J. Biochem. 93, 1129-1135 (1983)

Use of New Synthetic Substrates for Assays of Cathepsin L

and Cathepsin B 1

Nobuhiko KATUNUMA,* Takae TOWATARI,* Masaharu TAMAI,** and Kazunori HANADA**

*Department of Enzyme Chemistry , Institute for Enzyme Research, School of Medicine, The University of Tokushima, Tokushima, Tokushima 770, and **Research Center of Taisho Pharmaceutical Co., Ltd., Yoshinocho, Ohmiya, Saitama 330

Received for publication, November 27, 1982

Efficient methods were developed for synthesizing synthetic substrates for assays of cathepsin B and cathepsin L. Several 2-naphthylamide compounds with a

blocked NHS;-terminus, Suc-Tyr-Met-NA, ƒÀ-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met- NA, were specific and sensitive substrates for cathepsin L and cathepsin B; they were not specific for cathepsin L only, because all of them were also hydrolyzed

by cathepsin B. Some kinetic constants for the hydrolyses of these three synthetic substrates by cathepsin B and cathepsin L are given.

Cathepsin B [EC 3.4.22.1], cathepsin L [EC 3.4. (8). Cathepsin H is also assayed with BANA 22.-], and cathepsin H [EC 3.4.22.-] are lysosomal (9, 10), but with this substrate it is not possible thiol proteases found in the tissues of many ani to distinguish clearly between cathepsin B and mals (1-4), where they are considered to be impor cathepsin H. Direct fluorimetric assays based on tant in intracellular protein degradation. Cathep the use of Z-Phe-Arg-MCA for cathepsin B and sin B has normally been assayed with either BA-PA Arg-MCA for cathepsin H have also been devel or BANA as a substrate (5-7), but recently, oped (11). However, cathepsin L has little activity McDonald and Ellis reported that Z-Arg-Arg-NA toward synthetic substrates, although there is was a better substrate than BANA for this enzyme strong evidence that it has an important physio- logical role (2, 3). 1This work was supported by a Grant-in-Aid for For detailed studies on the biological prop Scientific Research (No. 548370) from the Ministry of erties of these proteases, specific assay methods Education, Science and Culture of Japan. are required for these proteases in various tissues Abbreviations: Symbols for amino acid residues and extracts. protecting groups are according to the IUPAC-IUB Our data have shown that cathepsin L cleaves Commission [J. Biol. Chem. 241, 2491 (1966)]; Suc, succinyl; TfaOH, trifluoroacetic acid; NA, 2-naphthyl peptide bonds with an amino acid residue such as amide; MCA, methyl coumarylamide; Boc, butyl Leu, Phe, or Tyr in position P,; for example, it oxycarbonyl; Z, benzyloxycarbonyl; BANA, ƒ¿-N- cleaved the bond between Met and Arg in the benzyol-DL-arginine-2-naphthylamide; BAPA, ƒ¿-N- synthetic hexapeptide, Leu-Trp-Met-Arg-Phe-Ala benzoyl-DL-arginine p-nitroanilide. (Towatari, T. & Katunuma, N., manuscript in

Vol. 93, No. 4, 1983 1129 1130 N. KATUNUMA, T. TOWATARI, M. TAMAI, and K. HANADA preparation). Thus it should be possible to de physicochemical properties are summarized in velop new specific synthetic substrates with an Tables I and ‡U. Except where otherwise noted, appropriate amino acid sequence for assay of all the derivatives used in this study contained cathepsin L. This paper describes studies on the L-amino acids.

