Hanoune J, Stengel D, Lacombe M L, Feldmann G, Coudrier E
J Biol Chem. 1977 Mar 25;252(6):2039-45.
Treatment of rat liver plasma membranes with various commercial preparations of crude collagenase from Clostridium histolyticum at concentrations as low as 1 mug/ml, resulted in activation of the adenylate cyclase system. Maximal activation occurred at 50 to 100 mug/ml of collagenase, and promoted a 2- to 3-fold increase in the basal activity as well as in the activities stimulated by catecholamines, glucagon, fluoride, or GTP. This was due to an increase in the maximal velocity of the cyclizing reaction without any increase in the affinity of the enzyme for its substrate. Treatment of plasma membranes with crude collagenase did not induce gross structural modifications as judged by electron microscopic examination. 5'-Nucleotidase activity was slightly inhibited and ATPase activity remained unaffected. The stimulatory substance was nondialyzable, thermolabile, and inhibited by both EDTA and -SH reagents, thus appearing to be a protein. The following observations suggest the effects observed were due to other protease(s) present in crude collagenase: (a) only crude collagenase was active on liver adenylate cyclase: treatment with purified collagenase from C. histolyticum or from Achromobacter iophagus gave no stimulation; (b) the stimulatory activity was irreversible since washing of the membranes after treatment was without effect; (c) crude collagenase contained no lecithinase or sphingomyelinase activity under our conditions of adenylate cyclase assay; (d) after chromatography on Sephadex G-100, the activator appeared as a peak in the 30,000-dalton region and was clearly separated from the collagenase and clostripain peaks, but coincident with elastolytic and caseinolytic activities; (e) the effect of crude collagenase could be prevented by addition of elastin in vitro and was mimicked by purified elastase from hog pancreas. It remains to be seen whether the effects observed result from an increase in the catalytic constant of adenylate cyclase, or an unmasking of new catalytic sites.
用来自溶组织梭菌的各种粗制胶原酶商业制剂，以低至1微克/毫升的浓度处理大鼠肝细胞膜，会导致腺苷酸环化酶系统的激活。在50至100微克/毫升的胶原酶浓度下出现最大激活，并且促进基础活性以及儿茶酚胺、胰高血糖素、氟化物或GTP刺激的活性增加2至3倍。这是由于环化反应的最大速度增加，而酶对其底物的亲和力没有任何增加。通过电子显微镜检查判断，用粗制胶原酶处理细胞膜不会引起明显的结构改变。5'-核苷酸酶活性略有抑制，而ATP酶活性不受影响。刺激物质不可透析、不耐热，并且被EDTA和-SH试剂抑制，因此似乎是一种蛋白质。以下观察结果表明观察到的效应是由于粗制胶原酶中存在的其他蛋白酶：(a) 只有粗制胶原酶对肝腺苷酸环化酶有活性：用来自溶组织梭菌或食酸无色杆菌的纯化胶原酶处理没有刺激作用；(b) 刺激活性是不可逆的，因为处理后洗涤膜没有效果；(c) 在我们的腺苷酸环化酶测定条件下，粗制胶原酶不含有卵磷脂酶或鞘磷脂酶活性；(d) 在Sephadex G-100上进行色谱分离后，激活剂在30,000道尔顿区域出现一个峰，并且与胶原酶和梭菌蛋白酶峰明显分开，但与弹性蛋白酶和酪蛋白酶活性一致；(e) 在体外添加弹性蛋白可以防止粗制胶原酶的作用，并且猪胰腺纯化的弹性蛋白酶可以模拟这种作用。观察到的效应是由于腺苷酸环化酶的催化常数增加，还是由于新催化位点的暴露，还有待观察。
