THE JOURNAL OF BIOLOGICAL CHEMISTRY ( 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 25, Issue of September 5, pp. 16715-16721, 1991 Printed in U. S. A.

Purification and Amino Acid Sequence of a Bitter Gourd Inhibitor against an Acidic Amino Acid-specific Endopeptidase of Streptomyces griseus”

(Received for publication, April 29, 1991)

Fusahiro OgataS, Toshiyuki MiyataQ, Nobuyoshi Fujii, Norio Yoshida, Kosaku Nodall, Satoru Makisumi, and Akio Ito From the Departments of Chemistry and §Biology, Faculty of Science, Kyushu University, Fukuoka 812, Japan and the llDepartment of Human Life Sciences, Fukuoka Women’s University, Fukuoka 813, Japan

An inhibitor (BGIA) against an acidic amino acid- specific endopeptidase of Streptomyces griseus (Glu s. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. gri- seus protease with an inhibition constant (Ki ) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:l complex with this protease. However, two other acidic amino acid-specific endopeptidases, pro- tease VS from Staphylococcus aureus and Bacillus sub- tilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, bar- ley subtilisin-chymotrypsin inhibitor CI- 1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corre- sponding amino acid in BGIA is alanine.

The presence of endopeptidases with selectivity for the carbonyl bond of the acidic amino acids glutamic acid and aspartic acid has been detected by several groups of investi- gators (Garg and Virupaksha, 1970a, 1970b; Drapeau et al. 1972; Edwards et al. 1977; Yoshida et al., 1988; Niidome et al., 1990). The number of such peptidases is limited, and little information on structural and functional properties is avail- able compared with trypsin-like, chymotrypsin-like, and elas- tase-like enzymes. However, there are reports of the partici- pation of enzymes cleaving peptide bonds after acidic amino acid residues in physiologically important reactions such as the germination of sorghum (Garg and Virupaksha, 1970a, 1970b), hatching of the sea urchin (Edwards et al., 1977), processing of precursors such as dog mast cell chymase (Caughey et al., 1990), human lymphocyte granzymes (Jenne e t al., 1988), and human interleukin l(3 (Sleath et al., 1990). In most of these, proteases directly participating in the reac- tions have either not been identified or have not been well characterized. A number of studies on serine and thiol pro- teases have shown that the use of specific inhibitors yields

* The costs of Publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed.

important information on the structure around the active site and on the reaction mechanism of the proteases.

We attempted to isolate and characterize a specific protein inhibitor of acidic amino acid-specific endopeptidases. We searched for inhibitory activity using Glu Streptomyces griseus protease from S. griseus as representative of acidic amino acid-specific proteases; the highest inhibitory activity was seen in seeds of the bitter gourd. The purification, character- ization, and primary structure of the inhibitor from bitter gourd seeds (BGIA)’ are described herein.

EXPERIMENTAL PROCEDURES~

RESULTS

Purification of BGIA-In order to obtain an inhibitor against an acidic amino acid-specific endopeptidase, we searched for a protein with such activity against Glu S. griseus protease, an acidic amino acid-specific endopeptidase from S. griseus, from various plant tissues. We found the highest inhibitory activity in the seeds of the bitter gourd (data not shown). The inhibitor (BGIA) was isolated from the seeds as described under “Experimental Procedures.” In the first chro- matography on Sepabeads FP-CM, inhibitory activity against Glu S. griseus protease was eluted as a single peak, whereas trypsin-inhibitory activity was separated into several peaks, the largest being eluted in the same fractions as BGIA. As shown in Fig. 1, however, BGIA was clearly separated from contaminating inhibitors by CM-Toyopearl 650M column chromatography. After further purification by hydrophobic chromatography on a butyl-Toyopearl column, the inhibitor fraction was applied to a Sephadex G-50 column (Fig. 2). BGIA activity eluted from this column as a single symmetric peak, and the elution pattern was identical with that of protein.

A summary of a typical purification experiment, starting from 100 g of the acetone powder of the seeds, is shown in Table 1. BGIA was purified about 220-fold from the crude extracts with an overall recovery of about 38%. The final

The abbreviations used are: BGIA, bitter gourd inhibitor against an acidic amino acid-specific endopeptidase (Glu S. griseus protease); PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sul- fate; pNA, p-nitroanilide; Boc, t-butyloxycarbonyl; SUC, succinyl; Cm, carboxymethyl; PTH, phenylthiohydantoin; HPLC, high perform- ance liquid chromatography. All optically active amino acids are of the L configuration.

