Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen

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Abstract
Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH3.5 and at 28.degree. C; mainly .alpha.-chain components were released into solution. At 28.degree. C the telopeptides in soluble skin collagen were also cleaved to yield .alpha.-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these 2 enzymes exhibited a synergistic effect. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzymes responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. Leupeptin, which is a structural analog of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against .alpha.-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The 2 enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26,000 for cathepsin B1 and 20,000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulfhate/polyacrylamide-gel electrophoresis.

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