a column (2.2 x 33 cm) of DEAE-cellulose which had been previously equilibrated with 50 mM Tris-HC1, pH 8.0, containing 10 mM CaCI:, and the column was washed with the same buffer. The activity of the enzyme passed through this column and the active fractions were pooled. Ammonium sulfate was added to the pooled fractions to 50% saturation, and the precipitate formed was removed by centrifugation. The supernatant was then adjusted to 80% saturation with ammonium sulfate, and the precipitate was collected by centrifugation. It was dissolved in 10mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 0.5 mM dithiothreitol, dialyzed against the same buffer, and applied to a column (3. I × 52 cm) of DEAE-Sephacel which had been equilibrated with the same buffer, and then the column was washed with the same buffer. The endogenous inhibitor of prolyl endopeptidase passed through the column, while the prolyl endopeptidase was adsorbed to the column and eluted with 21 of 0 ~ . 2 M linear gradient of NaC1 in the buffer. The active fractions eluted at about 60 mM NaCI were pooled, dialyzed against 10 mM sodium phosphate, pH 6.9, containing 0.5 mM dithiothreitol, and applied to a column (2.2 × 16 cm) of hydroxylapatite (BioGel HTP) previously equilibrated with the same buffer. After washing the column, the enzyme was eluted with 100ml of a 10-250raM linear gradient of sodium phosphate, pH 6.9, containing 0.5 mM dithiothreitol. The active fractions eluted at about 180 mM sodium phosphate were pooled, concentrated with an Amicon PM-10 membrane, and placed on a column (2.7 x 53 cm) of Sephadex G-150 previously equilibrated with 10 mM Tris-HC1, pH 7.0, containing 1 mM EDTA and 0.5 mM dithiothreitol. The column was developed with the same buffer (Fig. l(a)). Pooled fraction containing the activity were dialyzed against 10mMTris-HC1, pH7.0, containing 1 mM EDTA and 0.5 mM dithiothreitol, cooled to 0°C, and adsorbed to a column (10 ml in volume) of Z-Gly-Pro-Leu-Glyaminohexyl-Sepharose previously equilibrated with the same buffer at 0°C. The column was washed with the same buffer, and the enzyme was eluted with the same buffer containing 0.1 M NaC1 (Fig. l(b)). Active fractions were pooled and used as the final preparation of the purified enzyme. Protein concentration was measured spectrophotometrically at 280 nm or 225 rim, or according to the method of Lowry et al. (1951) with bovine serum albumin as a standard. Alternatively, it was estimated by measuring the fluorescence owing to the tryptophan residues of the protein with excitation at 285 nm and emission at 340 nm with bovine serum albumin as a standard.

Molecular weight determination The apparent molecular weight of the prolyl endopeptidase was estimated by gel filtration through a column (I .9 x 73 cm) of Sephadex G- 150, which had been calibrated with gamma-globulin (ICN, molecular weight, 150,000), bovine serum albumin (Sigma, 76,000), and bovine chymotrypsinogen A (Worthington, 25,000) in 25 mM sodium phosphate, pH 6.8, containing 1 mM EDTA, 0.1 mM NaCI and 0.5 mM dithiothreitol. Electrofocusing The enzyme was subjected to isoelectric focusing in 1% ampholine (LKB, pH 3.5-10) and a sucrose density gradient in a 110 ml column at 2°C for a total period of 48 hr at a constant current of 800 V. After completion of the run, 1.0 ml fractions were collected and assayed for activity. The pH of each fraction was determined at 2°C. Hydrolysis of peptides with prolyl endopeptidase Neurotensin, angiotensin II and luteinizing hormone-

811

Prolyl endopeptidase Table 1. Purificationof prolyl endopeptidasefrom ascidianeggs Steps

Total protein (mg)

Total

Specific

Purification

activity a activity b

Recovery

(-fold)

(%)

Extract

2,900 c

1,400

0.48

I

DEAE-cellulose

1,300 c

340

0.26

0.54

100 24

Ammonium sulfate

1,000 c

500

0.50

1.0

36

fractionation DEAE-Sephacel

4.7 d

Hydroxylapatite

0.16 d

91

19

40

6.5

110

690

1,400

7.9

Sephadex G-150

0.023 d

90

3,900

8,100

6.4

Z-Gly-Pro-Leu-Gly-

0.016 d

77

4,800

10,000

5.5

aminohexyl-Sepharose

'nmol 7-amino-4-methylcoumarin/hr.bnmol/hrper mg protein. CDeterminedby the method of Lowry et al. (1951)with bovineserum albuminas a standard. dDeterminedby measuringthe fluorescenceowingto tryptophanresiduewith bovineserumalbumin as a standard. releasing hormone at concentrations of 50 #M were incubated with the enzyme at 25°C in 25 mM sodium phosphate, pH6.8, containing 0.1 M NaC1. The extent of hydrolysis was monitored by measuring fluorescencegenerated by the reaction of amino groups with fluorescamine(Udenfriend et al., 1972).