substrate specificities of purified cathepsin L, ca Assays of Cathepsin L, Cathepsin B, and thepsin B and cathepsin H on a wide variety of Cathepsin H-The activities of cathepsin L, ca synthetic 2-naphthylamide substrates. Of these thepsin B, and cathepsin H were assayed at pH substrates, Suc-Tyr-Met-NA, ƒÀ-Ala-Tyr-Met-NA, 6.0 or pH 5.0 with BANA or the new peptide 2- and D-Leu-Tyr-Met-NA were the best for specific naphthylamide substrates as described by Barrett assay of cathepsin B and cathepsin L in various (6). The incubation buffer was 0.1 M potassium tissue extracts. phosphate or sodium acetate of appropriate pH containing 0.2 % Brij, 1 mm EDTA, and 2 mm cysteine. Routinely, cathepsin L, cathepsin B, MATERIALS AND METHODS and cathepsin H were assayed with 5 mm BANA. Animals-Male Wistar-strain rats of 200 g The new peptide 2-naphthylamide substrates were body weight were used. used at 1 mm or 2 mm and Z-Arg-Arg-NA at 0.2 Chemicals-Cathepsin B and cathepsin L were mm. All assays were carried out at 37"C after prepared from rat liver as described previously preincubation for 5 min with the substrate. (12, 3). Cathepsin H was purified as described Determination of Protein-Protein concen by Schwartz and Barrett (13). BANA and Z- tration was determined by the method of Lowry Arg-Arg-NA were supplied by Sigma Chemical et al. (18) with bovine serum albumin as a standard Co. and Bachem Feinchemikalien, respectively. or by measurement of A280. All other chemicals were of analytical grade and were obtained from Wako Pure Chemical Industry RESULTS (Tokyo) or Sigma Chemical Co. Syntheses of Peptide 2-Naphthylamide Sub Specifrcities of Cathepsin L, Cathepsin B, and strates-All peptide 2-naphthylamide substrates Cathepsin H from Rat Liver on Peptide 2-Naph except BANA and Z-Arg-Arg-NA were synthesized thylamide Substrates-Table ‡T shows the absolute

as follows. The substrates newly obtained in this and relative rates of cleavage at pH 5.0 of the study were prepared by the stepwise elongation 2-naphthylamide bond in various peptide sub method in solution using 2-naphthylamine as the strates by cathepsin L, cathepsin B, and cathep

starting material. All amino groups, the guanidyl sin H. These rates were compared with those

group of Arg and the ƒÀ-carboxyl group of Asp on BANA, the arylamide substrate usually used were protected with Boc, nitro and benzyl groups, for cathepsin B. Cathepsin L hydrolyzed 2- respectively. The hydroxyl group of Ser was pro naphthylamide substrates containing amino acids

tected with a t-butyl or benzyl group. The coupling such as Phe, Leu, Trp, and Tyr in position P2, reactions were carried out by the standard mixed as shown by the selective action of cathepsin L anhydride method or by the dicyclohexylcarbodi on various polypeptide substrates, such as syn

imide-N-hydroxysuccinimide method (14). The thetic hexapeptide, luteinizing hormone releasing hormone, neurotensin, and insulin A chain (Towa protecting groups of the amino and hydroxyl tari, T. & Katunuma, N., manuscript in prepara groups were removed by formic acid or TfaOH treatment. The deprotection of the guanidyl tion). For example, the rate of hydrolysis of

group, the methylation of Tyr and succinylation Leu-Met-NA was 6.8 times greater than that of the amino group were carried out according to for BANA, and the rates for Phe-Met-NA, Tyr- the methods of Yajima et al. (15), Neeman and Met-NA, and Try-Met-NA were 6.5 times, 2.6

Hashimoto (16), and Nakajima et al. (17), respec times, and 1.6 times, respectively, greater than tively. that for BANA. Surprisingly, however, the rate The purities of all the compounds were of hydrolysis of Ile-Met-NA was the same as that checked by thin layer chromatography and the of BANA. Cathepsin B did not enhance the identities were established by PMR analysis. The rates of hydrolysis of these 2-naphthylamide sub-

