Parker A, Gockerman A, Busby W H, Clemmons D R
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
Endocrinology. 1995 Jun;136(6):2470-6. doi: 10.1210/endo.136.6.7538463.
Smooth muscle cells (SMC) secrete insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) and an IGFBP-4 protease. The purpose of this study was to determine the characteristics of this IGFBP-4 protease and to compare its inhibitor profile to those of IGFBP-5 and IGFBP-2 proteases, which are also present in SMC-conditioned medium. Cultured SMC were exposed to serum-free medium for periods of 24-72 h, and the amount of proteolytic activity in the conditioned medium was assessed by its capacity to degrade pure IGFBP-4. Minimal activity (e.g. < 20% of IGFBP-4 degraded in 24 h at 37 C) was present in conditioned medium unless IGF-I or IGF-II was added. This resulted in more than 60% of the intact IGFBP-4 being degraded in 14 h. The activity was a calcium-dependent serine protease and was inhibited by EDTA or 3,4-dicloroisocoumarin. Calcium, but not zinc, could restore proteolytic activity. Heparin alone inhibited IGFBP-4 proteolysis by more than 60%. When heparin cofactor-II and antithrombin-III (AT-III) were added alone, they each had an effect. The combination of heparin plus AT-III was no more active than heparin alone, but the combination of heparin cofactor-II and heparin resulted in near complete inhibition. Peptides that contained the active sites of AT-III or alpha 1-antichymotrypsin were potent inhibitors of the IGFBP-4 protease. The medium also contained proteolytic activities for IGFBP-2 and IGFBP-5. Comparison of the inhibitor profiles for the IGFBP-4 and IGFBP-5 proteolytic activities revealed major differences, but the IGFBP-2 proteolytic activity was very similar to that of the IGFBP-4 protease. IGFBP-4 zymography showed a band with a molecular mass estimate of 48 kilodaltons. In contrast, when IGFBP-2 was used as the substrate, a single band at 36 kilodaltons was visualized. These data taken together with the protease inhibitor results suggest that the IGFBP-2, IGFBP-4, and IGFBP-5 proteases are members of a similar family of calcium-dependent serine proteases, but they are distinct proteases. As IGFBP-4 is a potent inhibitor of IGF action, and the activity of this protease is regulated by IGF exposure, the protease represents a novel system for regulating the actions of IGF-I in this cell type.
平滑肌细胞（SMC）分泌胰岛素样生长因子（IGF）结合蛋白-4（IGFBP-4）和一种IGFBP-4蛋白酶。本研究的目的是确定这种IGFBP-4蛋白酶的特性，并将其抑制剂谱与SMC条件培养基中也存在的IGFBP-5和IGFBP-2蛋白酶的抑制剂谱进行比较。将培养的SMC暴露于无血清培养基中24 - 72小时，通过其降解纯IGFBP-4的能力评估条件培养基中的蛋白水解活性。除非添加IGF-I或IGF-II，条件培养基中存在的活性极小（例如在37℃下24小时内降解的IGFBP-4 < 20%）。这导致在14小时内超过60%的完整IGFBP-4被降解。该活性是一种钙依赖性丝氨酸蛋白酶，可被EDTA或3,4 - 二氯异香豆素抑制。钙而非锌可恢复蛋白水解活性。单独使用肝素可抑制IGFBP-4蛋白水解超过60%。单独添加肝素辅因子-II和抗凝血酶-III（AT-III）时，它们各自都有作用。肝素加AT-III的组合活性并不比单独使用肝素时更高，但肝素辅因子-II和肝素的组合导致近乎完全抑制。包含AT-III或α1 - 抗糜蛋白酶活性位点的肽是IGFBP-4蛋白酶的有效抑制剂。该培养基中还存在针对IGFBP-2和IGFBP-5的蛋白水解活性。对IGFBP-4和IGFBP-5蛋白水解活性的抑制剂谱进行比较发现存在主要差异，但IGFBP-2蛋白水解活性与IGFBP-4蛋白酶的非常相似。IGFBP-4酶谱显示一条分子量估计为48千道尔顿的条带。相反，当使用IGFBP-2作为底物时，可见一条36千道尔顿的单一条带。这些数据与蛋白酶抑制剂结果一起表明，IGFBP-2、IGFBP-4和IGFBP-5蛋白酶是钙依赖性丝氨酸蛋白酶相似家族的成员，但它们是不同的蛋白酶。由于IGFBP-4是IGF作用的有效抑制剂，且该蛋白酶的活性受IGF暴露调节，该蛋白酶代表了一种在这种细胞类型中调节IGF-I作用的新系统。