Inhibition studies The enzyme was preincubated at 25°C for 30 min in the presence or absence of various concentrations of each inhibitor (diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, N-ethylmaleimide, p-chloromercuribenzoate, iodoacetamide, Z-Gly-Pro-chloromethyl ketone, or prolinal-containing peptide), and the remaining activity toward succinyl-Gly-Pro-MCA was measured as described above.

RESULTS

Purification o f prolyl endopeptidase from eggs The results of purification of prolyl endopeptidase from ascidian eggs are summarized in Table 1. The enzyme, together with trypsin-like enzyme which is responsible for the expansion of perivitelline space of egg during fertilization of the ascidian (Sawada et aL, 1985), was extracted from frozen and thawed eggs with artificial seawater. The former enzyme passed through the column of DEAE-cellulose, while the latter was adsorbed. Although the activity of prolyl endopeptidase diminished during dialysis of the extract and the subsequent DEAE-cellulose chromatography, in a similar manner to the case of the sperm extract (Yokosawa et al., 1983), it was restored in the following purification steps of ammonium sulfate fractionation and DEAE-Sephacel chromatography. The results suggest the preseence of endogenous inhibitor of prolyl endopeptidase. In fact, the inhibitory activity toward the enzyme was detected in the pass-through fractions obtained from DEAE-Sephacel chromatography (data not shown). Three additional chromatographic processes including affinity chromatography on immobilized Z-Gly-Pro-Leu-Gly yielded a highly purified enzyme preparation. The activity of the prolyl endopeptidase

was finally enriched 10,000-fold over that in the original egg extract with a recovery of 5.5%. The specific activity of the enzyme thus purified from eggs was approximately a half of that of the sperm enzyme purified by the similar procedure (Yokosawa et al., 1983).

Properties o f egg prolyl endopeptidase The molecular weight of the purified egg enzyme was estimated to be 66,000 by gel filtration. The enzyme showed a single peak at an isoelectric point of 5.0 on eleetrofocusing. The optimum pH of the enzymatic activity was determined to be 7.0-7.5

100

4

5

6

I

I

I

I

7 DH

8

9

10

Fig. 2. The dependence on pH of the hydrolysis of succinylGIy-Pro-MCA with prolyl endopeptidase from eggs (O) or spermatozoa (0) of H. roretzi. The activity was measured at 25°C in buffers with various pH values (4-10). The buffers consisted of 50 mM Tris, 50 mM 3-(N-morpholino) propanesulfonic acid, 50mM 2-(N-morpholino)ethanesulfonic acid, 50 mM acetic acid, 50 mM glycine, 0.1 M NaC1, 0.5 mM dithiothreitol, and appropriate amounts of NaOH or HC1 to adjust pH values.

812

HIDEYOSHI YOKOSAWA et al. Table 2. Effects of various reagents on prolyl endopeptidas¢ from ascidian gametes

~eagents

Concentration

(mM)

Diisopropylphosphorofluoridate

Phenylmethylsulfonyl fluoride

0.01

Inactivation

(%)

egg e n z y m e

sperm enzyme

95

91

0.I

100

99

0.1

61

52 98

1 ,0

98

N-Ethylmaleimide

I ,0

47

47

p-Chloromercuribeezoate

1 ,0

100

100

Iodoacetamide

I ,0

62

58

Z-Gly-Pro-chloromethyl ketone

0.001

100

100

Z-Val-prolinal

0,001

100

100

(Fig. 2, a similar value was obtained with the sperm enzyme). The K~ value toward succinyl-Gly-ProMCA was determined to be 0.38 mM at pH 7.0 in the presence of 0.1 mM NaCI and 0.5 mM dithiothreitol from a Lineweaver-Burk plot of the hydrolysis of the substrate (the value for the sperm enzyme was also 0.38mM). The enzyme exhibited little activities (less than 0.2% of the succinyl-Gly-ProMCA hydrolyzing activity) toward Gly-Pro-MCA and pyroglutamic acid-MCA. The purified enzyme was found to hydrolyze neurotensin, angiotensin II and luteinizing hormone-releasing hormone. Thus, these properties of the enzyme purified from eggs are almost similar to those of the sperm enzyme (Yokosawa et al., 1983). Comparison o f egg enzyme with sperm enzyme

To obtain further evidence of similarity between the egg and sperm enzymes, the effects of various reagents on the activity of the enzymes were exam-