Portions of this paper (including “Experimental Procedures,” Figs. 1-3 and 5-9, and Tables I-IX) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

16715

16716 Plant Inhibitor against Glu-specific Protease

preparation gave a single protein band on PAGE without SDS (Fig. 2). The homogeneity of BGIA was confirmed by SDS- PAGE in the presence and absence of 2-mercaptoethanol and HPLC analyses using a reversed-phase column (Asahipak ODP-50) and a gel filtration column (TSK gel G2000SWxL). SDS-PAGE showed BGIA is composed of a single polypeptide chain.

Physicochemical Properties-The apparent molecular weight of BGIA was estimated to be 6500, by SDS-PAGE, both in the presence and the absence of 2-mercaptoethanol and by gel filtration on TSK gel 2000SWxL. As described later, however, the amino acid sequence of BGIA revealed a molecular weight of 7419. The isoelectric point of BGIA was higher than pH 10.5, and the exact value could not be deter- mined by isoelectric focusing.

The stability of BGIA at elevated temperatures was inves- tigated in 0.1 M Tris-HC1 buffer, pH 8.8. When BGIA was incubated at 60 "C for 15 min, no loss of the inhibitory activity was observed, but at over 70 "C the activity was gradually lost. At 25 "C, BGIA was stable in a wide pH range from pH 1 to 11.

Formation of Glu S. griseus Protease-BGZA Complex-The formation of the complex of BGIA and Glu S. griseus protease was studied by gel filtration on an Asahipak GS310 column (Fig. 3). When Glu S. griseus protease with a molecular weight of 20,000 was applied to the column, the elution time was unexpectedly late compared with that of BGIA, thereby sug- gesting a selective interaction between the protease and the gel (Fig. 3A). A mixture of Glu S. griseus protease and a 2- fold molar excess of BGIA gave two peaks (Fig. 3C). Since the second peak (elution time of 13.2 min) was BGIA, the first peak (elution time of 12.1 min) is the complex of Glu S. griseus protease and BGIA. The area ratio of the first peak (the complex) to the second peak (excess free BGIA) was calculated to be 57 to 43. Since the absorptivity of Glu S. griseus protease at 280 nm was about one-fifth that of BGIA, the ratio suggests the formation of an equimolar complex between Glu S. griseus protease and BGIA.

Inhibitory Actiuity-Dixon and Lineweaver-Burk analyses showed that BGIA competitively inhibits the activity of Glu S. griseus protease (58 nM) with inhibition constants (Ki) of 65 and 70 nM, respectively. Table I1 shows the effect of BGIA on several other proteinases. The activities of protease V8 and Glu Bacillus subtilis protease, both of which are acidic amino acid-specific endopeptidases, were not inhibited by BGIA. While BGIA had no inhibitory activity against bovine trypsin and chymotrypsin, porcine pancreatic elastase, and papain, it strongly inhibited the activity of subtilisin Carls- berg. The inhibition constant of BGIA for this protease was estimated to be on the order of 0.1 nM, as determined by the method of Green and Work (1953).

Amino Acid Composition-Table I11 shows the amino acid composition of BGIA. The inhibitor is rich in arginine; hence, the high PI value. No free cysteine was detected by Ellman's method (Ellman, 1959), suggesting that the 2 cysteine residues in BGIA make a disulfide bond.

Amino Acid Sequence of BGZA-The primary structure of BGIA was elucidated by determination of the amino acid sequences of peptides generated by digestion of BGIA with lysyl endopeptidase, endoproteinase Asp-N, and thermolysin. The complete amino acid sequence of BGIA is shown in Fig. 4. Both native and carboxymethylated (Cm-) BGIA were resistant to Edman degradation, thereby indicating a blocked amino terminus. Since hydrazinolysis of BGIA yielded 0.14 nmol of glycine/nmol of the inhibitor (uncorrected) and no

1 10 20 30 40 50 60 68 I I I I I I ,

A c S O C O G I ( R S W W L V G S T G A M M V l E R g N P R V R A V I Y R V G P P A 1 G k""-( K1 " K 3

- -- - " " -I 'Klni

C------------------------------------------iH D 3 D l -

C"""""l THl2

~"t"""-(""-t~~"~~"~"-~~""-"~~"""--( TH9 - TH5 THlTH4 TH2 TH6 T H 3 TH14

b"""-( THlO

+""-( T H l 1

FIG. 4. Summary of the data used to establish the complete primary structure. One disulfide bond links Cys-3 to Cys-48. AcS, N-acetylserine. Peptides are numbered according to the order of elution from each HPLC column. Bracketed solid lines indicate the complete sequence determined by automated Edman degradation.

other amino acid residues were detected, the carboxyl termi- nus is glycine.

