Studies on the characteristics of aminopeptidase from barley classified flour.

本研究的目的是明确大麦分类粉中氨基肽酶的特性。氨基肽酶是由上述研究人员从大麦分类面粉中获得的。酶的纯化方法如下。1)冷丙酮处理，2)乙酸缓冲液（pH 5.5）萃取，3)sepphacryl S-200HR凝胶过滤，4)聚乙烯-亚胺离子交换层析，5)羟基磷灰石层析，6)sepphacryl S-100HR凝胶过滤层析。用各种合成底物考察了酶活性的最适pH。纯化酶的最适pH值为7.0 ~ 8.0。以亮氨酸-对硝基苯胺为底物，最佳pH为7.0，Km值为0.22mM。苯丙氨酸-对硝基苯胺的水解速度是亮氨酸-对硝基苯胺的2.4倍，而对亚胺酸和芳香胺酸的水解速度约为1/3。该酶不能水解酸性氨基酸对硝基苯胺。苯丙氨酸- β -萘酰胺的最佳pH为8.0，Km为0.047mM。大麦氨基肽酶对-萘基酰胺的亲和力高于对硝基苯胺。对异亮氨酸- β -萘酰胺和丝氨酸- β -萘酰胺的Km值分别为0.15和0.57 mM。该酶不受巯基肽酶抑制剂白细胞介素的抑制。由此可见，大麦分类粉中的氨基肽酶是一种中性氨基肽酶。

The aim of this study is to make clear the caracteristics of aminopeptidase from barley classified flour. Aminopeptidase was obtained from barley classified flour by the investigators described above. The enzyme was purified as follows. 1) cold aceton treatment, 2)extraction with acetic acid buffer(pH 5.5), 3) Sephacryl S-200HR gel filtration, 4)Polyethylene-imine ion exchange chromatography, 5) hyclroxyapatite chromatography, and 6) Sephacryl S-100HR gel filtration chromatography.Optimum pH of enzyme activity was examined using various synthesized substrates. The purifled enzyme showed optimum pH between 7.0 and 8.0. Using leucine-p-nitroanilide as a substrate, optimum pH was 7.0 and Km value 0.22mM.Phenylalanine-p-nitroanilide was hydrolyzed so quickly with the velosity of 2.4 fold compared with that to leucine-p-nitroanilide, whereas the hydrolysis velosities to imino acid and aromatic aminoacis was about 1/3. The enzyme did not hydrolyze p-nitroanilide of acidic amino acid. In the case of phenylalanine- beta-naphtylamide, optimum pH was 8.0 and Km 0.047mM.Aminopeptidase from barley classified had higher affinity to beta-naplitylamide than to p-nitroanilide. The Km values to isoleucine-beta-naphtylamide and serine-beta-naphtylamide were 0.15 and 0.57 mM, respectively. The enzyme was not inhibited by leupeptin, which was an inhibitor to thiol peptidase.It is concludeed that amninopeptidase from barley classified flour is a neutral aminopeptidase.