(a)

ined (Table 2). The two enzymes were strongly inactivated with diisopropylphosphorofluoridate and phenylmethylsulfonyl fluoride, indicating that the egg enzyme should be also classified as a serine protease. Sulfhydryl-specific reagents including p-chloromercuribenzoate, iodoactamide, and N-ethylmaleimide inhibited the two enzymes to the similar extent to each other. Z-Gly-Pro-chloromethyl ketone, as well as Z-Val-prolinal, was also a potent inhibitor for the two enzymes. We next compared the inhibitory effects of four prolinal-containing peptides on the activities of the two enzymes (Fig. 3). Each of all the prolinalcontaining peptides examined showed almost identical strength of inhibition against the two enzymes. Z-Pro-prolinal was the most potent inhibitor for both of the enzymes 0c50 = 0.2 nM with the two enzymes). The next was Z-Val-prolinal (Ics0 = 2 nM), followed by Z-Phe-prolinal (ICs0= 40 nM). Z-Ala-prolinal was the weakest inhibitor (zcs0 = 200 nM). Thus, the two

(b)

100

O

o

lo4210-" lo-'° 159 16' 167 16616s INHIBITOR (M)

161215'110.'010-9 15s 10<' 10-eIO-s INHIBITOR (M)

Fig. 3. Inhibition of prolyl endopcptidase from (a) eggs or (b) spermatozoa of 11. roretzi by prolinalcontaining peptidcs. The enzyme was preincubatcd with various concentrations of Z-Pro-prolinal (C)), Z-Val-prolinal (O), Z-Phe-prolinal (Z~), or Z-Ala-prolinal (&) in 25 mM sodium phosphate, pH 6.8, containing 0.1 M NaCI and 0.5 mM dithiothreitol at 25°C for 30 rain, and the remaining activity toward 50 pM succinyl-Gly-Pro-MCA was measured.

813

Prolyl endopeptidase Table 3. Properti¢~of prolyl endopeptidases

Property

Ascidian

Ascidian

Lamb

Lamb

sperm

egg

brain a

kidney a

Gel filtration

66,000

66,000

74,000

74,000

Isoelectric point

5.5

5.0

4.9

Molecular weight

Optimum pH Inhibitor

7.0-7.5 DVP b, PCMB c

7.0-7.5 DFP, PCHB

7.0 DFP, PCMB

Z-GIy-Pro-

Z-Gly-Pro-

Z-Gly-Pro-

CH2CI d CH2CI CH2CI Z-Val-prolinal Z-Val-prolinal

4.8 7.7 DFP, PCMB Z-GIy-ProCH2CI

"T. Yoshimoto et al. (1981). bDiisopropylphosphorofluoridate. Cp-Chloromercuribenzoate. ~Z-Gly-Pro-chloromethyl ketone.

enzymes may recognize the residue proximal to proiinal in the almost identical manner to each other.