When the digest of BGIA obtained with lysyl endopeptidase was applied to Asahipak ODP-50, three peptide peaks (Kl, K2, and K3) were obtained (Fig. 5). Since peptide K1 formed no PTH on Edman degradation, it was considered to be the amino-terminal peptide. On the other hand, peptide K3 had no lysine residues (see Table IV) and was considered to be the carboxyl-terminal peptide.

Peptide K1 was then treated with acylamino acid-releasing enzyme or L-pyroglutamyl peptidase. Only acylamino acid- releasing enzyme attacked K1 and generated two peaks KlAl and K1A2 (Fig. 8). Both had the same amino acid composition (Table V). The amino acid sequence of peptide KlAl was determined to be QCQGK, but K1A2 resisted Edman degra- dation. These results suggest that the amino-terminal amino acid of BGIA is serine and is acetylated and that the amino terminus of K1A2 was cyclized to a pyroglutamyl residue during purification of the peptide by HPLC after releasing acetyl serine. Acetylation of the amino-terminal amino acid of K1 was confirmed by fast bombardment mass spectrometry (data not shown).

When Cm-BGIA was digested with endoproteinase Asp-N followed by separation of the peptides with Asahipak ODP- 50, three peptide peaks (Dl, D2, and D3) were obtained (Fig. 6). The amino acid compositions (Table VI) and amino acid sequences of these peptides, together with data on .the amino acid sequences of the peptides obtained by lysyl endopeptidase digestion, indicate that in BGIA these peptides are in the order of D3, Dl, and D2. Acylamino acid-releasing enzyme did not attack peptide D3, probably because the peptide is too long for action of the enzyme.

To confirm the alignment of peptides described above, Cm- BGIA was then digested with thermolysin. Several peptides were obtained (Fig. 7), and the amino acid compositions were determined (Table VI1 and VIII). Of these peptides, TH9 and TH12 were sequenced. Peptide TH12 was treated with acy- lamino acid-releasing enzyme and the amino acid composition (Table IX) and amino acid sequence of the resultant peptide (Fig. 9) were determined. The results obtained by thermolysin digestion confirmed data obtained by digestion with lysyl endopeptidase and endoproteinase Asp-N.

DISCUSSION

We attempted to obtain a protein inhibitor of acidic amino acid-specific endopeptidases using Glu S. griseus protease as a representative of these endopeptidases. We purified an inhibitor, BGIA, from seeds of the bitter gourd, Momordica charantia. BGIA forms an equimolar complex with Glu S. griseus protease and competitively inhibits protease activity with a K, value of about 70 nM. However, two other acidic amino acid-specific endopeptidases examined, protease V8 from Staphylococcus aureus and Glu B. subtilis protease from

Plant Inhibitor against Glu-specific Protease

1 10 20 30 4 0 5 0 60 6 8

16717

B G I A

PC I

EGLIN-C

SCI-2

SCI-1

EVS

E FE

TEFGS

SKKPEGVNTGAGDRHN

YPEPTEGSIGASG

FIG. 10. Comparison of the amino acid sequences of five inhibitors. Enclosing lines indicate residues identical with the amino acid sequence of the five inhibitors. PCI, potato chymotrypsin inhibitor I; EGLIN-C, inhibitor from leech; SCI-I, subtilisin-chymotrypsin inhibitor CI-1 from barley; SCI-2, subtilisin-chymotrypsin inhibitor CI-2 from barley. Gaps are introduced into the sequences for maximal alignment of the five inhibitors. Stars indicate the putative reactive sites of the inhibitors.

B. subtilis, were not inhibited (Table 11). The substrate spec- ificity of Glu s. griseus protease differs from those of protease V8 and Glu B. subtilis protease. Glu S. griseus protease can hydrolyze peptides containing aspartic acid at the P1 position as well as those containing glutamic acid, whereas the sub- strate specificity of protease V8 and Glu B. subtilis protease is stricter, and peptides with aspartic acid are extremely poor substrates for these enzymes (Niidome et al., 1990). Among peptides with glutamic acid at the P1 position, Glu S. griseus protease favors a peptide with proline at the P2 position, but for protease V8 and Glu B. subtilis protease a peptide with leucine at this position is the best substrate (Niidome et al., 1990). Moreover, Glu S. griseus protease is inhibited by bovine serum a1-antitrypsin, but the two proteases are not.3 These data suggest that the structure around the active site of Glu S. griseus protease clearly differs from those of protease V8 and Glu B. subtilis protease, and that this structural difference might explain the difference in susceptibility to BGIA among the three acidic amino acid-specific endopeptidases.

