Some properties of cathepsin B and a-N-benzoylarginine-~-
naphthylamide (BANA) hydrolase from bovine lymph nodes have
been studies. a-N-benzoylarginine-~-naphthylamide was a sensitive
substrate for both enzymes. Leucine-2-naphthylamide was cleaved
only by BANA hydrolase. Degradation of low molecular weight
substrates was optimal at pH = 6.0. At this pH value, the enzymes
were most stable. Cathepsin B inactivated aldolase, was inhibited
by 1 μM leupeptin and by thiol blocking compounds. BANA hydrolase
was not inhibited by 1 μM leupeptin but showed that it required
thiol compounds and EDTA for full activation. It was concluded
that BANA hydrolase is very similar or identical to cathepsin
H from rat liver lysosomes

I. Kregar

T. Lah

T. Zvonar-Popovič

V. Turk

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CROATICA CHEMICA ACTA CCACAA 53 (3) 509-517 (1980) CCA-1232 YU ISSN 0011-1643 UDC 577.15 Original Scientific Paper Some Characteristics of Cathepsin Band a-N-Benzoylarginine--B-Naphthylamide Hydrolase From Bovine Lymph Nodes T . Zvonar-Popovic, T. Lah, I. Kregar, and V. Turk Department of Biochemistry, J. Stefan Institute, 61000 Ljubljana, Yugoslavia Received February 26, 1980 Some properties of cathepsin B and a-N-benzoylarginine-~­-naphthylamide (BANA) hydrolase from bovine lymph nodes have been studies. a-N-benzoylarginine-~-naphthylamide was a sensitive substrate for both enzymes. Leucine-2-naphthylamide was cleaved only by BANA hydrolase. Degradation of low molecular weight substrates was optimal at pH = 6.0. At this pH value, the enzymes were most stable. Cathepsin B inactivated aldolase, was inhibited by 1 µM leupeptin and by thiol blocking compounds. BANA hydro-lase was not inhibited by 1 µM leupeptin but showed that it requi-red thiol compounds and EDTA for full activation. It was con-cluded that BANA hydrolase is very similar or identical to ca-thepsin H from rat liver lysosomes. Intracellular proteinases of bovine lymph nodes have been studied in our laboratory in light of the important role of lymph tissue in defense mechanisms within mammalian organisms. In addition to cathepsins D (EC 3.4.23.5) and S (EC 3.4.22.-), cathepsin B (EC 3.4.22.1) has also been found in lymph nodes1•2• The last, along with other thiol dependent lysosomal pro-teinases, are thought to play an important role in the degradation of native and denatured proteins3• Cathepsin B may also be involved in the generation of active peptides from their precursors4•5• Further studies of thiol proteinases from lymph nodes have led to a novel method which has enabled us to simul-taneously isolate pure cathepsin B and another a-N-benzoylarginine-B-naphthy-lamide (BANA) hydrolase6• The aims of the present work were to determine some characteristics of those enzymes not previously isolated from lymph tissues and to identify BANA hydrolase. MATERIALS AND METHODS Bovine lymph nodes were used for the isolation of enzymes as previously described6. Enzymatic Assay Cathepsin B activity was measured by the method of Barrett7 with a-N-benzoyl--DL-arginine-~-naphthylamide. HCl (Bz-Arg-2-NNap, Sigma, USA) was used as substrate. Free naphthylamine was determined colorimetrically, after coupling with Fast Garnet GBC (Sigma, USA). One unit of activity hydrolyses 1 nmol of sub-strate per minute under the conditions of the reaction. Enzyme activity was also measured toward Leu-2-naphthylamide and the amount of released 2-naphthylamine 510 T. ZVONAR-POPOVIC ET AL. was determined as previously mentioned. When a-N-benzoyl-DL-arginine-4-nitro-anilide (Bz-Arg-NPhN02, Sigma, USA) was used as the substrate, the modified method of Otto8 was employed. 