Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil elastase from normal donors.
Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil elastase from normal donors.
复制标题
来自正常供体的人中性粒细胞弹性蛋白酶的分离、表征和氨基末端氨基酸序列分析。
DOI：
10.1016/0003-2697(85)90488-9
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发表时间：
1985
影响因子：
2.9
通讯作者：
Bennett,JC
中科院分区：
文献类型：
--
作者：
Heck,LW;Darby,WL;Hunter,FA;Bhown,A;Miller,EJ;Bennett,JC
用氯化钠提取、抑肽酶-琼脂糖亲和层析、CM-纤维素离子交换层析和AcA44凝胶过滤层析等步骤从正常供体中分离纯化了人中性粒细胞弹性蛋白酶。最后一个纯化步骤的加入对于从中性粒细胞弹性蛋白酶的活性形式中分离出非活性的低分子量物种是至关重要的，并导致最终制剂的比活性更高。十二烷基硫酸钠-聚丙烯酰胺梯度凝胶电泳法测定纯化蛋白的相对分子质量为31,000，28,000和27,500。用酸性凝胶电泳法对四种多肽进行了分离，每种多肽都具有酰胺化活性。此外，对金黄色葡萄球菌V8蛋白酶切产物进行的多肽分析表明，这些多肽在结构上是相互关联的，代表了纯化蛋白的微观异质性。用双向电泳法测定这四种形式的表观等电点在6.1~6.7之间。利用微测序技术，测定了中性粒细胞弹性蛋白酶的前40个残基，并与已报道的从白血病髓系细胞中分离的弹性蛋白酶序列进行了比较。此外，中性粒细胞弹性蛋白酶的氨基末端与丝氨酸蛋白酶、猪弹性蛋白酶、牛胰凝乳酶、人因子D和纤溶酶的β链具有高度的同源性。
Human neutrophil elastase from normal donors has been purified using an isolation procedure which included sequential sodium chloride extraction, Aprotinin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil elastase and resulted in a higher specific activity of the final preparation. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis of the reduced purified protein demonstrated three polypeptides of Mr31,000, 28,000, and 27,500. Four polypeptides were resolved on acid gel electrophoresis; each of the four possessed amidolytic activity. Furthermore, peptide analysis of Staphylococcus aureus V8 protease digests indicated that these polypeptides are structurally related to aach other and represent microheterogeneity of the purified protein. The apparent isoelectric points of these four forms as determined by two-dimensional electrophoresis range from 6.1 to 6.7. By utilizing microsequencing techniques, the first 40 residues of neutrophil elastase have been determined and compared with the reported sequence of elastase isolated from leukemic myeloid cells. In addition, a high degree of homology was found within the amino-terminal regions of neutrophil elastase and the serine proteinases porcine elastase, bovine chymotrypsin, human factor D, and the β chain of plasmin.