PURIFICATION OF INSULIN-SPECIFIC PROTEASE BY AFFINITY CHROMATOGRAPHY
PURIFICATION OF INSULIN-SPECIFIC PROTEASE BY AFFINITY CHROMATOGRAPHY
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DOI：
10.1073/pnas.69.12.3698
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发表时间：
1972-01-01
影响因子：
11.1
通讯作者：
KITABCHI, AE
中科院分区：
文献类型：
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作者：
DUCKWORT.WC;HEINEMAN.MA;KITABCHI, AE
从大鼠骨骼肌中分离出一种蛋白水解降解胰岛素的酶。该酶通过一系列步骤纯化1000倍，包括在B链的NH 2-末端苯丙氨酸处与琼脂糖结合的胰岛素上进行亲和层析。在B-29赖氨酸残基处与琼脂糖连接的胰岛素不与酶结合，因此不适合纯化程序。连接在苯丙氨酸残基上的胰岛素是酶的底物，并被其降解;连接在赖氨酸残基上的琼脂糖上的胰岛素不被酶降解。纯化的酶制剂在聚丙烯酰胺凝胶电泳上产生一条主带，洗脱该区域的凝胶产生胰岛素降解活性。纯化的酶降解胰岛素但不降解胰岛素原，胰岛素的aKm为22 nM，胰岛素原的aKi为40 nM。该酶是巯基依赖性的，具有生理最适pH。
A single enzyme that proteolytically degrades insulin was isolated from rat skeletal muscle. This enzyme was purified 1000-fold by a series of steps, including affinity chromatography on insulin bound to agarose at the NH2-terminal phenylalanine of the B chain. Insulin linked to agarose at the B-29 lysine residue did not bind the enzyme and, therefore, was not suitable for purification procedures. Insulin linked at the phenylalanine residue was a substrate for the enzyme and was degraded by it; insulin attached to agarose at the lysine residue was not degraded by the enzyme. The purified enzyme preparation yielded one major band on polyacrylamide gel electrophoresis, and elution of this area of the gel yielded insulin-degrading activity. The purified enzyme degraded insulin but not proinsulin, with aKmfor insulin of 22 nM and aKifor proinsulin of 40 nM. The enzyme is sulfhydryl-dependent, with a physiological pH optimum.