0.1 ml of preactivated enzyme with cysteine and EDTA was incubated for 60 min with 1 ml of substrate solution (0.6 mM substrate in phosphate buffer, pH = 6.0). Reaction was stopped by the addition of 0.2 ml of Tris-phosphate buffer, pH= 10. The amount of released 4-nitroaniline was deter-mined by measuring the absorbance at 410 nm. Proteolytic activity toward hemoglobin was measured by the method of Anson9 and toward casein by the method of Langner10. Assay of Other Enzymes. Aldolase (Sigma, USA) from r abbit muscle was measured by the Beisenherz method11 using Test Combination Aldolase Kit No. 123838 (Behringer, GFR) . Urease (Merck, GFR) was assayed by the method of Berthelot12 • Lactate dehydrogenase (Behringer, GFR) was measured using LDH Monotest Kit No. 12488513, glutamate dehydrogenase, according to Test Combination GLDH Kit No. 1243201\ and galactose dehydrogenase by Test Combination Galactose Kit No. 15921 (all kits from Behringer, GFR)13• Inactivation was determined following the incubation of the substrate enzyme with cathepsin B for 15 min at 37 °c in a citrate buffer of pH 5.2, containing 0.2 mM EDTA and 0.1 mM DTE. Nitrogen was determined by the modified Kjeldahl method'\ The amount of protein was estimated by multiplying the value by 6.25. Specific Extinction Coefficient. Pure enzyme preparations were lyophilized, dried in a desiccator over CaCh and weighed. They were then dissolved in a 1 M acetate buffer, pH = 2.5, or in 0.8 M NaHC03, pH = 8.0, at an approximate concentration of 0.25 mg/ml. Absor-bance of the solution was measured at 280 nm and the specific extinction coefficient was calculated : Isoelectric Focusing. A l'lo 280 A 280 in 1 cm cuvette · 10 cone. of protein in mg/ml This method was performed on the Desaga apparatus (Desaga, GFR) as described by the manufacturer. 50/o polyacrylamide gel plates (9 X 16 cm) containing carrier Ampholines (LKB, Sweden) hawing pH ranges of 2-11 were used. Voltage was gradually increased from 200 to 1180 V during the 90 min focusing time, whereas current dropped from an initial 50 mA to 7 mA. Gels were washed with 100/o trichlo-roacetic acid in order to remove Ampholines, and were subsequently stained with Coomassie brilliant blue G 250 (Serva, GFR). A mixture of 8 standard proteins with known isoelectric points (Serva, GFR) was run parallel in the same gel. pl values of proteins were determined by measuring the pH in the gel using a microcombination electrode (Desaga, GFR). N-terminal Amino Acid Analysis. N-terminal amino acid was determined by the dansylation method (15). After the hydrolysis of dansylated proteins, amino acid derivatives were identified by thin layer chromatography on micropolyamide F-1700 sheets (Schleicher-Schilll, GFR). Chromatograms were viewed under UV light at 254 nm and 366 nm (Camag, Switzerland). Dansyl amino acid standards were obtained from Calbiochem, Switzer-land. Amino Acid Analysis. Amino acid composition was determined by the method of Moore and Stein16 on a Jeol JLC-BC2 amino acid analyzer. LYMPH NODE THIOL PROTEINASE 511 Molecular Weight Molecular weight was determined by the method of Weber and Osborn17 using sodium dodecyl sulphate gel electrophoresis. Circular Dichroism Measurements. CD spectra were recorded on a Jobin Yvon Dichrograph III (France), using 0.5 mm cuvettes. Amounts of alpha-helix, beta structure and random coil were calculated using the method of Chen et al18• RESULTS The summarized purification procedure yielding electrophoretically pure cathepsin B and BANA hydrolase is shown in Figure 1. Isoelectric focusing of cathepsin B showed the presence of one strong band of pl between pH = 5.0 and 5.1, with minor impurities, whereas BANA hydrolase consisted of two equally intense bands of pl = 7.1 and 7.35 (Figure 2). The specific extinction coefficient of cathepsin B is 18.5, and 14.0 for BANA hydrolase. Leucine was ic\entified as the N-terminal amino acid of cathepsin B. The amino acid com-position of both enzymes is given in Table I. The number of residues was calculated by assumming a molecular weight of 26,300 for cathepsin B and HOMOGENIZATION . ACIDIFICATION CENTRIFUGATION AMONSULPHATE PRECIPITATION SEPHADEX G-75 THIOL SEPHAROSE CM CELLULOSE CATHEPSIN B SANA HYDROLASE CATHEPSIN H Figure 1. Flow diagram summarizing the purification procedure of cathepsin B and of BANA hydrolase . 