Although BGIA has no inhibitory activity against trypsin, chymotrypsin, and pancreatic elastase, it strongly inhibits the activity of subtilisin Carlsberg with an inhibition constant on the order of 0.1 nM, much lower than that for Glu S. griseus protease (Table 11). Trypsin, chymotrypsin, and pancreatic elastase demonstrate strict substrate specificities and have narrow pockets for substrates at their active sites, whereas the specificity of subtilisin is broad and its active site cleft is rather wide (Kraut, 1971). It is, therefore, likely that the binding site of BGIA is too bulky to form a stable complex with trypsin, chymotrypsin, and pancreatic elastase, but that BGIA can form an effective complex with subtilisin. At least two possibilities can explain why BGIA can inhibit the two different proteases Glu S. griseus protease and subtilisin. BGIA may interact with the two enzymes at a single reactive site or BGIA may have two distinct sites reacting with each enzyme. Since the activity of Glu S. griseus protease is not inhibited by the subtilisin-BGIA complex,4 the presence of two separate reactive sites, as found in the soybean Bowman- Birk inhibitor, can be ruled out.

When the primary structure of BGIA is compared with those of other proteinase inhibitors (NBRF data base, Sept. 26, 1990 release), it is similar to members of the eglin C superfamily (Fig. 10). The amino acid sequence of BGIA is about 43, 35, 31, and 31% identical with those of potato chymotrypsin inhibitor I (A chain) (Richardson and Cossins, 1975), barley subtilisin-chymotrypsin inhibitor CI-2 (Svend- son et al., 1982), CI-1 (Svendson et al., 1980), and leech eglin C (Seemuller et al., 1980). The sequences around the reactive site of these inhibitors, especially of potato chymotrypsin

’’ F. Ogata, I. Hirashiki, N. Yoshida, and A. Ito, unpublished obser- vation.

F. Ogata, N. Yoshida, and A. Ito, unpublished result.

inhibitor I (A chain) and eglin C, are highly homologous to a part of the sequence (from the 39th to 53rd residue) of BGIA. The amino acid residues (marked with a star in Fig. 10) at the putative reactive sites of these inhibitors are leucine or methionine, but the corresponding amino acid in BGIA is alanine instead of the acidic amino acids glutamic acid or aspartic acid expected from the substrate specificity of Glu S. griseus protease. However, BGIA has an aspartic acid next to the alanine residue. Since the reactive sites of BGIA for Glu S. griseus protease and subtilisin appear to be in close prox- imity as discussed above, this aspartic acid may function as a reactive site for Glu S. griseus protease. The low affinity of BGIA for Glu S. griseus protease might be due to the low activity of Glu S. griseus protease on aspartic acid, as com- pared with glutamic acid. The protease could not form a stable complex with BGIA with aspartic acid as the reactive site. Thus, BGIA appears to have multiple specificities for Glu S. griseus protease and subtilisin through interactions at adja- cent sites, as in a2-antiplasmin (Potempa et al., 1988).

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Crestfield, A. M., Moore, S., and Stein, W. H. (1963) J. Biol. Chem.

Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121,404-427 Drapeau, G. R., Boily, Y., and Houmard, J. (1972) J. Biol. Chem.

Edwards, B. F., Allen, W. R., and Barret, D. (1977) Arch. Biochem.

Ellman, G. L. (1959) Arch. Biochem. Biophys. 8 2 , 70-77 Garg, G. K., and Virupaksha, T. K. (1970a) Eur. J. Biochern. 17, 4-

Garg, G. K., and Virupaksha, T. K. (1970b) Eur. J. Biochem. 17,13-

Green, N. M., and Work, E. (1953) Biochern. J. 5 4 , 347-352 Jenne, D., Rey, C., Haefliger, J.-A., Quio, B.-Y., Groscurth, P., and

Kraut, J. (1971) in The Enzymes (Boyer, P. D., ed.) Vol. 3, pp. 547-

Laemmli, U. K. (1970) Nature 227,680-685 Niidome, T., Yoshida, N., Ogata, F., Ito, A., and Noda, K. (1990) J.

Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 6 0 ,

Potempa, J., Shieh, B.-H., and Travis, J. (1988) Science 241, 699-

Richardson, M., and Cossins, L. (1975) FEBS Lett. 5 2 , 161 Seemuller, U., Eulitz, M., Fritz, H., and Strobl, A. (1980) Hoppe-

Seyler’s Z. Physiol. Chem. 361, 1841-1846 Sleath, P. R., Hendrickson, R. C., Kronheim, S. R., March, C. J., and

Black, R. A. (1990) J. Biol. Chem. 265, 14526-14528 Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner,

F. H., Provenzzano, M. D., Fujimoto, E. K., Goeke, M. N., Olson, B. J., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85

Svendson, I., Boisen, S., and Heigaard, J. (1980) Carlsberg Res. Commun. 45, 79-85

