Irvine J W, Roberts S F, Lindberg I
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70112.
Anal Biochem. 1990 Oct;190(1):141-6. doi: 10.1016/0003-2697(90)90147-2.
A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.
本文描述了一种新方法，用于在含有共聚放射性标记蛋白质底物（如[35S]甲硫氨酸标记的前脑啡肽或125I标记的胰岛素原）的十二烷基硫酸钠-聚丙烯酰胺凝胶中进行蛋白酶的酶谱分析。电泳后，酶被重新激活，并将放射性标记的原位底物切割成较小的肽段。这些小肽段能够扩散出凝胶，通过放射自显影观察时，在深色背景下会留下清晰区域。该技术仅使用50 ng（1.25微居里）的[35S]前脑啡肽就能检测低至200 fg的胰蛋白酶。从牛肾上腺嗜铬颗粒中分离出可溶性和膜结合的肾上腺类胰蛋白酶。这两种蛋白酶都能切割[35S]甲硫氨酸标记的前脑啡肽，但不能切割125I标记的胰岛素原。此外，两者的分子量约为30,000。讨论了该技术的普遍应用潜力。还描述了另一种使用合成荧光底物叔丁氧羰基-Glu-Lys-Lys-氨基甲基香豆素的方法。
