Luaces A L, Barrett A J
Biochemistry Department, Strangeways Research Laboratory, Cambridge, U.K.
Biochem J. 1988 Mar 15;250(3):903-9. doi: 10.1042/bj2500903.
We report a one-step method for the purification to homogeneity of a cysteine proteinase of Entamoeba histolytica (histolysin) by affinity chromatography of the soluble extract of the parasite on immobilized phenylalanyl(2-phenyl)aminoacetaldehyde semicarbazone. The enzyme has an apparent Mr of 26,000 by SDS/polyacrylamide-gel electrophoresis and 29,000 by gel chromatography. Its pH optimum varies widely, from 5.5 with azocasein to approx. 7 with other protein substrates and benzyloxycarbonylphenylalanyl-L-citrullylaminomethylcourmarin++ + (Z-Phe-Cit-NHMec), and to 9.5 with benzyloxycarbonylphenylalanylarginylaminomethylcoumarin (Z-Phe-Arg-NHMec) and benzyloxycarbonylarginylarginylaminomethylcourmarin (Z-Arg-Arg-NHMec). Values of Km, kcat. and kcat/Km are 1.5 microM, 130 s-1 and 87 X 10(6) M-1.s-1 for Z-Arg-Arg-NHMec, and 32 microM, 0.4 s-1 and 0.012 x 10(6) M-1.s-1 for Z-Phe-Arg-NHMec, respectively, at pH 7.5 and 37 degrees C. The enzyme is inhibited by leupeptin and such inhibitors of cysteine proteinases as L-transepoxysuccinyl-L-leucylamido-4-(guanidino)butane, peptidyldiazomethanes, iodoacetic acid and chicken cystatin. The tentative N-terminal amino acid sequence of the enzyme closely resembles that of papain. Histolysin does not degrade type I collagen or elastin, but it is active against cartilage proteoglycan and kidney glomerular basement-membrane collagen. It also detaches cells from their substratum in vitro, and could well play a role in tissue invasion.
我们报道了一种一步法，通过将溶组织内阿米巴（组织溶素）的可溶性提取物在固定化苯丙氨酰（2-苯基）氨基乙醛半卡巴腙上进行亲和层析，将其半胱氨酸蛋白酶纯化至同质。通过SDS/聚丙烯酰胺凝胶电泳，该酶的表观分子量为26,000，通过凝胶色谱法为29,000。其最适pH值变化很大，以偶氮酪蛋白为底物时为5.5，以其他蛋白质底物和苄氧羰基苯丙氨酰-L-瓜氨酰胺甲基香豆素（Z-Phe-Cit-NHMec）为底物时约为7，以苄氧羰基苯丙氨酰精氨酰胺甲基香豆素（Z-Phe-Arg-NHMec）和苄氧羰基精氨酰精氨酰胺甲基香豆素（Z-Arg-Arg-NHMec）为底物时为9.5。在pH 7.5和37℃下，Z-Arg-Arg-NHMec的Km、kcat和kcat/Km值分别为1.5 microM、130 s-1和87×10(6) M-1·s-1，Z-Phe-Arg-NHMec的分别为32 microM、0.4 s-1和0.012×10(6) M-1·s-1。该酶被亮肽素以及半胱氨酸蛋白酶抑制剂如L-反式环氧琥珀酰-L-亮氨酰胺-4-（胍基）丁烷、肽基重氮甲烷、碘乙酸和鸡半胱氨酸蛋白酶抑制剂所抑制。该酶的暂定N端氨基酸序列与木瓜蛋白酶的非常相似。组织溶素不降解I型胶原或弹性蛋白，但对软骨蛋白聚糖和肾小球基底膜胶原具有活性。它还能在体外使细胞从其基质上脱离，很可能在组织侵袭中起作用。
