Isolation and Characterization of Cathepsin B from Bovine Brain

Overview

Journal Neurochem Res

Specialties Chemistry
Neurology

Date 1986 Jun 1

PMID 2426610

Citations 3

Authors

J D Bradley

J N Whitaker

Affiliations

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Abstract

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75-5.2 with the major band at pH 5.1-5.2, all of which were capable of degrading N alpha-carbobenzoxy-L-arginyl-L-arginine 4-methoxy-beta-naphthylamide. The cathepsin B activity against N alpha-benzoyl-DL-arginine beta-naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.

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References

Schwartz W, Barrett A . Human cathepsin H. Biochem J. 1980; 191(2):487-97. PMC: 1162239. DOI: 10.1042/bj1910487. View

Whitaker J, Seyer J . The sequential limited degradation of bovine myelin basic protein by bovine brain cathepsin D. J Biol Chem. 1979; 254(15):6956-63. View

WILKINSON G . Statistical estimations in enzyme kinetics. Biochem J. 1961; 80:324-32. PMC: 1244002. DOI: 10.1042/bj0800324. View

Whitaker J, Heinemann M, Uzman B . The renal degradation of myelin basic protein peptide 43-88 by two enzymes in different subcellular fractions. Biochem J. 1982; 201(3):543-53. PMC: 1163681. DOI: 10.1042/bj2010543. View

Whitaker J, Seyer J . Isolation and characterization of bovine brain cathepsin D. J Neurochem. 1979; 32(2):325-33. DOI: 10.1111/j.1471-4159.1979.tb00355.x. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

GOUNARIS A, Slater E . Cathepsin B from human renal cortex. Biochem J. 1982; 205(2):295-302. PMC: 1158481. DOI: 10.1042/bj2050295. View

Honn K, CAVANAUGH P, Evens C, Taylor J, Sloane B . Tumor cell-platelet aggregation: induced by cathepsin B-like proteinase and inhibited by prostacyclin. Science. 1982; 217(4559):540-2. DOI: 10.1126/science.7046053. View

Kopitar M, Stern F, Marks N . Cerebrocystatin suppresses degradation of myelin basic protein by purified brain cysteine proteinase. Biochem Biophys Res Commun. 1983; 112(3):1000-6. DOI: 10.1016/0006-291x(83)91717-5. View

10.

Olstein A, Liener I . Comparative studies of mouse liver cathepsin B and an analogous tumor thiol proteinase. J Biol Chem. 1983; 258(18):11049-56. View

11.

Barrett A, KIRSCHKE H . Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981; 80 Pt C:535-61. DOI: 10.1016/s0076-6879(81)80043-2. View

12.

Stracher A, McGowan E, SHAFIQ S . Muscular dystrophy: inhibition of degeneration in vivo with protease inhibitors. Science. 1978; 200(4337):50-1. DOI: 10.1126/science.635570. View

13.

Suhar A, Marks N . Purification and properties of brain cathepsin B. Evidence for cleavage of pituitary lipotropins. Eur J Biochem. 1979; 101(1):23-20. DOI: 10.1111/j.1432-1033.1979.tb04211.x. View

14.

Katunuma N, Kominami E . Structures and functions of lysosomal thiol proteinases and their endogenous inhibitor. Curr Top Cell Regul. 1983; 22:71-101. DOI: 10.1016/b978-0-12-152822-5.50007-5. View

15.

Ostensen M, Morland B, Husby G, Rekvig O . A serum antibody in patients with rheumatoid arthritis stimulates cathepsin B activity in peritoneal mouse macrophages. Clin Exp Immunol. 1983; 54(2):397-404. PMC: 1535904. View

16.

DEIBLER G, Martenson R, KIES M . Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep Biochem. 1972; 2(2):139-65. DOI: 10.1080/00327487208061467. View

17.

Hashimoto F, Horigome T, Kanbayashi M, Yoshida K, Sugano H . An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. Anal Biochem. 1983; 129(1):192-9. DOI: 10.1016/0003-2697(83)90068-4. View

18.

Recklies A, Tiltman K, Stoker T, Poole A . Secretion of proteinases from malignant and nonmalignant human breast tissue. Cancer Res. 1980; 40(3):550-6. View

19.

Mort J, Leduc M . A simple, economical method for staining gels for cathepsin B-like activity. Anal Biochem. 1982; 119(1):148-52. DOI: 10.1016/0003-2697(82)90678-9. View

20.

Takio K, Towatari T, Katunuma N, Titani K . Primary structure study of rat liver cathepsin B -- a striking resemblance to papain. Biochem Biophys Res Commun. 1980; 97(1):340-6. DOI: 10.1016/s0006-291x(80)80173-2. View