Bone, calvaria and dentine
collagens were incubated with crude preparations of lysosomal
cathepsins obtained from liver, spleen and bone cells. Degradation was most rapid near or below pH 4 and the rate of degradation was increased two-to four-fold in the presence of 50-75 mM CaCl2. This concentration of Ca2+
ions was close to the saturating level of
ions released from
calcium hydroxyapatite in the pH range 3.5-4.0. Purified
cathepsins B and L were very much less effective than the
crude extracts in degrading the hard-tissue
collagens.
Cathepsin B was equally sensitive to the inclusion of 50 mM CaCl2 but
cathepsin L activity was only slightly increased. The activating effect of Ca2+
ions was not specific as Mg2+
ions were equally effective. A partially-purified preparation of
cathepsin N gave results similar to those obtained for the crude, mixed
enzyme preparations. Spleen and bone
cell extracts were much more effective than those of liver despite a lower content of
cathepsins B and L. These findings suggest that a third
enzyme,
cathepsin N, which is known to be more abundant in spleen than liver, had contributed more of the collagenolytic activity in the spleen and bone
cell extracts. Therefore in osteoclastic
bone resorption the major
collagen-degrading enzyme could be
cathepsin N. Tendon
collagen was degraded very rapidly by the crude and pure preparations of lysosomal
cathepsins in the CaCl2-free
buffers. However, when 50 mM CaCl2 was included in the incubation mixtures the reaction was strongly inhibited. The effect of the added CaCl2 appeared to be on the substrate since the activity of
cathepsins B and L, was depressed by CaCl2 in the fluorimetric
peptidase assays for these
enzymes.