一种能水解琥珀酰丙氨酰-4-硝基苯胺的大鼠肾脏中性内肽酶的部分纯化及特性研究

Partial purification and characterization of a rat kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide.

作者信息

Katayama K, Kuwada M

出版信息

Biochim Biophys Acta. 1984 Jun 14;787(2):138-45. doi: 10.1016/0167-4838(84)90072-4.

Abstract

The activity for the hydrolysis of succinyl trialanine-4-nitroanilide was higher in kidney homogenates of female rats and mice than in those of male rats and mice. An enzyme hydrolyzing the above substrate was extracted from female rat kidney homogenate and partially purified by means of gel filtration on Sepharose 4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme cleaved the bond between succinyl dialanine and alanine-4-nitroanilide of the substrate and showed a Km value of 3.3 mM at the optimal pH of 7.5. The activity was increased by Ca2+ and Mg2+, but inhibited by EDTA. With oxidized insulin B chain as a substrate, the enzyme cleaved the carbonyl bonds of Ala-14, Tyr-16 and Gly-23 efficiently, and those of His-5 and His-10 less efficiently.

摘要

雌性大鼠和小鼠肾脏匀浆中琥珀酰三丙氨酸 - 4 - 硝基苯胺水解活性高于雄性大鼠和小鼠。从雌性大鼠肾脏匀浆中提取了一种水解上述底物的酶，并通过在Sepharose 4B上进行凝胶过滤、在DEAE - Sepharose CL - 6B上进行阴离子交换色谱以及在苄氧羰基 - L - 丙氨酸 - L - 丙氨酸 - D - 丙氨酸 - 聚赖氨酸 - 琼脂糖上进行亲和色谱进行了部分纯化。纯化后的酶可裂解底物中琥珀酰二丙氨酸与丙氨酸 - 4 - 硝基苯胺之间的键，在最适pH 7.5时Km值为3.3 mM。该活性可被Ca2+和Mg2+增强，但被EDTA抑制。以氧化胰岛素B链为底物时，该酶可有效裂解Ala - 14、Tyr - 16和Gly - 23的羰基键，对His - 5和His - 10羰基键的裂解效率较低。