Berri M, Rouchon P, Zabari M, Ouali A
Muscle Biochemistry Laboratory, SRV, INRA, St Genès Champanelle, France.
Comp Biochem Physiol B Biochem Mol Biol. 1998 Feb;119(2):283-8. doi: 10.1016/s0305-0491(97)00326-x.
Four papain-inhibiting peaks, labeled F-I, F-II, F-III, and F-IV, were fractionated from a crude bovine muscle extract by gel filtration chromatography on Sephadex G100, and the F-III fraction was analyzed. From F-III, a cysteine proteinase inhibitor was purified by two successive anionic exchange chromatography steps on Q-Sepharose and Mono-Q columns. This inhibitor has a molecular weight of about 30 kDa. Regarding its specificity toward different proteinases, the purified 30 kDa inhibitor was inactive against serine (trypsin and chymotrypsin) and aspartyl (pepsin) families. In contrast, cathepsin L, H, B, and papain, four enzymes of the cysteine class were strongly inhibited suggesting that this inhibitor was specific to the cysteine proteinase group. However, no inhibitory activity was shown against calpains. Kinetic parameters, including inhibition constants (Ki), rate constant for association (kass) and time required for almost complete inhibition of proteinase in vivo were determined. The values are consistent with a possible physiological function for this inhibitor protein in controlling in vivo cathepsin L activity.
通过在葡聚糖G100上进行凝胶过滤色谱，从粗制牛肌肉提取物中分离出四个木瓜蛋白酶抑制峰，分别标记为F-I、F-II、F-III和F-IV，并对F-III组分进行了分析。从F-III中，通过在Q-琼脂糖和Mono-Q柱上连续两步阴离子交换色谱法纯化出一种半胱氨酸蛋白酶抑制剂。该抑制剂的分子量约为30 kDa。关于其对不同蛋白酶的特异性，纯化的30 kDa抑制剂对丝氨酸（胰蛋白酶和糜蛋白酶）和天冬氨酸（胃蛋白酶）家族的蛋白酶无活性。相反，半胱氨酸类的四种酶，组织蛋白酶L、H、B和木瓜蛋白酶受到强烈抑制，这表明该抑制剂对半胱氨酸蛋白酶组具有特异性。然而，对钙蛋白酶没有显示出抑制活性。测定了动力学参数，包括抑制常数（Ki）、结合速率常数（kass）和体内蛋白酶几乎完全抑制所需的时间。这些值与该抑制剂蛋白在体内控制组织蛋白酶L活性的可能生理功能一致。
