Park H, Yamanaka N, Mikkonen A, Kusakabe I, Kobayashi H
Institute of Applied Biochemistry, University of Tsukuba, Ibaraki, Japan.
Biosci Biotechnol Biochem. 2000 May;64(5):931-9. doi: 10.1271/bbb.64.931.
Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.
通过在胃蛋白酶抑制剂A - EAH琼脂糖柱上进行亲和层析以及在Mono Q柱上进行层析，从向日葵种子提取物中纯化天冬氨酸蛋白酶。最终制剂包含三个纯化组分。SDS - PAGE显示其中一个组分由二硫键连接的亚基（29 kDa和9 kDa）组成，另外两个组分包含非共价结合的亚基（29 kDa和9 kDa）。这些纯化的酶在pH 3.0时对血红蛋白分解活性表现出最佳pH值，并且像其他典型的天冬氨酸蛋白酶一样被胃蛋白酶抑制剂A完全抑制。与其他天冬氨酸蛋白酶相比，向日葵酶对氧化胰岛素B链和胰高血糖素的特异性更有限。编码一种天冬氨酸蛋白酶的cDNA被克隆并测序。推导的氨基酸序列表明成熟酶由440个氨基酸残基组成，分子量为47,559 Da。纯化酶和成熟酶分子大小之间的差异是由于纯化酶是成熟酶经过蛋白水解加工形成的异二聚体。该酶推导的氨基酸序列与其他天冬氨酸蛋白酶的序列一致性为30 - 78%。
