Yamada R, Mizutani S, Kurauchi O, Okano K, Imaizumi H, Narita O, Tomoda Y
Department of Obstetrics and Gynaecology, Nagoya University School of Medicine, Japan.
Enzyme. 1988;40(4):223-30. doi: 10.1159/000469167.
Human placental aminopeptidase A (AAP) was purified 3,900-fold from human placenta and characterized. The enzyme was solubilized from membrane fractions with Triton X-100, then subjected to trypsin digestion, zinc sulfate fractionation, chromatographies with DE-52, Sephacryl S-300, and hydroxylapatite, affinity chromatography with Bestatin-Sepharose 4B, and finally immunoaffinity chromatography with the antibody against microsomal leucine aminopeptidase (LAP). Aminopeptidase A was completely separated from leucine aminopeptidase by the immunoaffinity chromatography. The apparent relative molecular mass (Mr) of the enzyme was estimated to be 280,000 by gel filtration. The purified enzyme was most active at pH 7.1 with L-aspartyl-beta-naphthylamide (L-Asp-NA) as substrate; the Km value for this substrate was 4.0 mmol/l in the presence of Ca2+. Human placental aminopeptidase A was markedly activated by alkaline earth metals (Ca2+, Sr2+, Ba2+), but strongly inhibited by metal chelating agents such as EDTA and o-phenanthroline. The highest activity was observed with L-glutamyl-beta-naphthylamide, while only minimal hydrolysis was found with some neutral and basic amino acid beta-naphthylamides.
人胎盘氨肽酶A（AAP）从人胎盘中纯化出来，纯化倍数达3900倍，并对其进行了特性鉴定。该酶用Triton X-100从膜组分中溶解出来，然后进行胰蛋白酶消化、硫酸锌分级分离、用DE-52、Sephacryl S-300和羟基磷灰石进行层析、用Bestatin-Sepharose 4B进行亲和层析，最后用抗微粒体亮氨酸氨肽酶（LAP）抗体进行免疫亲和层析。通过免疫亲和层析，氨肽酶A与亮氨酸氨肽酶完全分离。通过凝胶过滤法估计该酶的表观相对分子质量（Mr）为280,000。以L-天冬氨酰-β-萘酰胺（L-Asp-NA）为底物时，纯化后的酶在pH 7.1时活性最高；在Ca2+存在的情况下，该底物的Km值为4.0 mmol/l。人胎盘氨肽酶A被碱土金属（Ca2+、Sr2+、Ba2+）显著激活，但被金属螯合剂如EDTA和邻菲罗啉强烈抑制。用L-谷氨酰-β-萘酰胺时观察到最高活性，而用一些中性和碱性氨基酸β-萘酰胺时仅发现极少的水解作用。
