Feinstein G, Janoff A
Biochim Biophys Acta. 1975 Oct 22;403(2):477-92. doi: 10.1016/0005-2744(75)90076-5.
A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.
一种类胰凝乳蛋白酶（EC 3.4.21.-）从人嗜中性粒细胞（多形核白细胞）的颗粒中纯化得到。分离过程包括对颗粒进行分级盐提取，然后在4-苯基丁胺-琼脂糖凝胶上进行亲和层析。这种快速纯化方法在短时间内以相对较高的产率获得了纯酶。根据其氨基酸组成计算，纯化的粒细胞类胰凝乳蛋白酶的最小相对分子质量为22378。通过十二烷基硫酸钠凝胶电泳获得的相对分子质量值为20000 - 23000。该酶不与对粒细胞弹性蛋白酶具有单特异性的抗体发生反应。粒细胞类胰凝乳蛋白酶被二异丙基氟磷酸酯（Dip-F）以及氯甲基酮衍生物Z-苯丙氨酸氯甲基酮（Z-PheCH2Cl）和Z-（甘氨酸）2-苯丙氨酸氯甲基酮（Z-(Gly)2-PheCH2Cl）灭活，但不被甲苯磺酰苯丙氨酸氯甲基酮（Tos-PheCH2Cl）灭活。因此，该酶在其活性位点似乎具有丝氨酸和组氨酸侧链，类似于胰凝乳蛋白酶。粒细胞酶的底物特异性与对甲苯磺酰-L-酪氨酸对硝基苯胺（pac-Tyr-Nan）和乙酰基-L-苯丙氨酸对硝基萘酯（Ac-Phe-1-ONap）相似。它对一些经典的弹性蛋白酶底物如叔丁氧羰基-L-丙氨酸对硝基苯酯（Boc-Ala-ONp）和乙酰基-DL-丙氨酸对硝基萘酯（Ac-DL-Ala-1-ONap）也具有内在的微弱水解活性。粒细胞酶受到人血清和人α1-抗胰蛋白酶的抑制。它对α1-抗胰蛋白酶的亲和力比对相同抑制剂的粒细胞弹性蛋白酶的亲和力弱。该酶在37℃中性pH条件下稳定，但在pH 3.5和高温下不稳定。
