Jackson M C, Choudry Y, Bourne A, Woodley J F, Kenny A J
Department of Biological Sciences, University of Keele, Staffs, U.K.
Biochem J. 1988 Jul 1;253(1):299-302. doi: 10.1042/bj2530299.
A novel two-step enzyme-linked assay for aminopeptidase W is described and validated by comparison with other assays. L-alpha-Glutamyl-L-tryptophan (Glu-Trp) is a favoured substrate for this enzyme. With the use of glutamate dehydrogenase (EC 1.4.1.2) in a second step, the assay measured the release of free glutamate from L-alpha-glutamyl-L-tryptophan by the increase in NADH fluorescence. In the presence of 5 mM-1,10-phenanthroline and 50 microM-cilastatin the contribution of other membrane peptidases, in particular aminopeptidases N and A and microsomal dipeptidase in kidney, was very small. Residual cytosolic activities hydrolysing Glu-Trp were sensitive to inhibition by 2.5 mM-N-ethylmaleimide. The activity of aminopeptidase W was unaffected by these inhibitors. There was good correlation between the fluorimetric assay and those in which the free tryptophan released by kidney membrane fractions was determined by h.p.l.c. or the aminopeptidase W was measured immunoradiometrically with a monoclonal antibody.
本文描述了一种用于氨肽酶W的新型两步酶联测定法，并通过与其他测定法比较进行了验证。L-α-谷氨酰-L-色氨酸（Glu-Trp）是该酶的首选底物。在第二步中使用谷氨酸脱氢酶（EC 1.4.1.2），该测定法通过NADH荧光的增加来测量L-α-谷氨酰-L-色氨酸中游离谷氨酸的释放。在存在5 mM 1,10-菲咯啉和50 μM西司他丁的情况下，其他膜肽酶，特别是肾脏中的氨肽酶N和A以及微粒体二肽酶的贡献非常小。水解Glu-Trp的残余胞质活性对2.5 mM N-乙基马来酰亚胺的抑制敏感。氨肽酶W的活性不受这些抑制剂的影响。荧光测定法与通过高效液相色谱法测定肾膜组分释放的游离色氨酸或用单克隆抗体免疫放射测定法测量氨肽酶W的方法之间具有良好的相关性。
