J. Biochem. 107, 273-279 (1990)

Purification and Characterization of an Aminopeptidase from Sperm of the Sea Urchin, Strongylocentrotus intermedius. Ca"-Dependent Substrate Specificity as a. Novel Feature of the

Toshimasa Yasuhara,' Hideyoshi Yokosawa, and Shin-ichi Ishii

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University , Kita-ku, Sapporo, Hokkaido 060

Received for publication, September 11, 1989

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (M, = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Nil', Cull, Zn2+, and Hg2t, as well as 1,10 phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Call, Mn2+, Mgt+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as -MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolyis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single of the enzyme. Furthermore, the enzyme showed an identical K, value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.

Aminopeptidases (a-aminoacyl-peptide ) [EC gylocentrotus intermedius, and examined the inhibitory 3.4.11 ] have been reported to exist in various animal effects of bestatin (12) and its methyl ester on the activity tissues (1). Although some of them are well characterized, of this enzyme. Unexpectedly, the enzyme was completely their biological roles have not been elucidated yet except insensitive to bestatin when it was in a cell-associated for a proposed significance in inactivation of bioactive form; however, it was markedly sensitive to the reagent if peptides such as angiotensin (2), enkephalin (3), and it was in a form solubilized from the cell. Bestatin methyl tuftsin (4). ester, on the other hand, showed strong inhibition of the In animal spermatozoa, several have enzyme in either of the two forms. Fertilization of the sea been isolated and suggested to play some important roles in urchin was inhibited only by bestatin methyl ester (11). the process of fertilization (5). isolated from Furthermore, we have recently found a good correlation mammalian sperm (5, 6), a -like enzyme between the inhibitory effects of the methyl ester deriva from sea urchin (7) and frog (8) sperm, and two tives of four bestatin analogs on the fertilization of sea like (acrosin and spermosin) from ascidian sperm urchin and on the activity of spermatozoon-associated (9) have been reported to be involved in sperm penetration aminopeptidase (manuscript in preparation). These find through egg investment. However, functions of amino ings suggest that the aminopeptidase present in sper peptidases in spermatozoa (10, 11) have scarcely been matozoa has an important role in fertilization of the sea investigated. urchin. We (11) have previously found aminopeptidase activity The present paper deals with purification of the amino associated with spermatozoa of the sea urchin, Stron peptidase solubilized from spermatozoa of S. intermedius and several of its properties including susceptibility to ' Present address: Meiji Institute of Health Science , Odawara, typical aminopeptidase inhibitors and some cations. Un Kanagawa 250. usual effects of Cal' on the substrate specificity of this Abbreviations: MCA, 4-methylcoumaryl-7-amide; AMC, 7-amino enzyme were found. 4-methylcoumarin; FMOC-, 9-fluorenylmethyloxycarbonyl-; EDC, 1-ethyl -3-(3-dimethylaminopropyl)carbodiimide hydrochloride; AH-, aminohexyl-; BSA, bovine serum albumin; EGTA, ethylene EXPERIMENTALPROCEDURES glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; PCMB, Enzyme Source-Sperm was collected from the sea p-chloromercuribenzoic acid; MSH, melanocyte -stimulating hor mone; DFP, diisopropylphosphorofluoridate; Sar, sarcosine residue. urchin, S. intermedius (harvested in Akkeshi Bay, Hokkai

