PURIFICATION OF DIFFERENT AMPHIPHILIC FORMS OF A MICROVILLUS AMINOPEPTIDASE FROM PIG SMALL-INTESTINE USING IMMUNOADSORBENT CHROMATOGRAPHY
PURIFICATION OF DIFFERENT AMPHIPHILIC FORMS OF A MICROVILLUS AMINOPEPTIDASE FROM PIG SMALL-INTESTINE USING IMMUNOADSORBENT CHROMATOGRAPHY
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DOI：
10.1111/j.1432-1033.1978.tb12476.x
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发表时间：
1978-01-01
期刊：
影响因子：
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通讯作者：
CHRISTIANSEN, L
中科院分区：
文献类型：
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作者：
SJOSTROM, H;NOREN, O;CHRISTIANSEN, L
用一种快速简便的免疫吸附技术纯化了溶于去污剂的猪肠微绒毛氨肽酶（微粒体氨肽酶，EC 3.4.11.2）约150倍，收率约20%。交叉免疫电泳和十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示纯化的酶是同质的。它也基本上不含其他已知的肽酶和二硫键合酶。通过电荷位移交叉免疫电泳，在更耗时的纯化过程中以及在储存过程中连续产生亲水形式。快速纯化程序的开发允许制备不含亲水性的两亲性形式，其可以通过0.5 mM HgCl 2稳定。从处死前3天结扎胰管的猪中分离的肠氨肽酶在聚丙烯酰胺凝胶电泳中迁移为分子量为162，000的单链多肽。当在Kunitz胰蛋白酶抑制剂存在下纯化时，来自具有完整胰管的猪的酶由3条多肽链组成（MW 162，000、123，000和61，000）。体外胰蛋白酶处理将大的多肽链转化为2个较小的多肽链。生物合成后的膜结合酶可以被胰蛋白酶和/或胰凝乳蛋白酶修饰。两亲性形式向亲水性形式的转化和大多肽链向2个较小多肽链的水解代表不同的反应。
A detergent-solubilized pig intestinal microvillus aminopeptidase (microsomal aminopeptidase, EC 3.4.11.2) was purified approximately 150 times with a yield of about 20% by a rapid and simple immunoadsorbent technique. The purified enzyme was shown to be homogeneous by crossed immunoelectrophoresis and by polyacrylamide gel electrophoresis in dodecyl sulfate. It was also essentially free from other known peptidases and disaccharidases. By charge-shift crossed immunoelectrophoresis, the hydrophilic form was continuously generated during a more time-consuming purification procedure and also during storage. The development of a rapid purification procedure allowed the preparation of an amphiphilic form free from the hydrophilic one, which could be stabilized by 0.5 mM HgCl2. Intestinal aminopeptidase, isolated from a pig having the pancreatic duct ligated 3 days before sacrifice, migrated in polyacrylamide gel electrophoresis as a single polypeptide chain with a MW of 162,000. The enzyme from a pig with intact pancreatic duct, when purified in the presence of Kunitz trypsin inhibitor consisted of 3 polypeptide chains (MW 162,000, 123,000 and 61,000). Trypsin treatment in vitro converted the large polypeptide chain into the 2 smaller ones. The membrane-bound enzyme after biosynthesis may be modified by trypsin and/or chymotrypsin. The conversion of the amphiphilic form to the hydrophilic one and the hydrolysis of the large polypeptide chain into the 2 smaller ones represented different reactions.