Yamazaki Y, Furukawa F, Nishikawa A, Takahashi M, Oka S
National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba, Ibaraki, Japan.
Biotech Histochem. 1998 Jan;73(1):23-31. doi: 10.3109/10520299809140503.
Esterases in normal hamster pancreas and pancreatic tubular adenocarcinoma of ductal origin induced by N-nitrosobis(2-oxopropyl)amine were stained in cryostat sections with mixtures of a diazonium salt (fast blue RR) and with each of the enantiomers of alpha-naphthyl N-methoxycarbonylalaninate, N-methoxycarbonylvalinate, and N-acetylprolinate. Azo coupling of alpha-naphthol formed by enzymatic hydrolysis with the diazonium salt gives an azo dye that indicates the presence and amount of the enzyme activity in situ. Comparison between the color intensities obtained with each of the enantiomers of a chiral alpha-naphthyl ester shows the stereoselectivity, or enantiomeric preference, of the enzyme. Esterases in acinar cells of the normal pancreas showed slight stereoselectivity for N-methoxycarbonylalaninate, while esterases in fat cells scattered throughout the exocrine pancreas showed high stereoselectivity for (R)-N-acetylprolinate. These esterase activities were not found in the tumor, but another prominent esterase activity with high stereoselectivity for (S)-N-methoxycarbonylvalinate was found. Similar results were obtained by staining after polyacrylamide gel electrophoresis showing that the bands of esterases in the adenocarcinoma stained only with the S enantiomer of the N-methoxycarbonylvalinate. The present method is a valuable tool for designing anticancer prodrugs that are activated by tumor-specific esterases.
用重氮盐（固蓝RR）与α-萘基N-甲氧基羰基丙氨酸酯、N-甲氧基羰基缬氨酸酯和N-乙酰脯氨酸酯的对映体混合物，对正常仓鼠胰腺以及由N-亚硝基双（2-氧代丙基）胺诱导产生的导管源性胰腺管状腺癌的冷冻切片中的酯酶进行染色。酶促水解形成的α-萘酚与重氮盐发生偶氮偶联，生成一种偶氮染料，可原位指示酶活性的存在和量。比较手性α-萘基酯的每种对映体所获得的颜色强度，可显示该酶的立体选择性或对映体偏好性。正常胰腺腺泡细胞中的酯酶对N-甲氧基羰基丙氨酸酯表现出轻微的立体选择性，而分布于整个外分泌胰腺中的脂肪细胞中的酯酶对（R）-N-乙酰脯氨酸酯表现出高立体选择性。在肿瘤中未发现这些酯酶活性，但发现了另一种对（S）-N-甲氧基羰基缬氨酸酯具有高立体选择性的显著酯酶活性。通过聚丙烯酰胺凝胶电泳后染色也获得了类似结果，表明腺癌中酯酶条带仅用N-甲氧基羰基缬氨酸酯的S对映体染色。本方法是设计由肿瘤特异性酯酶激活的抗癌前药的有价值工具。
