The Influence of Dissolved Calcium Salts on the Degradation of Hard-Tissue Collagens by Lysosomal Cathepsins

David J. Etherington , Henning Birkedahl-Hansen
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引用次数: 28

Abstract

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.

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溶钙盐对溶酶体组织蛋白酶降解硬组织胶原的影响
骨、颅骨和牙本质胶原与从肝、脾和骨细胞中获得的溶酶体组织蛋白酶粗制剂孵育。在pH值接近或低于pH值时,降解速度最快,在50-75 mM CaCl2的存在下,降解速度提高了2 - 4倍。这一Ca2+离子浓度接近于pH值在3.5 ~ 4.0范围内羟基磷灰石钙离子释放的饱和水平。纯化的组织蛋白酶B和L在降解硬组织胶原方面的效果远远低于粗提物。组织蛋白酶B对50 mM CaCl2的加入同样敏感,但组织蛋白酶L的活性仅略有增加。Ca2+离子的激活作用不具有特异性,因为Mg2+离子同样有效。部分纯化的组织蛋白酶N的制备得到的结果与粗的混合酶制剂相似。尽管组织蛋白酶B和l的含量较低,但脾脏和骨细胞提取物比肝脏更有效。这些发现表明,第三种酶,组织蛋白酶N,在脾脏和骨细胞提取物中比肝脏更丰富,贡献了更多的胶原溶解活性。因此,在破骨细胞骨吸收中,主要的胶原降解酶可能是组织蛋白酶n .。在无cacl2缓冲液中,溶酶体组织蛋白酶的粗制和纯制都能迅速降解肌腱胶原。然而,当在孵育混合物中加入50 mM CaCl2时,反应被强烈抑制。在组织蛋白酶B和L的荧光肽酶测定中,CaCl2的加入对底物的影响似乎是由于CaCl2抑制了组织蛋白酶B和L的活性。
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