Vol. 107, No. 2, 1990 273 274 T . Yasuhara et al. do) by intracoelomic injection of 0.5 M KCI, as "dry" as from the results of amino acid analysis after hydrolysis. possible, and stored at -80°C until use. SDS-Polyacylamide Gel Electrophoresis-SDS-disc elec Chemicals-Leu-MCA, AMC, Leu-Gly, angiotensin II, trophoresis was performed in a slab of 7.5% polyacrylamide angiotensin III, [Sar', Ala8]-angiotensin II, Leu-enke gel according to the method of Laemmli (17). After phalin, and MSH-release inhibiting factor were obtained electrophoresis, proteins in the gel were visualized by the from the Peptide Institute (Osaka). Ala-MCA, Phe-MCA, silver-staining method of Oakley et al. (18). Arg-MCA, and Lys-MCA were purchased from Bachem Molecular Weight Estimation-The apparent molecular Feinchemikalien. 9-Fluorenylmethyloxycarbonyl chloride weight of the purified enzyme was estimated in 20 mM (FMOC-Cl), 1 -ethyl 3 (3 dimethylaminopropyl)carbodi Tris/HCI (pH 7.4) by gel filtration through a column (1.9 x imide hydrochloride (EDC), and 1,10-phenanthroline were 97 cm) of Sephadex G-200 which had been calibrated with obtained from Pierce Chemical, Nakarai Chemicals, and chymotrypsinogen A (Mr=25,000), ovalbumin (Mr= Wako Chemicals, respectively. RNA polymerase B was 45,000), BSA (Mr=67,000), and y-globulin (Mr= from Seikagaku Kogyo. Sephadex G-200 and Sepharose 4B 150,000). The molecular weight of the enzyme was also were purchased from Pharmacia Fine Chemicals AB. estimated from the mobility in SDS-polyacrylamide gel p-Chloromercuribenzoic acid (PCMB), leucine hydrox electrophoresis by using RNA polymerase B (subunit Mrs= amate, and arginine hydroxamate were from Sigma Chemi 39,000, 42,000, 100,000, 140,000, and 180,000) as a cal. Bestatin and amastatin were generous gifts of Dr. W. standard. Tanaka of Nippon Kayaku. Speract was donated by Dr. K. Electrofocusing-The enzyme was subjected to isoelec Nomura of Tokyo Metropolitan Institute of Gerontology. tric focusing on a 5% polyacrylamide-gel plate with 3% All amino acid derivatives and peptides used were of cross-linkage containing 2.4% Ampholine (LKB, pH 3.5 L-configuration. 9.5) with an LKB Multiphor apparatus. After electro Assay of Aminopeptidase Activity-Usually, amino phoresis for 2 h at 300 W under cooling (4°C), the gel plate peptidase activity was determined by using Leu-MCA or was cut into 18 pieces. The pH and the enzyme activity Arg-MCA as a substrate. An enzyme solution (5 pl) was toward Leu-MCA of the pieces were determined after added to a mixture of 10 u 1 of the substrate solution (1 mM extraction into 1 ml each of deionized water and of 50 mM in 5% dimethyl sulfoxide) and 0.5 ml of 50 mM Tris/HCl Tris/HCI (pH 7.5) containing 1 mM CaCl2j respectively. (pH 7.5) (containing 1 mM CaC12 only for Leu-MCA). Inhibitor Susceptibility-The enzyme in a volume of 5 ul After incubation at 25°C for 10 min, fluorescence due to was preincubated at O'C for 10 min with 0.5 ml of 50 mM 7-amino 4-methylcoumarin (AMC) produced was mea Tris/HC1 (pH 7.5) containing various concentrations of sured by excitation at 380 nm and emission at 460 rim with inhibitors. The remaining activity toward 20 u M Leu-MCA a Shimadzu RF-500 spectrofluorophotometer. One unit of or Arg-MCA was measured