The purification of bovine cathepsin B1 and its mode of action on bovine collagens

Abstract

1. Cathepsin B1 was isolated from bovine spleen by autolysis, (NH4)2SO4 fractionation and chromatography on Amberlite IRC-50. Two isoenzyme forms were purified to homogeneity by chromatography on CM-cellulose and DEAE-Sephadex. 2. A collagenolytic cathepsin was separated from cathepsin B1 during purification. The remaining collagenolytic activity of the purified cathepsin-B1 isoenzymes was no greater than 0.3 unit/unit of cathepsin B1 compared with about 5.0 unit/unit of cathepsin B1 in the autolysed spleen extracts. 3. The cathepsin B1 isoenzymes lowered the viscosity of gelatin at 37°C. Optimum activity was at pH4–5. 4. At 28°C the interchain cross-links in native tropocollagen were cleaved most effectively at pH4–5. Insoluble tendon collagen was digested at pH3.5 and 28°C to yield mainly α-chain components, with the loss of a short N-terminal sequence. 5. Electron-microscope studies of collagen fibrils showed that cathepsin B1 caused longitudinal splitting and dissociation of the protofilaments. The effect was not general but occurred at selected sites. 6. The isoenzymes of cathepsin B1 cleaved the telopeptide region of calf skin tropocollagen between the lysine-derived cross-link and the triple helix. The CB1 peptide fragments obtained from enzyme-degraded α1 chains were hydrolysed at Gly12-Ile13 and Ser14-Val15. The residual α2 CB1 peptides were hydrolysed at Ala8-Asp9 and Asp9-Phe10.