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Tschopp, J. (1988) PFOC. Natl. A c u ~ . Sci. U. S. A. 85,4814-4818

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16718 Plant Inhibitor against Glu-specific Protease Svendson, I., Boisen, S., and Heigaard, J. (1982) Curkberg Res. Yamamoto, A., Toda, H., and Sakiyama, F. (1989) J. Biochem. (To-

Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244,4406-4412 Yoshida, N., Tsuruyama, S., Hirayama, K., Noda, K., and Makisumi, Wrigley, D. W. (1971) Methods Enzymol. 22, 559-564 S. (1988) J. Biochem. (Tokyo) 104, 451-456

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Supplemenlory motrrlol to

Purification and Amino Acid Sequence of a Bitter Gourd Inhibitor against an Acidic Amino Acid.Specifie Endopeptidase of Strepromyces gr isous

Furahira Ogata, Toshiyuki Miyata, Nobuyoshi Fujii, Norio Yorhida, Kosaku Noda, Satoru Makisurni, and Akio 110

EXPERIMF.NTAL PROCEDURES

Maler ials - Bitter gourd seeds were obtamed from local market and stored at 4°C untd use. Bovine trypsln, endoproteinase Asp-N, and chymotryprm were purchased from

Staphyiococcur aureus were from Slgma Chemlcal Co. and Mlles Laboratones Inc., Boehrmger Mannhem Yamanouch1 Co. Porcme elastase and protease v8 from

respectively. Lysyl endopeptldare from Achromobocrer Iyttcus M497-I, thermolynin, and acylamlno.ac,d-releasing enzyme were from Wsko Pure Chemfal lndustrles Co., Nacalal Tesque Inc , and Takara Shuzo Co.. respectwely. Glu S g. protease, synthetK substrates and Glu B s. protease were prepared as descrnbed prewously (Yoshida et 81.. 1988; Nildome et a l , 1990). Sepabeads FP-CM was purchased from Mitubirhn Kanei Co. Sephadex G-50 and Pharmalyte were from Pharmacm LKB Bmtechnalogy. CM-Toyopearl. Butyl-Toyopearl, and TSK-gel G2000 SWXL were the products of Tosoh C o . Asahipak ODP-50 and Asahlpak GS310 columns were from Asah1 Chemical Industry Ca., LTD. All other chemicals were of reagent grade.

In four volumes of acetone (volumeiweight) and air-dried at room temperature. The Purlflcatlon of B C l A - Bntter gourd seeds (125 g) were ground wxth an electric mixer

resultant acetone powder (100 g) was extracted overnight wlth 1% N ~ C I (900 ml) under SIOW COntlnuOus Stlrrlng at 4'C followed by centrlfugation at 6,000 x g for 30 mnn. The supernatanr was pooled as the crude entract. The crude extract war rubjccted to ammonium sulfate fractionation ( 4 0 4 0 % saturation). The precipitate was dissolved rn a mmmum volume of dimlied water and dlalyzed against dtstilled water. and msoluble matter was removed by centnfugation.

50 mM Trir-HCI buffer. pH 8 0 and eluted w t h a h e a r gradient of NaCl from 0 to 300 mM The solutmn was applled to a Sepabeadr FP-CM column (3.5 x 50 cm) equlhbrated wlth

(500ml each) m the same buffer ( R g I ) The mhlbltor fractmns were pooled. concentrated and desalted wlth a Diaflo YM2 ultrafdtrauon membrane

equhbrated wlth 50 m M Tns-HCI buffer, pH 8.0 and eluted w t h a linear gradienr of NaCl The desalted fraction was then applied to B CM-Toyopearl 650M column (2.1 x 42 cm)

from 0 to 300 mM (500ml each) In the same buffer. The inhlbltor fracoon was brought to

cm) equlllbrarcd with 20 mM Tris-HCI buffer. pH 7.5. containmg 40% saturation of 40% S L I I U I I ~ I O D of ammonium sulfate and applned to B Butyl-Toyopearl column (1 5 x 13

ammomum sulfate The mhlbltor was eluted wlth a h e a r gradient of ammomum sulfate from 40% ID 0% raturatmn m the same buffer (300ml each). The inhibitory actwity war eluted as two peaks and the first large fraction was pooled, concentrated and desalted as described above. The concentrated fraction was fmally applied to il S e p h a d e ~ G - 5 0 column ( 2 6 x 100 cm) eqwllbratcd with 20 mM Tris-HCI buffer, pH 7 5 (Fig. 2). The inhibitor (BGIA) fractlonr were pooled. lyophilized after desalting and stored at -20°C until we.