J. Biochem. SYNTHETIC SUBSTRATES FOR CATHEPSINS L AND B 1131

TABLE ‡T. Substrate specificities of cathepsin L, cathepsin B, and cathepsin H from rat liver on peptide 2-naphthyl amide substrates. Activities were measured in 0.1 M acetate buffer, pH 5.0, as described in "" MATERIALS AND

METHODS." BANA was used at 5 mm and other substrates at 1 mm. Activities were calculated as f

strates. However, when the amino acid residue a specific substrate for cathepsin L and cathepsin at the Pl position was Ser, as in Leu-Ser-NA, B, it is necessary to protect the substrate from cathepsin B caused marked hydrolysis. The ratio attack by intracellular aminopeptidases. The of the activity with cathepsin L to that with ca amino-terminus of Tyr-Met-NA was substituted thepsin B was highest with Tyr-Met-NA among with succinate, ƒÀ-Ala or D-Leu and the rates of the 2-naphthylamide substrates examined. Only hydrolysis of these NH.-blocked substrates by the rate of hydrolysis of Met-NA by cathepsin H cathepsin L, cathepsin B, and cathepsin H were was comparable to that of BANA. To develop compared with those of BANA and Z-Arg-Arg-

Vol. 93, No. 4, 1983 1132 N. KATUNUMA, T. TOWATARI, M. TAMAI, and K. HANADA

NA (Table ‡U). Cathepsin L and cathepsin B 2-naphthylamide substrates to cathepsin H were

showed enhanced rates of hydrolysis of the NH2- not greater than that of BANA. blocked substrates. Cathepsin B could also hy Kinetic Constants for Hydrolysis of NH2- drolyze these NH2-blocked substrates at rates Terminus Blocked 2-Naphthylamide Substrates-

comparable to that with cathepsin L. Therefore, The kinetic constants of rat liver cathepsin B and these NH2-blocked substrates can be used as spe cathepsin L for hydrolyses of the NHS terminus cific substrates for assays of cathepsin L and blocked 2-naphthylamide substrates are shown in

cathepsin B. However, the sensitivities of these Table ‡V. The Km values were calculated from

TABLE ‡U. Activities of cathepsin B, cathepsin L, and cathepsin H from rat liver on various amino-terminus- blocked peptide 2-naphthylamide substrates. Activities were measured in 0.1 M potassium phosphate buffer, pH

6.0, as described in " MATERIALS AND METHODS." Z-Arg-Arg-NA was added at 0.2 mm BANA at 5 mM, and other substrates at 2 mm. Activities were calculated as mol of substrate hydrolyzed (min-1•Emg protein-1).

Rates relative to that with BANA are shown in parentheses.

TABLE ‡V. Kinetic constants for hydrolysis of substrates by cathepsin B and cathepsin L. Reactions were carried

out at 37•Ž in 0.1 M potassium phosphate buffer, pH 6.0, as described in" MATERIALS AND METHODS." The concentrations of rat liver cathepsin B and cathepsin L were 2 jig and 0.82 jig, respectively. The kinetic parameters

are defined by the equation

where Io=initial velocity, V=maximum velocity, [S]o=initial substrate concentration, Km=Michaelis constant= (k-1+kcat)/k, E=enzyme, P=hydrolytic products. The values for kcat were calculated from V[E]. [E]=enzyme concentration.

a The data with this substrate,'containing DL-Arg, are corrected for the L-form.

J. Biochem. SYNTHETIC SUBSTRATES FOR CATHEPSINS L AND B 1133

Lineweaver-Burk plots with various concentrations and lysosomal fraction from rat liver was sub of substrates. The kcat values were calculated jected to three cycles of freezing and thawing. from the V values and enzyme concentrations, Then the mixture was centrifuged at 100,000 •~ g taking the molecular weight of rat liver cathepsin for 30 min and the supernatant was passed through B as 29,000 and that of rat liver cathepsin L as a Sephadex G-25 column (6 •~ 20 cm) equilibrated 23,000. Values of kcat/Km showed that rat liver with 20 mm acetate buffer, pH 5.0, containing 50 cathepsin B hydrolyzed Suc-Tyr-Met-NA, ƒÀ-Ala-