at 25'C as described above. K, the activity was defined as the amount required to hydro values were determined by plotting the initial reaction rate lyze 1 nmol of substrate per min. data according to the method of Dixon (19). Measurement of Protein Concentration-Protein con Effect of Ca2+ on the Activity-The activity of the centration was determined according to the method of enzyme toward Leu-, Ala-, Arg-, Lys-, or Phe-MCA in the Lowry et al. using bovine serum albumin (BSA) as a presence of various concentrations of Ca 21 was measured as standard (13). Alternatively, it was estimated by measur described above. Km and Vmax values in the presence of ing the fluorescence due to tryptophan residues of proteins various concentrations of Ca 2+were determined by plotting by excitation at 287 nm and emission at 348 nm using BSA the initial reaction rates according to the method of as a standard. Lineweaver and Burk (20). The K, value of Ca 2+ for Preparation of Leu-Gly-AH-Sepharose and Bestatin-AH Arg-MCA was determined according to the method of Sepharose-Aminohexyl-Sepharose (AH-Sepharose) was Dixon (19). prepared by coupling 1,6-diaminohexane (15 g) to Sepha Hydrolyses of Peptides-Each peptide, at a final concen rose 4B (100 ml in wet volume) previously activated with tration of 50 pM, was hydrolyzed with the enzyme (90 ng) cyanogen bromide (15 g) according to the method of March at 25°C in 0.1 ml of 8 mM sodium borate (pH 7.5) in the et al. (14). Leu-Gly-AH-Sepharose was prepared by cou presence or absence of 0.8 mM CaC12. After the reaction pling Leu-Giy (0.8 g) to AH-Sepharose (50 ml) at pH 4.5 in was stopped by the addition of 20,u M bestatin, the released the presence of EDC (0.6 g) according to the method of amino acids were analyzed as their dansyl derivatives by Basha et al. (15). Bestatin-AH-Sepharose was prepared as reversed-phase HPLC according to the procedure of follows. First, the amino group of bestatin (84 mg) was Weiner (21). The chromatographic equipment used con coupled with FMOC-Cl (64 mg) according to the method of sisted of a Toyo Soda HPLC-803D liquid chromatograph, a Carpino and Han (16). Second, dried AH-Sepharose (15 ml Toyo Soda GE-4 gradient unit, and a Shimadzu RF-530 in wet volume) was added to a solution containing FMOC spectrofluorophotometer. Analytical conditions are de bestatin (0.145g) and EDC (0.096g) in dimethylform scribed in the legend to Fig. 5S. amide-dioxane (1 : 1) (20 ml), shaken for 20 h at room temperature, and then filtered. FMOC-bestatin-AH-Se RESULTS pharose thus obtained was extensively washed with di methylformamide-dioxane (1 : 1), 1 M NaCl, and water. Purification of Aminopeptidase from Sea Urchin Sperm Third, a suspension of FMOC-bestatin-AH-Sepharose (12 -Aminopeptidase was effectively extracted from frozen ml) in 28% aqueous ammonia (20 ml) was shaken for 5 h at and thawed spermatozoa of the sea urchin and purified as room temperature to remove the FMOC-protecting group. follows. All procedures were carried out at 4°C. A thawed Bestatin-AH-Sepharose, the final product, was estimated sperm suspension (110 g) was mixed with 1 liter of 10 mM to contain 0.25 umol of bestatin per 1 ml of settled volume Tris/HCI (pH 7.4) and homogenized with a Teflon homog

