Seltzer J L, Eschbach M L, Winberg J O, Bauer E A, Eisen A Z, Weingarten H
Division of Dermatology, Washington University School of Medicine, St. Louis, MO 63110.
Coll Relat Res. 1987 Dec;7(6):399-407. doi: 10.1016/s0174-173x(87)80038-9.
The intracellular degradation of interstitial collagen is accomplished by two neutral metalloproteases, collagenase and gelatinase. Both enzymes are inhibited by metal chelating agents, by certain sulfhydryl reagents, and by similar protein inhibitors. Here, we demonstrate that the dye eriochrome black T (EBT) appears to be unique in its capacity to inhibit collagenase but not gelatinase. Using native reconstituted helical collagen in gel form at 37 degrees C, half-maximal inhibition of collagenase activity by EBT occurs at approximately 45 microM. EBT more effectively inhibits the breakdown of native collagen in solution, with a KI of approximately 8 microM. Using a newly-developed spectrophotometric substrate, AcProLeuGly-S-LeuLeuGly-OC2H5, a KI of 1.4 microM was calculated for EBT on collagenase. Although this same thiopeptolide serves as a substrate for gelatinase with kinetics similar to those of collagenase, no inhibition by EBT was observed. EBT also did not inhibit the gelatinase-mediated breakdown of the natural substrate, gelatin. The data suggest that EBT may have significant potential for allowing the differentiation in biological fluids of two metalloproteases with similar cleavage site specificities.
间质胶原的细胞内降解是由两种中性金属蛋白酶——胶原酶和明胶酶完成的。这两种酶都受到金属螯合剂、某些巯基试剂以及类似蛋白质抑制剂的抑制。在此，我们证明染料依来铬黑T（EBT）在抑制胶原酶而非明胶酶的能力方面似乎是独特的。在37℃下使用凝胶形式的天然重组螺旋胶原，EBT对胶原酶活性的半数最大抑制浓度约为45微摩尔。EBT能更有效地抑制溶液中天然胶原的降解，其抑制常数（KI）约为8微摩尔。使用新开发的分光光度法底物AcProLeuGly - S - LeuLeuGly - OC2H5，计算得出EBT对胶原酶的KI为1.4微摩尔。尽管这种相同的硫肽内酯作为明胶酶的底物时，其动力学与胶原酶相似，但未观察到EBT对其有抑制作用。EBT也不抑制明胶酶介导的天然底物明胶的降解。这些数据表明，EBT在区分生物体液中具有相似切割位点特异性的两种金属蛋白酶方面可能具有巨大潜力。
