培养中的骨外植体同时释放以及原胶原酶和一种能降解软骨蛋白聚糖和变性胶原的潜在中性蛋白酶的平行激活。

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen.

作者信息

Vaes G, Eeckhout Y, Lenaers-Claeys G, François-Gillet C, Druetz J E

出版信息

Biochem J. 1978 May 15;172(2):261-74. doi: 10.1042/bj1720261.

DOI:10.1042/bj1720261

PMID:208518

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1185692/

Abstract

A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4 degrees C and prolonged preincubation at 25 degrees C. Its activation often followed that of the procollagenase present in the same media. 3. Activation of neutral proteinase (as does that of procollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes (60 000-70 000) by 20 000-30 000, as determined by gel filtration of media after removal of heparin. 6. The latency of both enzymes could be due either to a zymogen or to an enzyme-inhibitor complex. A thermostable inhibitor of both enzymes was found in some media. However, combinations of either enzyme with that inhibitor were not reactivated by trypsin, indicating that this inhibitor is unlikely to be the cause of the latency.

摘要

在小鼠骨外植体的培养基中发现了一种潜在的中性蛋白酶。其在培养过程中的积累与原胶原酶的积累密切平行；两者都需要培养基中存在肝素。2. 已知几种能激活原胶原酶的培养基处理方法可激活潜在的中性蛋白酶，如用胰蛋白酶、糜蛋白酶、纤溶酶或激肽释放酶进行有限的蛋白水解、在4℃下用3M-NaSCN透析以及在25℃下长时间预孵育。其激活通常跟随同一培养基中原胶原酶的激活。3. 胰蛋白酶或纤溶酶对中性蛋白酶的激活（与原胶原酶的激活一样）涉及两个连续步骤：激活培养基中存在的潜在内源性激活剂，然后由该激活剂激活中性蛋白酶本身。4. 该蛋白酶在中性pH下可降解软骨蛋白聚糖、变性胶原（偶氮胶原）和酪蛋白；它被EDTA、半胱氨酸或血清抑制。胶原酶不受酪蛋白或偶氮胶原抑制，并且比该蛋白酶对热或胰蛋白酶的抵抗力更弱。通过对培养基进行凝胶过滤可实现两种酶的部分分离，但通过分级硫酸铵沉淀、离子交换或在琼脂糖-胶原上进行亲和层析则无法实现。这些分级分离不会激活潜在的酶。5. 如通过去除肝素后对培养基进行凝胶过滤所测定，胰蛋白酶激活会使两种潜在酶（60000-70000）的分子量降低20000-30000。6. 两种酶的潜伏性可能是由于酶原或酶-抑制剂复合物。在一些培养基中发现了两种酶的热稳定抑制剂。然而，任何一种酶与该抑制剂的组合都不会被胰蛋白酶重新激活，这表明该抑制剂不太可能是潜伏性的原因。