J. Biochem. Sea Urchin Sperm Aminopeptidase 275

'Estimated from the fluorescence intensity due to tryptophan residues .

TABLE II. Effects of reagents on the activity of aminopep tidase from sea urchin sperm.

Fig. 1. Effects of Call on the activity of sea urchin amino peptidase. Hydrolysis of Arg-MCA (0), Lys-MCA (.), Phe-MCA (o), Ala-MCA (L), or Leu-MCA (o) at the concentration of 18.5 p M was measured at 25°C for 10 min in 50 mM Tris/HCI (pH 7.5) containing various concentrations of CaC12.

'No effect: elastatinal (1 pM) , pepstatin (1 pM), antipain (1 ,OM), chymostatin (1 pM), leupeptin (1 pM), N-a-tosyl-L-lysylchloro methyl ketone (10 p M). bN-Tosyl-L-phenylalanylchloromethyl ketone. affinity chromatography on a column (0.42 x 10 cm) of `Phenylmethanesulfonyl fluoride . 'Dithiothreitol. 'N-Ethylmale bestatin-AH-Sepharose under the conditions described in imide. the legend to Fig. 1S. The active fractions eluted from the column were pooled as shown in Fig. 1S and dialyzed against 500 ml of 20 mM Tris/HC1 (pH 7.4) for 6 h. The enizer at 1.300 rpm (5 strokes). The homogenate was preparation was rechromatographed on the same bestatin centrifuged at 17,000 x g for 40 min, and the resulting AH-Sepharose column under the same conditions. The supernatant fluid was again centrifuged at 70,000 x g for 60 active fractions (22.4 ml) were pooled, concentrated to 1.2 min. The supernatant fluid thus obtained was used as a ml with an Amicon PM-10 membrane, and subjected to gel sperm extract. The extract (1 liter) was concentrated to 30 filtration through a column (1.9 x 97 cm) of Sephadex ml with an Amicon PM-10 membrane and placed on a G-200 under the conditions described in the legend to Fig. column (4.6 x 85 cm) of Sephadex G-200 previously equili 2S. The enzyme emerged from the column as a symmetri brated with 10 mM Tris/HC1 (pH 7.4). The column was cal peak. Fractions under the peak were pooled (see Fig. 2S) developed with the same buffer at a flow rate of 20 ml/h and and used as a final enzyme preparation. 20-m1 fractions of the effluent were collected. The enzyme Results of the enzyme purification are summarized in activity appeared in fractions Nos. 40-45 as a symmetrical Table I. From 110 g of sperm, approximately 200,ug of the enzyme was isolated with a yield of 14%. The final enzyme peak. These fractions were pooled and applied to a column (1.6 x 21 cm) of Leu-Gly-AH-Sepharose previously equili preparation (2 pg) gave a single silver-stained band on brated with 10 mM Tris/HC1 (pH 7.4). The column was SDS-polyacrylamide gel electrophoresis (see Fig. 3). The washed with 220 ml of the same buffer, and the adsorbed elution pattern of Leu-MCA and Arg-MCA-hydrolyzing enzyme was eluted with a 0-0.4 M linear gradient of NaCl activities from the second gel filtration column were super in 240 ml of the buffer. The flow rate was 3.7 ml/h, and imposable on the protein concentration peak (see Fig. 2S), 3.7-m1 fractions were collected. The active six fractions also indicating apparent homogeneity. eluted at about 0.25 M NaCl were pooled, dialyzed against Molecular Weight and Isoelectric Point-The molecular 1 liter of 20 mM Tris/HCl (pH 7.4) for 5 h, and subjected to weight of the purified enzyme was estimated to be 107,000

Vol. 107, No. 2, 1990 276 T . Yasuhara et al.