Assay of l n h b i l n r y A c t w r y - The inhibitory act~vnty of BGlA was estimated from the residual activity of Glu S g. protease in the mmture of mhlbltor and the protease. After preincubation of the mixture of Glu S a. protease (about 100 m units) and inhibitor ~n 1.9 ml of 0 I M Tris-HCI buffer. pH 8.8. contamng 5 mM CaC12 at 3 7 T for 5 mi". 0.1 ml of 2

mubation for 10 mm at 37°C. the reactton was terminated by adding 1.0 rnl of 30% acetie mM Boc-Ala-Pro-Glu-pNA in dunethyl sulfoxide was added to start the rcactmn. After

acid and the resldual activity was measured ~n terms of the absorbance at 410 nm One unit of thc mhlbitary actwtty for Glu S g. protease was defined as the amount of inhlbmr which suppressed the liberatmn of I p m o l e of p-nltroanilme per min by the actlye enzyme

Assay for mhtbltory actlwty agalnsr other proteases was done as essentlally the same as described above. The concentrations of BGIA and proteases were adjusted to 140 nM and 20 nM. respectively. Boe-Ala-Ala-Leu-Glu-pNA. Boc-Thr-Leu-Leu-pNA. Bz-Arg-pNA. Suc- Phe-Pro-Phe-pNA, and Suc-Ala-Ala-Ala-pNA were used as the substrate for protease V8 and Glu 8 s protease. subtilism Carlsberg. trypsin and papain, chymotrypsm. and porclne pancreauc elastase. respectively The buffers used were 0.1 M phosphate buffer. pH 7.8

B I protease, 0.1 M T~IS-HCI buffer. pH, contaming 10 mM CaC12 for trypsin and for protease v8. 0 I M Tris-HCI buffer. pH 8.0. contamng 5 mM CaC12 for subolirin and Glu

chymorrypsm. 0.05 M Tris-HCI buffer, pH 7 5, contaming 5 mM L-cysteine and 2 mM EDTA for papain. and 0.2 M tnethanolamme-NaOH buffer. pH 8.0, for porcine pancreatic elastase.

Analysts of Complex Formol lon - After pretncubation of Glu S g. protease (2.7 nmol) wlth two-fold molar excess of the purified BGlA for 5 m m at room temperature in 0.12 ml of 0.1 M TrmHCl buffer, pH 8.8, contatnmg 5 mM CaCll and 0 I M NazSO4, the mixture (0.1 ml) was subjected to gel filtrauon HPLC usmg a Asahipak GS310 column (7.5 x 500 mm) equilibrated wlth the same buffer. BGIA and Glu S g. protease were also applied separately to the same column.

R e d u c t i o n a n d C o r b o x y m e t h y l o t i o n of B G l A - BGlA was reduced with 2- mercaptoethanol and S-carboxymethylated ( C m ~ ) by the method of Crestfield et ai. (1963). The reaction mixture was desalted by parsmg through a reversed-phase chromatography using Asahipak ODP-50 column.

Eniymotzc Cleovoge of BClA - Cm-BGIA (90 mg. 12 nmol) was digested with lysyl endopeptidase (0.2 mg) in 50 mM Tris-HCI buffer. pH 9.0. containing 4 M urea at 37'C for 30 min. Cm-BGIA ( 1 0 0 mg. 13 nmol) was also digested with cndopeptidasc Asp-N (1 mg) ~n 50 mM phosphate buffer. pH 8.0, at 37'C for 8 h. Thennolysin (1 mg) digestton of Cm- BGlA (180 mg. 24 nmol) war carried out in 0.2 M N-erhylmorphallne-acctate buffer. pH 8.0, contatnmg I mM CaClz at 37°C for 2 h. Treatmcnt of pepndes. K1 (8.1 nmol) and THl2

done in 10 mM phosphate buffer. pH 7.2. containing 1 mM 2mercaptoethanol at 37°C for (11.5 nmol). wlth acylammo acid-releasing enzyrnc (0.05 and 0,037511. respecouely) were

42.5 h

enzymatic dlgcstion of BGlA were purified on a Hmchi HPLC apparatus (L-6200 intelligent lsolotion of Peprtdrs offer D ~ g e s r i o n of E G l A - Peptide mixtures resulting from

pump. L-4000 UV detector. and D-2000 Chromatointegrator) by use of a Arahlpak ODS-50 column (6 x 150 mm). Solvent A was 0.05% trifluoroaeetic acid in water. and solvent B was 0.05% trifluoroacctic acid I" 80% acetonitrile. The elutmn was run at a flaw rate of 0.8 mllmm at 25°C. Peptides were eluted from the column with a linear gradlent of solvent B (0.50%. O.K%/min) followed by second h e a r gradlent (50-100%. 7.1%/min). Absorbance at 215 nm was measured and peptides were collected manually. concentrated. I f necessary, and used far sequence analysis. Peptides were numbered according to the order of elution from a HPLC column. Peptides derived from lysyl endopeptidase, endoprotemasc Asp-N,

thermolysin. and acylamino acid-releasmg enzyme digestions were deslgnated as K, D, TH. and A, respectively.

