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<mol of substrate hydrolyzed (min-1.mg protein-1) and rates relative to that with BANA are shown in parentheses. 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.