Fig. 2. Effect of ionic strength on the activity of sea urchin aminopeptidase. (A) Hydrolyses of substrates (Leu-MCA and Arg-MCA) were mea sured in 10 mM Tris/HC1 (pH 7.5) containing various concentrations of NaCI (o and • , respec tively), KCI (o and A), and Na2SO, (o and 0). (B) Hydrolyses of substrates (Leu-MCA and Arg-MCA) were measured in 10 mM Tris/HCl (pH 7.5) con taining various concentrations of CaC12 in the pres ence (• and A, respectively) or the absence (0 and o) of 0.125 M NaCI.

TABLE III. Effects of Cal' on the release of N-terminal amino acids from bioactive peptides with aminopeptidase of sea urchin sperm.

as a strong inhibitor. The effect of 0.1 mM PCMB, which Fig. 3. Time course of release of amino acids from Leu-enke had lowered the activity (Leu-MCA as the substrate) to 9% phalin with sea urchin aminopeptidase. The released amino acids of the original level, was completely reversed by the were determined by reversed-phase HPLC after dansylation as shown addition of 1 mM dithiothreitol. Hg", Zn2+, Cue+, and Ni2+ in Fig. 5S. Open and closed symbols indicate the releases of amino acids in the presence and absence of Cal' (0.8 mM), respectively. Tyr, inhibited the enzyme irrespective of the substrate used. On a or • ; Gly, a or .; Phe, o or • ; Leu, 7 or v. the other hand, Ca', Mn2+ Mg2+, and Colt stimulated the Leu-MCA hydrolyzing activity and depressed the Arg MCA-hydrolyzing activity. The effect of Ca` was most by SDS-polyacrylamide gel electrophoresis under reduc remarkable. tive conditions and 110,000 by gel filtration through a Effects of Ca' on the Activity-The dual effect of Ca` on column of Sephadex G-200 in native form, suggesting that the aminopeptidase activity was further examined by using the enzyme consists of a single polypeptide chain. The five kinds of MCA substrates (Fig. 1). On the basis of these isoelectric point of the enzyme was determined to be 5.2± results, the substrates used could be divided into three 0.1 by isoelectric focusing on polyacrylamide gel. The groups. The first group consisted of Leu-MCA and Ala enzyme activity toward either Leu-MCA or Arg-MCA MCA, toward which the enzyme activity was stimulated by showed a pH optimum around 7.0-7.5. Cal'. Lineweaver-Burk plots of the rates of Leu-MCA Effects of Inhibitors on the Activity-Table II shows the hydrolysis at various Cal' concentrations (Fig. 4S-A) effects of various reagents on the enzyme activity toward showed that only the Vmaxvalue is altered by Ca2+ (Km = 5.4 Leu-MCA and toward Arg-MCA. Typical aminopeptidase ,uM). Furthermore, hydrolysis occurred even in the pres specific inhibitors such as puromycin, amastatin, bestatin, ence of EGTA, suggesting that the presence of Ca2+ is not leucine hydroxamate, and arginine hydroxamate inhibited essential for the activity. The activity toward the second both of the activities. On the other hand, various endopep group of substrates, Arg-MCA and Lys-MCA, was inhib tidase-specific inhibitors of microbial origin such as elas ited by Cal'. Lineweaver-Burk plots for the activity (Fig. tatinal, pepstatin, and antipain had little effect. 1,10 4S-B) indicated that Ca2+ is a competitive inhibitor with a Phenanthroline, a metal chelating reagent, showed a strong K, value of 0.6 mM. The Km value for Arg-MCA was 1.9 inhibitory effect, but EGTA and EDTA did not. Among /cM without addition of Ca'. Cal' showed a negligible sulfhydryl-directed reagents tested, only PCMB behaved effect on the enzyme activity toward Phe-MCA, one of the