and spermatozoa can be estimated to be 3.5 #g per ml and 22#g per ml, respectively. Assuming that diameters of an egg and a sperm head in H. roretzi are 280#m and 5gm, respectively, it can be estimated that the content of prolyl endopeptidase in one egg DISCUSSION is about 30,000-fold higher than that in one sperWe described for the first time the presence of a matozoon. Since normal fertilization in ascidians prolyl endopeptidase in eggs of the ascidian, H. undergoes in a mammer of monospermy, that is, one roretzi. Similar to the sperm enzyme, this enzyme was spermatozoon is incorporated into one egg, almost efficiently extracted from ascidian eggs with artificial all of the prolyl endopeptidase in fertilized eggs may sea water, together with trypsin-like enzyme (Sawada be originated from the egg enzyme rather than from et al., 1985) and chymotrypsin-like enzyme (Saito and the sperm enzyme. Yokosawa, unpublished). Since an endogenous inThe purified enzyme from ascidian eggs showed hibitor of prolyl endopeptidase was also extracted similar physical and enzymatic properties to those of with the same medium, the activity of prolyl endo- prolyl endopeptisae from ascidian sperm (Yokosawa peptidase detected immediately after extraction was et al., 1983) and also from various mammals (Koida diminished during dialysis and the first chro- and Walter, 1976; Yoshimoto et al., 1977, 1981, 1983; matography, suggesting that the enzyme may interact Orlowski et al., 1979; Rupnow et al., 1979; Andrews with the inhibitor during these operations. The sepa- et al., 1980, 1982; Hersh, 1981). These properties ration of the enzyme from the inhibitor was achieved included molecular weight, isoelectric point, optiby the subsequent purification steps, ammonium mum pH, substrate specificity, and susceptibility to sulfate fractionation and DEAE-Sephacel chro- inhibitors. Comparison of the properties among the matography. While the precise localization of the enzymes from ascidian gametes and lamb tissues enzyme and the inhibitor, as well as the physiological (brain and kidney) are shown in Table 3. With respect role of the inhibitor, remains to be resolved, the to the subsite substrate specificity of prolyl endoenzyme and the inhibitor would be present in peptidase, it has been reported that the enzyme from different regions in the egg. lamb kidney (Walter and Yoshimoto, 1978), lamb The enzyme was purified from eggs by the similar brain (Yoshimoto et al., 1981), and bovine brain procedure to that used for the sperm enzyme. It (Yoshimoto et al., 1983) prefer alanine to glycine as exhibited almost the same chromatographic behavior the P2-subsite residue of substrates. Here, the amino as the sperm enzyme in each step of purification. 1"he acid residues of substrates are numbered PI, 1'2, etc., specific activity of the final preparation was about a in the N-terminal direction from the scissile bond, as half of that of the sperm enzyme preparation. Protein usually defined (Schechter and Berger, 1967). Our concentration was determined fluorometricaily with results, shown in Fig. 3, on the susceptibility of bovine serum albumin as a standard in the former the ascidian enzymes to four prolinal containing case, while that was measured spectrophotometrically peptides further suggest that the favorable P2-subsite in the latter case by assuming that ~a2s01°/_° 10.0 (Yok- residues are ranked in the following order, osawa el al., 1983). Taking account that the value of proline > valine > phenylalanine > alanine. Stronger A~'/° 2s0 of bovine serum albumin is 6.6, the difference in inhibition by Z-Pro-prolinal than by Z-Val-prolinal their specific activities is not considered to be mean- has been already reported in prolyl endopeptidases ingful. Approximately 16/~g of the enzyme was ob- from microorganism and bovine brain (Yoshimoto et tained from 83 ml of eggs with a recovery of 5.5%, al., 1985). while approximately 100/~g from 65 ml of packed We have previously found that ascidian fertilspermatozoa with a recovery of 9% (Yokosawa et al., ization measured on the basis of chorion elevation (at 1983). Therefore, the enzyme concentrations in eggs 30 min after insemination) is inhibited by Z-Gly-Pro-

814

HIDEYOSHI YOKOSAWAet al.

chloromethyl ketone (Yokosawa et al., 1983). The fertilization measured on the basis of cell division (at 2 hr after insemination) was also inhibited by the same reagent and the prolinal-containing peptides used in this study at the concentrations lower than that required for the inhibition of fertilization measured on the basis of chorion elevation (Yokosawa, unpublished date). This preliminary finding suggests that prolyl endopeptidase in fertilized eggs is responsible for some biochemical event required between chorion elevation and cell division. In fact, we have preliminarily observed that, when Z-Gly-Prochloromethyl ketone or prolinal-containing peptides was added to the egg cells at 3 0 m i n after insemination, either of the compounds inhibited the cell division measured at 2 hr after insemination. Because the enzyme in fertilized eggs is mainly originated from unfertilized eggs as discussed above, the enzyme purified from eggs in this study may have a role in the process leading to cell division. Experiments on the precise action of prolyl endopeptidase inhibitors are now in progress to clarify the physiological role of prolyl endopeptidase of gametes in the fertilization. SUMMARY A prolyl endopeptidase has been demonstrated to be present in eggs of the ascidian Halocynthia roretzi. The enzyme was extracted from frozen and thawed eggs and was highly purified by a m m o n i u m sulfate fractionation and five chromatographic operations using DEAE-cellulose, DEAE-Sephacel, hydroxylapaptite, Sephadex G-150 and Z-Gly-Pro-Leu-Glyarninohexyl-Sepharose. The molecular weight and isoelectric point of the enzyme were estimated to be 66,000 and 5.0, respectively. The p H optimum of the activity was 7.0-7.5. The enzyme was inactivated with diisopropylphosphorofluoridate, phenylmethylsulfonyl fluoride, Z-Gly-Pro-chloromethyl ketone and sulfhydryl-specific reagents; The susceptibility to these inhibitors was similar to that of the enzyme previously purified from spermatozoa of the same ascidian. Furthermore, the ranking of four prolinalcontaining peptides in their inhibitory potencies to the egg enzyme was in good agreement with that of the sperm enzyme. Possible involvement of the prolyl endopeptidase in fertilization of the ascidian was discussed. Acknowledgements--We are grateful to Dr. T. Numakunai of the Asamushi Marine Biological Station, Tohoku University, for his cooperation in collecting gametes of the ascidian, to Drs. T. Yoshimoto and D. Tsuru of Nagasaki University for the gift of Z-Gly-Pro-chloromethyl ketone. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. REFERENCES

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