performed with a Yanagimoto LC-8A automatic amino acid analyzer after hydrolysis with Amino Acld ond Sequence Analyses - Ammo acid analysts of native BGlA was

5.7 M HCI at llO°C for 24. 48. and 72 h. Tryptophan was determmcd by hydrolysis in 4 M mercaptocthanesulfonic acid (Penke et al.. 1974).

Amino acid analysis of peptldes was done with a Waters PICO-TAG system after hydrolysis with 5.7 M HCI at l l0"C for 20 h. Amino acid sequences of peptides were analyzed in an Applied Bmsystemr model 477A gas-phase sequencer with an Applied Biosystems model 120A PTH analyzer.

hydrazinalysis according to the method of Yamamoto et al. (1989). The carbory-terminal amino acld residue of BGlA was determined by vapor-phase

polyacrylamide gcl at pH 6.6 (Davis. 1964). SDS-PAGE in 15% gcl in the presence or Other Anolyrmrl Procedures - Published procedures were used for natlve PAGE ID 15%

absence of 2-mercaptocthanol (Weber and Osborn. 1969) or I" 15% gel in the presence of 8 M urea (Laemmli. 1970). The disc gel electrofocusing was performed at 4'C in 7.5% gel contaimng 2% of pH 3-10 range Pharmalytc (Wrigley. 1971). Protcins in the gel were stained with Coornarie Brilliant Blue G-250.

buffer was 0.1 M Tris-HCI buffer, pH 7.5, containing 0.1 M Na2S04. Bovinc serum albumin The gel filtration was carried out by HPLC on a TSKgel G2000SWx~ column. Thc runnmg

(Mr. 68.000). ovalbumin (Mr.45.000). carbomc anhydrase (Mr. 29.000). cytochrome c (Mr.

molecular weight standard. 12,400). aprotinin (Mr. 6,500). and bovme insulin B cham (Mr. 3.500) were used as the

Protein was measured wnh BCA method (Smith et a l , 1985) using bovine Serum albumin as the standard. Protem content of each fracuon from column chromatography was estimated from the absorbance at 280 nm 01 215 nm

P"rlflCafiO" Of BGIA from acetone powder 1100 g) of bitter gourd seeds Table I

Total Total Specific step Protein Activitya Activity ~ecovery Purification

( m 9 ) I I U I IIUlm91 l % l (fold)

Crude extract 9857 125 0.013 100 INH412S04

1 2198 89.3 0.041 71

Sepabeadn FP-CM 192 3

CM-Toyopearl 57.1 73.9 1.30 77.6 0.404 62.1 31

Butyl-Toyopearl 21.3 57.5 2.70 59.1 100

Sephadex G-50 16.5 46.9 2.85 17.7 208 37.5 219

Suppressed the liberatim of 1 y m l a r p-nitroanillne per by ~ 1 " inhibitor Unit (1 IUI was defrned as the amount of lnhibitor which

5. 9. protease.

Table 111

Values axe taken from average value of 24. 48. and 72 h hydrolysates and values in parentheses

are the nearest inteqer5.

Amino acid conpasition of BGIA

GlY 5.38 i 6 i 6 *la 7.50 18) 8

2 112cysb 2.49 I21 V a l 11.3OC1111 11 net I 0 1 I le Le"

3.5OC 1 4 1

Ty= 0.76 I l l

Phe 101

0.76 I l l

." -_ hydrolyals wrth

nic acid

Plant Inhibitor against Glu-specific Protease 16719

Amino acid compositions of pptides Obtained from Cm-BGIA by IYWI endowmxdase disestiona

Table 1V

Amino acid compositions of peptides obpined from Cm-BGIA by Table V I 1 1

themlvsin diaestion AA K l K2 K3 Totel

A.A. TI11 I TI112 TI114 TI118

IResidueslmoleculo) 1.42 1 1 ) 3.08 ( 2 1

2.95 1 3 1

CY% V a l 0.57 ( 1 ) 0.60 11) 9.15(11) Met

LC" I le 0.55 ( 1 ) 1.53 12) 2.90 ( 4 )

Tyr 1.01 ( I )

Phc LYS

1.00 ( 1 ) 1.00 ( 1 )

1115 2.16 ( 2 )

TrP n.d.'=(l) A T 9

n.d. ( 2 ) 1.03 1 1 ) 0.99 ( 1 ) 1.00 1 1 ) 10.0 (101

Total 7 I 1 9 68

Position 60-66 1-11 60-68 1-68

*lues in parentheses a r ~ from the sequence data. Corboxymethylcysteine. Not determined.