J. Biochem. Sea Urchin Sperm Aminopeptidase 277

bioactive peptides. The effects can be divided into three categories, similar to the cases of the MCA substrates. The presence of Ca` was essential for the release of N-terminal proline from MSH-release inhibiting factor. Cal' inhibited the cleavage of arginine from angiotensin III, aspartic acid from angiotensin II, and sarcosine from [Sar', Alae] angiotensin II. The ion showed almost no effect on the release of glycine and phenylalanine from speract, as in the case of tyrosine liberation from Leu-enkephalin. However, Cal' was essential for the release of aspartic acid, the third amino acid of speract (data not shown). Substance P was not hydrolyzed by the enzyme in either the presence or the absence of Ca". These results indicate that the action of this aminopeptidase is affected by Call in different man ners depending on the nature not only of the amino terminal residue but also of the penultimate residue.

DISCUSSION Fig. 4. Competition between Leu-MCA and Arg-MCA (5 pM each) at the active site of aminopeptidase. o, the enzyme activity We previously reported that almost all the aminopeptidase (AMC-forming rate) measured when the enzymatic reaction mixture activity detectable in the sperm of S. intermedius is firmly contained both of the substrates, Leu-MCA and Arg-MCA; o and -7, associated with the intact cells, from which the enzyme is the activity measured when the reaction mixture contained only one most effectively solubilized by homogenizing with a hypoto of the two substrates, Leu-MCA and Arg-MCA, respectively; A, sum nic solution (10 mM Tris-HC1, pH.7.5) (11). In this report, of each pair of the data indicated by o and v; •, the activity we describe the purification and characterization of the calculated on the basis of the assumption that each of the two substrates behaves mutually as a competitive inhibitor for the other aminopeptidase solubilized from spermatozoa. Ion-ex substrate, by using the following equation, change and hydrophobic chromatography were not adopted in our purification process, because treatment with these chromatography media was found to result in a marked loss where the K, values of one substrate for the other was considered to of activity. Addition of some reagents, such as 5 mM metal correspond to the K0, value of the former. ions, 10 mM amino acids, 0.2 mM dithiothreitol, 1 mM DFP, 1 mM iodoacetamide, 0.005% Triton X-100, and 5% glycerol did not improve the recovery. In contrast, chro third-group substrates. As shown in Fig. 1, the enzyme matography on Leu-Gly-AH-Sepharose, bestatin-AH-Se preferred the first-group substrates to the second-group pharose, and Sephadex G-200 provided useful purification when Cal' was added at concentrations more than 1 mM, steps with reasonable recovery of activity. The step with but the order was reversed in the absence of Cal'. bestatin-AH-Sepharose was especially effective. In all the Effect of Ionic Strength on the Activity-Dependence of chromatographic operations, both Leu-MCA and Arg the enzyme activity toward Leu-MCA and Arg-MCA on MCA-hydrolyzing activities showed essentially the same ionic strength was examined in the presence of various elution profiles. The final enzyme preparation, showing concentrations of NaC1, KC1, or Na2SO, without addition of electrophoretic homogeneity, was obtained with almost Ca'. The hydrolysis rates of both of the substrates in identical yields with respect to the activities toward these creased with the ionic strength until it reached 0.125. When two (and several other) MCA substrates. the ionic strength passed this value, the rate of Leu-MCA The purified enzyme showed broad substrate specificity became constant and that of Arg-MCA decreased (Fig. 2A). and was inhibited by typical aminopeptidase-specific in In either the presence or the absence of 0.125 M NaCl, Ca` hibitors. The enzyme may be classified as a metalloprotease manifested a stimulative effect on the hydrolysis of Leu according to its susceptibility to the metal-chelating re MCA and an inhibitory effect on the hydrolysis of Arg-MCA agent, 1,10-phenanthroline, similarly to aminopeptidases (Fig. 2B). Thus, it seems that the effect of Cal' is not due from various mammals (22). EDTA and EGTA, however, to an increase of ionic strength by itself. showed little effect on the activity of the sea urchin enzyme. Hydrolyses of Peptides-The effect of Ca 2. on the en These results may suggest that the metal-binding locus zyme activity was also studied by using various peptides as of the enzyme prefers 1,10-phenanthroline to the latter substrates. HPLC analysis was used to determine the hydrophilic chelating reagents because of its hydrophobic successive release of amino acid residues from the amino nature. Hg", Zn2+, Cu", and Ni2+ inhibited the enzyme. A terminus of each peptide by aminopeptidase action. The sulfhydryl group seems to be indispensable for the mainte nance of full activity, since the enzyme that had been peptide fragment that seemed to be produced by endopep tidase action was not observed in any case. The results on inhibited with PCMB or heavy metal ions recovered its Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) are shown in Figs. original activity on treatment with dithiothreitol. The 5S and 3. The liberation rate of tyrosine from this peptide activity increased with the ionic strength of assay media was not affected by the addition of 0.8 mM Cal', whereas until the strength reached 0.125. Aminopeptidase B from that of glycine, phenylalanine, and leucine was stimulated. rat liver and from human erythrocytes has been reported to Table III summarizes the effects of Cal' on the release of show Cl--dependent activity (23, 24). The activity of the the respective N-terminal amino acids from six different sea urchin aminopeptidase, however, does not specifically