512 T. ZVONAR-POPOVIC ET AL. Figure 2. Isoelectric focusing of cathepsin B and of BANA hydrolase . 1. Cathepsin B (90 µg) 2. Standard proteins (pl 4.40-10.45) 3. BANA hydrolase (60 µg) 4. Standard proteins (pl = 4.40:-10.45) 30,800 for BANA hydrolase. The CD spectrum in .the far UV region for cathepsin B and BANA hydrolase is shown in Figure 3. From the experi-mentally determined elipticities, the percentage of the secondary structure was calculated and the results are shown in Table II. Cathepsin B degraded hemoglobin optimally at pH = 4.0, and BANA hydrolase at pH= 6.5-7.0. An approximately similar pH optimum (pH= 6) for both enzymes was found for the hydrolysis of Bz-Arg-2-NNap. The specific activity toward some substrates is shown in Table III. Both enzymes have a high and similar specific activity toward Bz-Arg-2-NNap. Cathepsin B did not hydrolyze Leu-2-naphthylamide, which, in turn, was a good substrate for BANA hydrolase. Cathepsin B (approximately 1 mg/ml) solution was rather stable on storage at -20 °c in an acetate buffer pH_= 5.0 in the presence of 1 mM EDTA. After 5 months, the initial activity was reduced by 25°/o. BANA hydrolase was less stable. At 5 months, 500/o of the activity was lost. Inactivation of the test enzyme was limited to a study with cathepsin B. More than 8Qll/o of the aldolase activity (8.5 mU/ml) was inactivated by purified cathepsin B. No inactivation of lactate dehydrogenase, glutamate dehydrogenase, galactose dehydrogenase and urease was noted. 0 -1000 ~ :!;'.-2 000 'E ~ -3 000 ~-4 000 -5000 -6000 -7000 -8000 LYMPH NODE THIOL PROTEINASE TABLE I Amino Acid Composition of Cathepsin B and Bana Hydrolase Amino acid Asx Thr Ser Glx Pro Gly Ala Cys Val Met lie Leu Tyr Phe Lys His Arg Catheps in B · Residues/molecule Cathepsin B 17 9 19 20 15 27 8 13 13 3 12 8 8 7 13 9 7 - 2000 0 E ~ 1000 "' E ~ 0 ~ -1 000 - 2000 - 3000 - 4000 -5000 BANA hydro lase 23 12 15 27 20 23 21 7 13 6 10 10 10 11 18 5 7 BANA Hydrolase 513 190 200 210 220 230 . 240 250 >. (nmJ 200 210 220 230 240 250 11! nm) Figure 3. CD spectrum of cathepsin B (21.9 mg/100 ml in 0.02 M Na-acetate buffer, pH = 5.0) and of BANA hydrolase (9.8 mg/100 ml in 0.02 M Na-acetate buffer, pH = 5.0). 514 T . ZVONAR-POPOVIC ET AL. TABLE II Secondary Structure of Cathepsin B and BANA Hydrolase a-helix ~-structure Unordered st. Cathepsin B BANA hydrolase O/o O/o 12 31 57 18 12 70 The pH stability of the enzymes following 1 h incubation at 37 °C is shown in Figure 4. Both enzymes are maximally stable at pH = 6. Cathepsin B sta-bility is decreased below pH= 5 and drops sharply above pH= 7, whereas BANA hydrolase is less sensitive to pH change. A 520 0.4 0.3 0.2 0.1 C~thepsin 8 2 4 6 8 A520 0.4 0.3 0.2 0.1 SANA Hydrolase Figure 4. Stability of cathepsin B (1) and of BANA hydrolase' at various pH values. The enzyme was . dissolved in Mc II vain buffer and incubated for 1 h at 37 •c. Residual proteolytic activity toward Bz-Arg-2-NNap was measured. Results are · expressed as absorbance at 520 nm. TABLE III Specific Activities of Cathepsin B and BANA Hydrolase Enzyme and Substrate were Incubated at 37 °c in a Phosphate Buffer pH = 6.0 in the Presence of 2 mM Cysteine and 1 mM EDT A. Cathepsin B BANA Substrate Cone. hydro lase E. U./mg E. U./mg Bz-Arg-2-NNAp 1 mM 2020 1960 Leu-2-NNap 1 mM 0 2150 Bz-Arg-NPhN02 0.6 mM 84 5 Azocasein 30/o 6 2 LYMPH NODE THIOL PROTEINASE 515 TABLE IV Effects of Inhibitors and Activators on Cathepsin B and BANA Hydrolase The Enzymes were Preincubated with Eeach Compound at Various Concentrations for 5 min at 37 °c and Assayed Toward Bz-Arg-2-NNap. The Effect of EDT A on Enzyme Samples Previously Dialyzed Against Redistilled Water was Determined. Results Shown are the Mean Values of 3 Experiments. Cone. Cath. B BANA Effector (mM) 0/o act. hydrol. 