The influence of dissolved calcium salts on the degradation of hard-tissue collagens by lysosomal cathepsins.
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.
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002403:Cathepsins / Q000378:metabolism*; D002417:Cattle; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D003804:Dentin / Q000378:metabolism; D005260:Female; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000201:enzymology; D013710:Tendons / Q000378:metabolism
D003094:Collagen; D002403:Cathepsins; D002118:Calcium