Tyr-Met-NA, and D-Leu-Tyr-Met-NA more effi ciently than BANA and that rat liver cathepsin L also hydrolyzed those synthetic substrates pre-

ferentially. Effects of pH on Rates of Suc-Tyr-Met-NA

Hydrolysis by Cathepsin B and Cathepsin L-As shown in Fig. 1, rat liver cathepsin B has a pH optimum of about 6.0 on Suc-Tyr-Met-NA, as on

BAPA (12). Cathepsin L has a pH optimum of 5.5-5.7 on Suc-Tyr-Met-NA, which is the same as that for its inactivation of G6PD (3). Paper chromatographic analysis of the reaction products

of Suc-Tyr-Met-NA and ƒÀ-Ala-Tyr-Met-NA showed that free 2-naphthylamide is cleaved from these 2-naphthylamide substrates (data not shown). Use of the NH2-Terminus-Blocked Peptide 2-

Naphthylamide Substrates Suc-Tyr-Met-NA, ƒÀ-Ala- Tyr-Met-NA, and D-Leu-Tyr-Met-NA for Assays of Cathepsin B and Cathepsin L in Crude Tissue

Extracts-A suspension of crude mitochondrial

Fig. 2. Separation of cathepsin H, cathepsin B, and

cathepsin L on CM-Sephadex C-50. A crude mito

chondrial and lysosomal lysate of rat liver (300 g) was

applied to a CM-Sephadex column (3.8 •~15 cm) which

had been equilibrated with 20 mm acetate buffer, pH

5.0, containing 50 mm sodium chloride, 1 mM EDTA,

and 5 mm 2-mercaptoethanol. The column was washed

Fig. 1. Effect of pH on the rate of Suc-Tyr-Met-2- sufficiently with the same buffer and then material was naphthylamide hydrolysis by cathepsin L and cathep eluted stepwise with 0.1 M and 0.5 M sodium chloride sin B. Reaction mixtures contained 2 ƒÊmol of Suc- in 20 mm acetate buffer, pH 5.0, containing 1 mm

Tyr-Met-NA, 1 ƒÊmol of EDTA, 2 ƒÊmol of cysteine, EDTA and 5 mm 2-mercaptoethanol. Fractions of 0.2% of Brij, and 2 ƒÊg of cathepsin L (•œ) or cathepsin 12.8 ml were collected and their enzymatic activities B (•›) in 1 ml of 0.1 M acetate buffer (below pH 5.5) were measured. A: Locations of BANA (•£) and or 0.1 M phosphate buffer (pH 5.7 to 7.5). All reac Z-Arg-Arg-NA (•¢) hydrolyzing activities. B: Locations tion mixtures were incubated for 10 min at 37•Ž as of Suc-Tyr-Met-NA (•›), ƒÀ-Ala-Tyr-Met-NA (•œ), and described under " MATERIALS AND METHODS." D-Leu-Tyr-Met-NA (•¡) hydrolyzing activities.

Vol. 93, No. 4, 1983 1134 N. KATUNUMA, T. TOWATARI, M. TAMAI, and K. HANADA mm sodium chloride, 1 m vt EDTA, and 5 mm bonds by cathepsin L and cathepsin B with hex

2-mercaptoethanol. This crude enzyme solution apeptide, luteinizing hormone releasing hormone, was then applied to a CM-Sephadex C-50 column neurotensin, and oxidized insulin A chain as model