0/o cat. None 1 19 Cysteine 2 100 100 EDTA 1 2 20 Cysteine + EDT A 2+1 100 100 Cysteine + EDT A 5+1 109 120 4-Chloromercuri- 0.1 72 90 benzoate 1 0 0 2.2'dipyridil disulphide 0.5 5 5 Tos-Phe-CH2Cl 0.01 20 80 Iodoacetamide 1 5 10 Iodoacetic acid 1 0 0 Leupeptin 0.001 30 98 The effect of various potential inhibitors and activators is shown in Table IV. Inhibition with 4-chloromercuribenzoate, iodoacetic acid and i!odoacetamide agrees well with the classification of cathepsin B and BANA hydrolase as thiol proteinases. Tos-PheCH2Cl inhibited cathepsin B to a much higher extent than BANA hydrolase. Leupeptin at 10-5 M concentration did not inhibit BANA hydrolase, whereas cathepsin B activity was lowered by 70'fJ/o. DISCUSSION Cathepsin B has been purified from a number of animal tissues, as well as from humans3• The existence of multiple forms of cathepsin B has been reported by many authors, although cathepsin B in a single form has been isolated from human placenta19 and from rat liver lysosomes20• In the present study we have isolated cathepsin B which displayed one major protein band and some minor impurities upon isoelectric focusing. The isoelectric point of the major band agrees with the value for the major form obtained from human liver21 • The specific extinction coefficient is very similar to the value reported by Barrett21 • Leucine was indentified as the N-terminal amino acid, as also reported by Franklin and Metrione22 , and by Keilova and Tomasek23• Comparing our results on secondary structure obtained by CD measurement with those for papain18 we can see that cathepsin B and BANA hydrolase contain less alpha-helix and beta structure. High proline content contributes to the low amount of alpha-helix. Our results on the specificity of cathepsin B toward low molecular weight substrates are in agreement with data reported in the literature24 •25. Zero activity toward Leu-2-'naphthylamide indicates the purity of our enzyme preparation26 • Aldolase inactivation by cathepsin B has been used for the identification of cathepsin B24• The failure of the enzyme to inactivate lactate dehydrogenase and glutamate dehydrogenase has also been observed by Towatari et al.27 whereas the effect upon urease and galactose 516 T. ZVONAR-POPOVIC ET AL. dehydrogenase has not been studied. The absolute requirements of free thiol groups for the enzyme activity were shown by the strong inhibitory effect of thiol blocking reagents, and natural aldehyde leupeptin. Additional BANA hydrolyzing activity of a higher molecular weight than that of cathepsin B, but without aldolase inactivation capability, has been noted by Distelmeyer et al.28 and investigated further by Davidson and Poole29• Kirschke et al.30 reported on a thiol enzyme from rat liver lysosomes having a moleculfir weight ·of 28,000 with exo- and endo-peptidase activity and named it cathepsin H. Our results show that BANA hydrolase from bovine lymph nodes has properties similar or identical to those of cathepsin H, such as the isoelectric point, molecular weight, pH optimum toward Bz-Arg-2-NNap and specificity toward synthetic and protein substrates. BANA hydrolase was also inhibited with thiol blocking reagents and was not inhibited with 1 µM leupeptin, as cathepsin H. Lymph node BANA hydrolase shows more similarity with cathepsin H than with BANA hydrolase from rabbit lung31• Acknowledgement. - We thank Mrs. A. Burkeljc and Mr. K. Lindie for their excellent technical assistance. This work was supported by the Research Council of Slovenia, and in part by the NSF USA grant no. F7F030Y. REFERENCES 1. T. Turns e k, I. Kr e gar, and D. Le be z, Croat. Chem. Acta 47 (1975) 59. 