Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence.
Proteinases in human polymorphonuclear leukocytes. Purification and characterization of an enzyme which cleaves denatured collagen and a synthetic peptide with a Gly-Ile sequence.
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人多形核白细胞中的蛋白酶。
DOI：
10.1111/j.1432-1033.1983.tb07541.x
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发表时间：
1983
期刊：
影响因子：
--
通讯作者：
Uitto,J
中科院分区：
文献类型：
--
作者：
Rantala-Ryhänen,S;Ryhänen,L;Nowak,FV;Uitto,J
多形核白细胞已被证明含有蛋白水解酶，能够降解结缔组织蛋白，如天然胶原蛋白。本研究从人多形核白细胞中提取蛋白水解酶，并对一种中性蛋白酶进行了广泛的纯化和表征。该酶的活性通过降解变性[3H]脯氨酸标记的I型胶原蛋白或通过裂解合成的具有Gly - Ile序列的二硝基苯化肽来监测。该酶通过刀豆蛋白- a - sepharose亲和层析从白细胞胶原酶中分离出来，并通过QAE - sehadex离子交换层析和Sephacryl S - 200凝胶过滤进一步纯化。纯化后的酶分子量约为105 000，最适pH值约为7.8，可被Na2EDTA和二硫苏糖醇抑制，但不受胎牛血清的抑制。该酶在非螺旋形态下降解基因上不同的I、II、III、IV和V型胶原，而在天然三螺旋形态下则不降解。Dansyl监测的端组分析，结合羧肽酶A的消化，表明该酶在Gly - Xaa序列上裂解变性I型胶原，其中Xaa可以是亮氨酸、异亮氨酸、缬氨酸、苯丙氨酸、赖氨酸或蛋氨酸。因此，这里提到的纯化酶Gly - Xaa蛋白酶是一种中性蛋白酶，它可能在炎症疾病过程中通过进一步降解胶原蛋白肽而发挥重要作用，这些胶原蛋白肽由于胶原酶裂解而呈现非螺旋状。
Polymorphonuclear leukocytes have been shown to contain proteolytic enzymes which are capable of degrading connective tissue proteins such as native collagen. In this study, proteolytic enzymes were extracted from human polymorphonuclear leukocytes and a neutral proteinase was extensively purified and characterized. The activity of this enzyme was monitored by degradation of denatured [3H]proline‐labeled type I collagen or by cleavage of a synthetic dinitrophenylated peptide with a Gly‐Ile sequence. The enzyme was readily separated from leukocyte collagenase by concanavalin–A–Sepharose affinity chromatography and further purified by QAE‐Sephadex ion‐exchange chromatography and gel filtration on Sephacryl S‐200. The purified enzyme had a molecular weight of approximately 105 000, its pH optimum was about 7.8, and it was inhibited by Na2EDTA and dithiothreitol, but not by fetal calf serum. The enzyme degraded genetically distinct type I, II, III, IV and V collagens, when in a non‐helical form, but not when in native triple‐helical conformation. Dansyl‐monitored end‐group analyses, combined with digestion by carboxypeptidase A, indicated that the enzyme cleaved denaturated type I collagen at Gly‐Xaa sequences, in which Xaa can be leucine, isoleucine, valine, phenylalanine, lysine, or methionine. Thus, the purified enzyme referred to here as Gly‐Xaa proteinase, is a neutral proteinase, which may be of importance in inflammatory disease processes by degrading further collagen peptides which have been rendered non‐helical as a result of collagenase cleavage.