(3.8 •~ 15 cm) equilibrated with 20 mm acetate substrates (Towatari, T. & Katunuma, N., manu buffer, pH 5.0, containing 50 mm sodium chloride, script in preparation). The results showed that 1 mm EDTA, and 5 mm 2-mercaptoethanol. The cathepsin L cleaves peptide bonds with an amino column was washed with the same buffer and then acid residue such as Leu, Phe, Trp, or Tyr in material was eluted stepwise with 0.1 M and 0.5 M position P2. These results are consistent with sodium chloride in 20 mm acetate buffer, pH 5.0, those of Kargel et al. (19). On the basis of these containing 1 mm EDTA and 5 mm 2-mercapto results, we studied whether various synthetic pep-

ethanol. Cathepsin H was eluted with 50 mm tide 2-naphthylamide substrates with a hydro- sodium chloride, cathepsin B with 0.1 M sodium phobic amino acid in position P2 are suitable for chloride and cathepsin L with 0.5 M sodium chlo use in assays of cathepsin L, cathepsin B, and

ride. Figure 2 shows the locations of cathepsin cathepsin H. As the synthetic peptide 2-naphthyl H, cathepsin B, and cathepsin L during chro amide substrates used in this study were synthe matography. The fractions with activities of sized after selective cleavage of the hexapeptide cathepsin H, cathepsin B, and cathepsin L were Leu-Trp-Met-Arg-Phe-Ala, the penultimate residue

each pooled. was methionine in almost all of them. Purified The proportions of activities toward these cathepsin L hydrolyzed peptide 2-naphthylamide NH.-terminus blocked 2-naphthylamide substrates substrates containing amino acids such as Phe, in the different fractions were determined. The Leu, and Tyr in position P2, as shown with model results showed that 26% of the total BANA polypeptide substrates except for that containing hydrolyzing activity and 7 % of the Z-Arg-Arg- Ile (Table I). Although the relative rates of NA hydrolyzing activity were due to cathepsin H cleavage of these substrates by cathepsin L were and the remaining activities to cathepsin L and greater than those by cathepsin B, cathepsin B cathepsin B. Moreover, 92% of the total Suc- also hydrolyzes these substrates slightly. How-

Tyr-Met-NA hydrolyzing activity, 92% of that ever, Ser-Met-NA was hydrolyzed by both rat for ƒÀ-Ala-Tyr-Met-NA and 97;0 of that for D- liver and bovine spleen cathepsin C at an excep

Leu-Tyr-Met-NA were due to cathepsin B and tionally high rate (20). Thus, when these amino cathepsin L, and 8.5 •~ 10 M E-64 inhibited these acid 2-naphthylamide substrates are used for assays

hydrolyzing activities toward Suc-Tyr-Met-NA, of cathepsin L and cathepsin B in crude tissue ƒÀ-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met-NA com extracts, their amino-terminus should be blocked.

pletely. Experiments using rat kidney as an en When the amino-terminus of Tyr-Met-NA zyme source gave similar results (data not shown). was substituted with succinate, ƒÀ-alanine or D-

These results show that the new 2-naphthylamide leucine (as shown in Table II), cathepsin L and substrates with a blocked NH2-terminus, Suc-Tyr- cathepsin B showed enhanced rates of hydrolysis.

Met-NA, ƒÀ-Ala-Tyr-Met-NA, and D-Leu-Tyr-Met- Thus it seems impossible to predict the rate of NA, are very useful for assays of both cathepsin attack on synthetic substrates by cathepsin B from

B and cathepsin L in crude preparations. studies on the hydrolyses of polypeptides, though the hydrolyses of polypeptides and synthetic sub

DISCUSSION strates by trypsin and chymotrypsin correlate well. Recently Barrett and Kirschke pointed out For detailed studies on the biological properties that Z-Phe-Arg-MCA is very sensitive to cathepsin of cathepsin L, cathepsin B, and cathepsin H, a L but it is not completely specific for cathepsin L specific and simple method is required for assay- because it is also hydrolyzed by cathepsin B (21). ing the activities in various tissue extracts. In Z-Phe-Arg-MCA gives a higher value of kcat/Km particular, a specific substrate for cathepsin L is than our synthetic substrates and shows no spon required, since this enzyme has very little activity taneous hydrolysis. A major disadvantage of the on usual synthetic substrates. use of our synthetic substrates is spontaneous We studied the selective cleavages of peptide hydrolysis. When we used our synthetic 2-naph-