2. T. Turns e k, I. Kr e gar, and D. Le be z, Biochim. Biophys. Acta 403 (1975) 514. 3. A. J. Barrett, Proteinases in mammalian cells and tissues, Ed. A. J . B a r -r et t. Elsevier/North Holland Biomedical Press, Amsterdam 1977, p. 181. 4. R. F. S m it h and R. M. V a n F r a n k, Lysosomes in biology and pathology, Eds. J . T. Dingle and R. T. De an. North Holland Pub!. Co., Amsterdam 1975, p. 193. 5. P. S . Quinn and J. D. Jud ah, Biochem. J. 172 (1978) 301. 6. T. Z v on a r, I. Kr e gar, and V. Turk, Croat. Chem. Acta 52 (1979) 411. 7. A. J . Barrett, Anal. Biochem. 47 (1972) 280. 8. K . 0 t to, Hoppe-Seyler's Z. Physiol. Chem. 348 (1967) 1449. 9. M. L. A n s o n, J. Gen. Physiol. 22 (1939) 79. 10. J. Langner, A. Wakil, M. Zimmermann, S. Ansorge, P . Bohley, H. Kirschke, and B. Wieder an de rs, Acta Biol. Med. Ger. 31 (1973} 1. 11. G. Beisenherz, H. J . Bolte, T. Bucher, R. Czok, K. H . Gar-bade, E . Meyer-Arendt, and G. Pfleiderer, Z . Naturforsch. Sb (1953) 555. 12. H. J. Richard, Clinical chemistry: principles and techniques. HIH Inter-national reprint, Harper and Row Pub!. Co., New York 1966, p . 266. 13. Deutsche Gesellschaft fii,r klinische Chemie, Z. Klin. Chem. Klin. Biochem., S (1970) 658; 9 (1971) 464; 10 (1972) 182. 14. C. Duma z er t and Y Marc e 1 et, Bull. Soc. Chim. Biol. 20 (1938) 201. 15. K . Nari ta, Protein sequence determination. Ed. S . B .. Needleman. Springer Verlag, Berlin, New York 1970, p. 38. 16. D. H . Spackman, W. H . Stein, and S. Moore, Anal. Chem. 30 (1958) 1190. 17. K. Weber and M. 0 s b or n, J. Biol. Chem. 244 (1969) 4406. 18. Y. H. Chen, J. T. Yang, and K. H . Ch an, Biochemistry 13 (1974) 3350. 19. P . Evans and D. J. Etherington, Eur. J. Biochem. 83 (1978) 87. 20. T. Tow at a r i and N. Kat u nu ma, Biochem. Biophys. Res. Commun. 83 (1978) 513. 21. A. J. Barrett, Biochem. J. 131 (1973) 809. 22. S . G. Franklin and R. M. Metrione, Biochem. J. 127 (1972) 207. 23. H. Keilova and V. Tomasek, FEBS Lett. 29 (1973) 335. 24. K. Otto and K. B ha k di, Hoppe-Seyler's Z. Physiol. Chem. 350 (1969) 1577. LYMPH NODE THIOL PROTEINASE 517 25. K. Takahashi, M. Is emu r a, and T. I ken aka, J . Biochem. 85 (1979) 1053. 26. J. K. Mc Don a 1 d, B. B. Z e it man, and S. E 11 is, Nature 225 (1970) 1048. 27. T. Tow at a ri, T. Tan aka, D. Yoshikawa, and N. Kat u nu ma, J. Biochem. 84 (1978) 659. 28. P . Dist e 1 me ye r, H. H ii b n er, and K. 0 t to, Enzymologia 42 (1972) 363. 29. E. Davidson and B. ' Po o 1 e, Biochim. Biophys. Acta 397 (1975) 437. 30. H. Kirschke, J . Langner, B. Wiederanders, S. Ansorge, P. Bo h 1 e y, and H. Hanson, Acta Biol. Med. Ger. 36 (1977) 185. 31. H. Singh and G. Ka 1 n its k y, J. Biol. Chem. 253 (1978) 4319. IZVLECEK Nekatere Iastnosti katepsina B in a-N-benzoilarginin-~-naftilamid hidrolaze iz govejih Iimfnih zlez T. Zvonar-Popovic, T. Lah, I. Kregar i V. Turk Studirali smo nekatere lastnosti katepsina B in BANA hidrolaze iz govejih bezgavk. Bz-Arg-2-NNap je bil obeutljiv substrat za oba encima. Leu-2-naftilamid je hidrolizirala le BANA hidrolaza. Optimalni pH za razgradnjo nizkomolekularnib substratov je bil pH= 6. Pri tej pH vrednosti sta bila encima tudi najbolj stabilna. Katepsin B je inaktiviral aldolazo. 1 µM leupeptin je inhibiral katepsin B, ne pa BANA hidrolaze. Oba encima so aktivirale tiolne spojine in EDTA, inhibirale pa so ju spojine, ki blokirajo -SH skupine. Ugotovili smo, da je BANA hidrolaza iz govejih limfnih zlez zelo podobna ali identicna s katepsinom H iz lisosomov podganjih jeter. ODDELEK ZA BIOKEMIJO INSTITUT JOZEF STEFAN JAMOVA 39, 61000 LJUBLJANA JUGOSLA VIJA Prispjelo 26. veljace 1980. 

Some Characteristics of Cathepsin Band a-N-Benzoylarginine- B-Naphthylamide Hydrolase From Bovine Lymph Nodes

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