Vol. 107, No. 2, 1990 278 T. Yasuhara et al. depend on the Cl concentration; stimulative effects were biological activity by 4-5 orders of magnitude (26). The also observed with other anions such as SO,'- , CH3COO-, sperm aminopeptidase may function in cleavage (or process and NO3 (data not shown). ing) of a bioactive peptide(s) such as speract. Studies on Cal' and some other divalent cations (Mn 21, Mg", and the natural substrate and on the regulation of its cleavage Cot+) affected the activity of sea urchin aminopeptidase in by Ca" will elucidate the precise role of this enzyme in dual ways. They stimulated the activity if Leu-MCA or fertilization of the sea urchin. Ala-MCA was used as a substrate and depressed it if Arg-MCA or Lys-MCA was the substrate. Thus , the We are indebted to Dr. Motonori Hoshi of Tokyo Institute of enzyme preferred the first substrate group to the second Technologyfor useful discussionsand encouragement throughout this when Cal' was added at a concentration of more than 1 mM, work, to Mrs. Akiko Tsuchida-Watanabe for her technical assistance, but the order was reversed in the absence of Ca". Diversity and to the staff of Akkeshi Marine Biological Station, Hokkaido University, where part of this work was carried out. We are also of the effect of these ions might be explained by assuming grateful to Drs. Wataru Tanaka and Kohji Nomura for their gifts of that the enzyme has plural active sites which correspond to bestatin and speract, respectively. the respective substrate groups. Alternatively, the enzyme might have a single active site in which the hydrolyses of REFERENCES different substrates are differently subjected to the effect of Ca". To ascertain which is the case, we designed the 1. McDonald, J.K. & Schwabe, C. (1977) in Proteases in Mam following two experiments. malian Cells and Tissues (Barrett, A.J., ed.) pp. 311-391, North-Holland Publishing, Amsterdam First, the K; values of aminopeptidase-specific inhibitors 2. Nagatsu, I., Nagatsu, T., Yamamoto, T., Glenner, G.G., & Mehl, for the enzyme were determined with each of the two J.W. (1970) Biochim. Biophys. Acta 198,255-270 distinct substrates, Leu-MCA and Arg-MCA, in 50 mm 3. Hui, K.S., Wang, Y.J., & Lajtha, A. (1983) Biochemistry 22, Tris/HC1 buffer (pH 7.5) containing 1 mM Cal'. Irre 1062-1067 spective of the type of substrate used, an identical K, value 4. Nagaoka, I. & Yamashita, T. (1981) Biochim. Biophys. Acta 675, was obtained for each of the inhibitors; amastatin ( K, = 9 85-93 5. Morton, D.B. (1977) in Proteases in Mammalian Cells and nM), bestatin (37 nM), leucine hydroxamate (4.8 pM), and Tissues (Barrett, A.J., ed.) pp. 455-500, North-Holland Publish arginine hydroxamate (54 pM). All of these compounds ing, Amsterdam behaved as competitive inhibitors. In the second experi 6. McRorie, R.A. & Williams, W.I. (1974) Annu. Rev. Biochem. 43, ment, we examined whether the two substrates competed 777-803 with each other in occupying a single active site of the 7. Yamada, Y., Mitsui, T., & Aketa, K. (1982) Eur. J. Biochem. 122, enzyme when they were both present in a single reaction 57-62 8. Iwao, T. & Katagiri, C. (1982) J. Exp. Zool. 219, 87-95 mixture (Fig. 4). The rate of release of AMC from the two 9. Sawada, H., Yokosawa, H., & Ishii, S. (1984) J. Biol. Chem. 259, substrates in the mixture was lower than the sum of the 2900-2904 rates that were separately determined when each of the 10. Meizel, S. & Cotham, J. (1972) J. Reprod. pert. 28, 303-307 substrates was allowed to react with the enzyme indepen 11. Yasuhara, T., Yokosawa, H., Hoshi, M., & Ishii, S. (1983) dently. Instead, the rate was coincident with the value Biochem. Int. 7, 593-598 calculated on the basis of the assumption that the two 12. Umezawa, H., Aoyagi, T., Suda, T., Hamada, M., & Takeuchi, T. (1976) J. Antibiot. 29, 97-99 substrates were hydrolyzed at a common active site. These 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. results suggest that the enzyme has a single active site (1951) J. Biol. Chem. 193, 265-275 common to various substrates, and that the site alters its 14. March, S.C., Parikh, I., & Cuatrecasas, P. (1974) Anal. Biochem. substrate specificity upon interaction with Ca` and some 60,149-152 other divalent cations. 15. Basha, S.M.M., Horst, M.N., Bazar, F.W., & Roberts, R.M. The multiple effects of Ca` were also observed on the (1978) Arch. Biochem. Biophys. 185, 174-184 hydrolysis of various bioactive peptides catalyzed by this 16. Carpino, L.A. & Han, G.Y. (1972) J. Org. Chem. 37,3404-3409 17. Laemmli, U.K. (1970) Nature 227, 680-685 enzyme. With these substrates, the rate of amino acid 18. Oakley, B.R., Kirsh, D.R., & Morris, S. (1980) Anal. Biochem. release was affected by Ca` in different (stimulative, 105,361-363 suppressive, and neutral) ways depending on the nature not 19. Dixon, M. (1953) Biochem. J. 55, 170 only of the amino-terminal residue but also of the penulti 20. Lineweaver, H. & Burk, D. (1934) J. Am. Chem. Soc. 56, 658 mate residue. We previously suggested that an amino 21. Weiner, S. (1981) J. Chromatogr. 213, 501-506 22. Delange, R.J. & Smith, E.L. (1971) in The Enzyms (Boyer, P.D., peptidase participates in some process of fertilization in the ed.) Vol. 3, pp. 81-118, Academic Press, New York sea urchin, S. intermedius, on the basis of parallel inhibi 23. Hopsu, V.K., Makinen, K.K., & Glenner, G.G. (1966) Arch. tory effects of bestatin methyl ester on fertilization of the Biochem. Biophys. 114, 567-575 sea urchin and on the activity of aminopeptidase present in 24. Soderling, E. (1982) Arch. Biochem. Biophys. 216, 105-115 the spermatozoon (11). In this study, aminopeptidase 25. Garbers, D.L., Watkins, H.D., Hansbrough, J.R., Smith, A., & isolated from the spermatozoon was found to hydrolyze Misono, K.S. (1982) J. Biol. Chem. 257, 2734-2737 speract, a sperm-activating peptide present in egg jelly of 26. Nomura, K., Suzuki, N., Ohtake, H., & Isaka, S. (1983) Biochem. the sea urchin (25). It has been reported that cleavage of Biophys. Res. Commun. 117, 147-153 the Phe-Asp bond of speract results in a decrease of its

