Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.
Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.
复制标题
牛脾组织蛋白酶 B1 和溶胶原组织蛋白酶。
作者：
D. Etherington
用Amberlite IRC-50柱层析分离牛脾组织蛋白酶B_1和胶原溶解性组织蛋白酶，用DEAE-Sephadex（A-50）柱层析部分纯化胶原溶解性组织蛋白酶。2.溶胶原组织蛋白酶降解不溶性肌腱胶原蛋白最大pH值为3.5和28摄氏度，主要是α链成分释放到溶液中。在28摄氏度下，可溶性皮肤胶原蛋白中的端肽也被切割以产生α链组分。因此，溶胶原蛋白的组织蛋白酶在其对天然胶原蛋白的作用方面类似于组织蛋白酶B1，但这两种酶的混合物表现出协同效应。3.加入巯基阻断化合物对胶原溶解性组织蛋白酶和组织蛋白酶B1产生类似的抑制作用。该酶的反应类似于所有其他化合物的测试，除了6-氨基己酸，当胶原蛋白溶解组织蛋白酶轻微激活和组织蛋白酶B1几乎不受影响。4.亮抑酶肽是一种含精氨酸的合成底物的结构类似物，其抑制胶原溶解性组织蛋白酶的效果与组织蛋白酶B1相同。胶原溶解性组织蛋白酶显示在纯化过程中保留对α-N-苯甲酰基-DL-精氨酸对硝基苯胺的低残留活性，其相当于组织蛋白酶B1活性的0.2%。5.组织蛋白酶B1和溶胶原组织蛋白酶不能通过有机汞-Sepharose 4 B亲和层析分离。这两种酶可在DEAE-Sephadex（A-50）上和在Ampholine pH梯度中通过等电聚焦分离。主要组织蛋白酶B1同工酶的pI为4.9，溶胶原组织蛋白酶的pI为6.4。6.从Sephadex G-75（超细级）上的层析，计算出组织蛋白酶B1的分子量为26000，胶原溶解组织蛋白酶的分子量为20000。通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳确认分子量的差异。
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). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees 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 two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue 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. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two 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. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.