J. Biochem. SYNTHETIC SUBSTRATES FOR CATHEPSINS L AND B 1135

thylamide substrates with a blocked amino-ter- 6. Barrett, A.J. (1972) Anal. Biochem. 47, 280-293 minus for assays of cathepsin B and cathepsin 7. Barrett, A.J. (1976) Anal. Biochem. 76, 374-376 L in a liver extract, more than 90% of the total 8. McDonald, J.K. & Ellis, S. (1975) Life Sci. 17, activities for their hydrolyses was due to cathepsin 1269-1276 9. Jarvinen, M. & Hopsu-Havu, U.K. (1975) Acta B and cathepsin L, compared with 74%. for BANA. Chem. Scand. Ser. 29, 772-780 From these results we conclude that it is very 10. Singh, H. & Kalnitsky, G. (1978) J. Biol. Chem. difficult to find a specific synthetic substrate for 253,4319-4326 cathepsin L in crude enzyme sources, but that the 11. Barrett, A.J. (1980) Biochem. J. 187, 909-912 2-naphthylamide substrates with a blocked amino- 12. Towatari, T., Kawabata, Y., & Katunuma, N. terminus, Suc-Tyr-Met-NA, ƒÀ-Ala-Tyr-Met-NA, (1979) Eur. J. Biochem. 102, 279-289 and D-Leu-Tyr-Met-NA, are very useful for assay 13. Schwartz, W.N. & Barrett, A.J. (1980) Biochem. of both cathepsin B and cathepsin L in crude J. 191, 487-497 enzyme sources. 14. Konig, W. & Geiger, H. (1970) Chem. Ber. 103, 788-798 We would like to thank Mrs. E. Inai for help in the 15. Yajima, H., Kawasaki, K., Kinomura, Y., Oshima, T., Kimoto, S., & Okamoto, M. (1968) Chem. preparation of this manuscript. Pharm. Bull. 16, 1342-1350 16. Neeman, M. & Hashimoto, Y. (1968) J. Ain. Chem. REFERENCES Soc. 84, 2972-2978 1. Barrett, A.J. (1977) in Proteinases in Mammalian 17. Nakajima, K., Powers, J.C., Ashe, B.M., & Zim Cells and Tissues (Barrett, A.J., ed.) pp. 181-208, merman, M. (1979) J. Biol. Chem. 254, 4027-4032 Elsevier/North-Holland, Amsterdam, New York 18. Lowry, O.H. Rosebrough, N.J., Farr, A.L., & 2. Kirschke, H., Langner, J., Wiederanders, B., An Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 sorge, S., & Bohley, R. (1977) Eur. J. Biochem. 19. Kargel, H.J., Dettmer, R., Etzold, G., Kirschke, 74,293-301 H., Bohley, P., & Langner, J. (1980) FEBS Lett. 3. Towatari, T., Tanaka, K., Yoshikawa, D., & 114,257-260 Katunuma, N. (1979) J. Biochem. 84, 659-671 20. McDonald, J.K., Zeitman, B.B., Reilly, T.J., & 4. Kirschke, H., Langner, J., Wiederanders, B., An Ellis, S. (1969) J. Biol. Chem. 244, 2693-2709 sorge, S., Bohley, P., & Hanson, H. (1977) Acta 21. Barrett, A.J. & Kirschke, H. (1981) Methods En Biol. Med. Germ. 36, 185-199 aymol. 80, part C, 535-561 5. Otto, K. (1971) in Tissue Proteinases (Barrett, A.J. & Dingle, J.T., eds.) pp. 1-28, North-Holland, Amsterdam, New York

Vol. 93, No. 4, 1983