J. Biochem. Sea Urchin Sperm Aminopeptidase 279

Supplemental Materials

Fig. 45. Lineweaver-Burk plots of the effects of Ca2~ on the hydrolyses of MCA substrates with sea urchin aminopeptidase. (A) The hydrolysis rata of Leu-MCA was determined at the substrate concentrations of 5, 7.5, 10, and 15 ), 0.7 (U), inuM the presence of 0 (0),0.025 (A), 0.05 )V), 0.075([] 0.5 (~/b or 1 mM1 A) Cccl;. The value of Km(5.4 59) was found to be conetan t in any Cat concentration. (B) The hydrolysis rate of Arg-MCA was determined at the concentrations of 5, 7.5, 10, 15, and 20 pM in the presence of 0 (•), 1 (A), 2.5 (0), or 10 mM (O) CaC12. The value of Ki (0.6 nMi was estimated for Cat' from a replot according to the method of Dixon 119).

Fig. IS. Affinty chromatography on a column of bestatin-AH-Sepharose (first) of the sea urchin aminopeptidase preparation obtained by Leu-Gly-AH-Sepharose chromatography. The enzyme preparation was applied to the column (0.42 x 10 cm) which had been equilibrated with 20 mM Tris/HC1 (pH 7.4). The column was washed with the same buffer, and the adsorbed enzyme was eluted with a linear gradient of NaCl and ethyleneglycol concentrations formed by mixing 55 ml of 20 mM Tris/HC1 (pH 8.0) containing 0.6 M NaCl and 10 a ethyleneglycol into the same volume of 20 mM Prim/Rd (pH 7.4). The flow rate was 4.4 mllh, and 2.2-m1 fractione were collected. --a--, abeorbance at 280 nm; -O- enzyme activity toward Leu-MCA; -.*-, enzyme activity toward Arg-MCA.

Fig. 2S. Gel filtration through a column (1.9 x 97 cm) of Sephadex G-200 (second) of the aminopeptidase preparation obtained by the second beetatin-AH-Sepharose chromatography. The effluent (20 mM Tris/HC1, pH 7.4) was collected in 2.6-m1 fractions at a flow rate of 4.7 ml/h. a--, protein concentration estimated from the fluorescence intensity; '-0-, activity toward Leu-MCA;-, activity toward Arg-MCA. Fig. 55. HPLC analysis. of release of amino acids from Leu-enkephalin (Tyr Gly-Gly-P.he-Leu) with sea urchin aminopeptidase. The substrate at the concentration of 50 pM we a incubated with the enzyme (90 ng) in 8 mM sodium borate (pH 7.5) in the absence (A) or the presence (B) of 0.8 MM Ca2° at 25°C for 3 h. After the reaction was stopped by adding 20 pM beetatin, the liberated amino acids were analyzed as their fluore'scent daneyl derivatives by HPLC on a reversed-phase column (ERC-ODS-1161, 6 x 100 mm. See EXPERIMEN TAL PROCEDURES for other details). Each peak was identified by comparing its retention time with those of standard daneyl amino acids. B indicates the peak of do neyl bestatin. The peak of Leu-enkephalin (retention time, 45 min) h ad disappeared almost completely in both chromatograma, (A) and (B).

Fig. 3S. SDS-polyacrylamide gel electrophoresis of the final preparation of sea urchin aminopeptidase. Lane b, purified enzyme (2) wthout 2-mercaptoethanol; c, purified enzyme (2 pg) with 2-mercaptoethanol) a, ANA polymerase B subunita (Mram 39,000, 42,006, 700,000, 140,000, 6 180,000) with 2-mercaptoethanol. Proteins were visualized by the silver staining method of Oakley et al. (18).

Vol. 107, No. 2, 1990