Cathepsin B1. A lysosomal enzyme that degrades native collagen

Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. At acid pH values cathepsin B1 released ¹⁴C labeled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4 chloromercuribenzoate, by the chloromethyl ketones derived from tosyl lysine and acetyltetra alanine and by human α₂ macroglobulin. Cathepsin B1 degraded collagen in solution, the pH optimum being pH 4.5-5.0. The initial action was cleavage of the nonhelical region containing the cross link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment long spacing crystalloids (measured as native collagen molecules aligned with N termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an α chain was attacked it was rapidly degraded to low molecular weight peptides. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH 3.0. Neither cathepsin B1 nor D cleaved Pz Pro Leu Gly Pro D Arg. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.