POsltiOn 1-6 7-21 22-68

AA TIU 2 Tl112A1 V112A7

1 . 4 2

1.67 2.82 1.03 0.91

&A K 1 KLAI RIA2

Lys 1.00 ( 1 ) 1.00 ( 1 ) 1.00 1 1 1

Total 6 5 5

Position 1-6 2-6 2-6

Amno acid ComwuSitionS of peptides Table VI

Obtained from Cm-8GlA bv endooroteinasc AS.-N'

AA D l D2 03 Total

ser 3.68 1 4 1 4 1.00 ( 1 1 4.81 i 5 i

2.04 ( 2 ) 3.90 ( 4 ) 2.10 ( 2 ) 3.27 0 )

7.10 ( 2 ) 5.71 ( 6 1

6 5 6 8 0

11 0 4 1 0 1 2 0 2 68 10

1.00 ( 1 )

n.d.ctl) 3.50 ( 4 )

120) FIG. I . CM-Toyopearl 650M Column Chromatography o t the Inhibitor-rich

described in EXPERIMENTAL PROCEDURES. Flow raw. 19 mllh: fraction volume. IO Fracl ion obtained by Sepabeads FP-CM Column Chromatography. Dctails arc

ml:-. absorbance at 280 nm; --*---. Glu S. 8. protease-inhibitory acuvily: "-A"- . vyprin inhlbilory activity: "x-, clectric conductivity.

Position 15-18 19-68 1-44

r 1 1 2 3 , 1 .o

E 0

N m

8 0.5

f! n a

C

u)

0

Amino acid compositions Of peptides Obtalned from CaBGIA by themlysin digestlona Table V I 1

I. n,, *"I T". TU5 T,46 n19 1 ii -

1.00 1 1 ) 1.00 1 1 )

Fraction No.

FIG. 2 Sephadex C-50 Column Chromatography of the inhibitor Fraction Obtained by Butyl-Toyopearl Column Chromatography. -, Absorbance at 280 nm:.-.*--. Glu S. 8 prolease-inhibitory activily. The

Scphadcx G-50 column chromatography was electrophoresed a1 pH 6.6 and a current of I O inset shows the rcsult of native PAGE a1 15% acrylamnde gel. The inhibitor obtained by

mA. Lane I ( 5 118). lane 2 (10 @E). lane 3 (15 Pg).

16720 Plant Inhibitor against Glu-specific Protease

0.04 t A

Elution Time ( min)

FIG. 3. Complex Formation Analyses of BGlA and Glu S . g. Protease by Gel

EXPERIMENTAL PROCEDURES. A. Glu S g. prolease (1 37 nmol): B. BGlA (5.42 nmol): C. Ihe Filtration Using a Asahipak GS 310 Column for HPLC. The details are desertbed I"

mixture of BGlA (5.42 "mol) and Giu S. g protease (2.7 mol) .

0 3

Elution T i m e l m i n )

FIG. 6 . Purification of Endoproteinasc Asp-N Peptides by Reversed-phase HPLC with a Gradient of Acetonitrile in 0.05% Trifluoroacetic Acid.

1 K 3

I 1 0 2 0 4 0 50

E l u t i o n Tirnc (mi")

FIG. 5 . Purification of Lysyl Endopeptidase Peptides by Reversed-phase HPLC with a Gradienl of Acetonitrile in 0.05% Trifluaroacetie acid. The expcrimental conditions arc descnbed m EXPERIMENTAL PROCEDURES.

TI

TI

T H l Z

THI

16

TH18

FIG. 7. Purification of Thermolysin Peptides by Reversed-phase HPLC with a Gradient of Acetonitrile in 0.05% Trinuoroacet ic Acid.

0.0

0

Plant Inhibitor against Glu-specific Protease

K l A l

KIA2

2 0 4 0

E l u t i o n T ~ m e (mi")

E l u t i o n T i m e ( r n i n ]

FIG. 9. Purification of Aeylamino-Aeid-Releasing Enzyme Peptides of THlZ

Trifluoroacelir Acid. Peptide by Reversed-phase HPLC with a Gradient of Acetonitrile in 0.05%

16721

FIG. 8. Purification of Acylamino-Aeid-Releasing Enzyme Peptides of K1 peptide

Trinuoroacelie Acid. by R e r e r s e d . p h s s e H P L C w i t h B G r a d i e n t o f A c e t o n i t r i l e i n 0.05%