Abstract
Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withK i value of 12.5 × l0−9M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.
Similar content being viewed by others
Abbreviations
- BANA:
-
α-N benzoyl-D, L-arginine β-naphthylamide
- Z-Arg-Arg-4mβNA:
-
benzyloxy-carbonyl-arginine-arginine-4-methoxy-β-naphthylamide
- DTE:
-
dithioerythritol
- SDS-PAGE:
-
sodium dodecyl sulphate Polyacrylamide gel electrophoresis
- E-64:
-
L-trans-epoxysuccinyl-leucylamido (4-guanidinoj-butane
- TLCK:
-
α-N-tosyl-lysine chloromethyl ketone
- TPCK:
-
α-N-tosyl-phenylalanine chloromethyl ketone
References
Barrett, A. J. (1973)Biochem J.,131, 809.
Barrett, A. J. (1977) inProteinases in mammalian cells and tissues (ed. A. J. Barrett) (Amsterdam: North-Holland) p. 181.
Bradley, J. D. and Whitaker, J. N. (1986)Neurochem. Res.,11, 851.
Burnett, D., Crocker, J. and Stockley, A. A. (1983)Am. Rev. Respir. Dis.,128, 915.
Davidson, E. and Poole, B. (1975)Biochim. Biophys. Acta,397, 437.
Davis, B. J. (1964)Ann. N. Y. Acad. Sci.,121, 404.
Dean, R. T. (1975)Eur. J. Biochem.,58, 9.
de Duve, C, Wittiaux, R. and Baudhuin, P. (1962)Adv. Enzymol.,24, 291.
Dixon, M. (1953)Biochem. J.,55, 170.
Ferren, L. G., Stauber, W. R. and Kalnitsky, G. (1978)Proc. Soc. Exp. Biol. Med.,159, 239.
Knight, C. G. (1980)Biochem. J.,189, 447.
Koenig, H. (1974)Methods Enzymol.,31, 457.
Marks, N. (1977) inPeptides in neurobiology, (ed. H. Gainer) (New York: Plenum Press) p. 221.
McGregor, R. R., Hamilton, J. W., Shofstall, R. E. and Cohn, D. V. (1979)J. Biol. Chem.,254, 4423.
Neurath, H. and Walsh, K. A. (1976)Proc. Natl. Acad. Sci. USA,73, 3825.
Noda, T., Isogai, K., Katunuma, N., Torumoto, Y. and Ohzeki, M. (1981)J. Biochem. (Tokyo),90, 893.
(Mensen, M., Morland, B., Husbr, G. and Rekvig, O. P. (1983)Clin. Exp. Immunol.,54, 397.
Otto, K. (1971) inTissue proteinases (eds A. J. Barrett and J. T. Dingle) (Amsterdam: North-Holland) p.1.
Poole, A. R., Tiltman, K. J., Recklies, A. D. and Stoker, T. A. M. (1978)Nature (London),273, 545.
Quinn, P. S. and Judah, J. D. (1978)Biochem. J.,172, 301.
Reisfeld, R. A., Lewis, U. J. and Williams, D. E. (1962)Nature (London),195, 281.
Segal, H. L. (1975) inLysosomes in biology and pathology (eds J. T. Dingle and R. T. Dean) (Amsterdam: North-Holland) p. 295.
Sher, J. H., Stracher, A., Shafiq, S. A. and Hardy-Stashin, J. (1981)Proc. Natl. Acad. Sci. USA,79, 7742.
Swkimi, T., Yamamoto, D. and Handa, M. (1987)J. Pharmacobio-Dynamics (Jpn.),10, 750.
Singh, H. and Kalnitsky, G. (1978)J. Biol. Chem.,253, 4319.
Singh, H. and Kalnitsky, G. (1980)J. Biol. Chem.,255, 369.
Sloane, B. F., Rozhin, J., Johnson, K., Taylor, H., Crissman, J. D. and Honn, K. V. (1986)Proc. Natl. Acad. Sci. USA,83, 2483.
’Smith, R. E. and Van Frank, R. M. (1975) inLysosomes in biology and pathology (eds J. T. Dingle and R. T. Dean) (Amsterdam: lsforth-Holland) p. 193.
Suhar, M. and Marks, N. (1979)Eur. J. Biochem.,101, 23.
Sylven, B. (1974) Schweiz.Med. Wochenschr.,104, 258.
Takio, K., Towatari, T., Katunuma, N. and Titani, K. (1980)Biochem. Biophys. Res. Commun.,97, 340.
Towatari, T., Kawabata, Y. and Katunuma, N. (1979)Eur. J. Biochem.,102, 279.
Uy, R. and Wold, F. (1977)Science,198, 890.
Ward, W. F., Cox, J. R. and Mortimore, G. E. (1977)J. Biol. Chem.,252, 6955.
Weber, K. and Osborn, M. (1969)J. Biol. Chem.,244, 4406.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kamboj, R.C., Pal, S. & Singh, H. Purification and characterization of cathepsin B from goat brain. J Biosci 15, 397–408 (1990). https://doi.org/10.1007/BF02702681
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02702681