Konishi Y, Mimura Y
Department of Ophthalmology, School of Medicine, Tokushima University, Japan.
Exp Eye Res. 1992 Oct;55(4):569-78. doi: 10.1016/s0014-4835(05)80170-8.
The major soluble protein of bovine cornea (BCP 54: bovine corneal protein 54 kDa) was isolated successively by gel filtration, anion-exchange chromatography and chromatofocusing. The amino acid sequence of a fragment of the purified BCP 54 obtained by lysyl-endopeptidase digestion showed marked homology with tumor-associated and 2,3,7,8-tetrachloro-dibenzo-p-dioxin-inducible aldehyde dehydrogenase (AIDH). From the high similarity of BCP 54 with tumor-associated AIDH in structural form, it is suggested that BCP 54 has AIDH activity. We confirmed a high AIDH activity of BCP 54 by immunoprecipitation using a mouse anti-BCP 54 monoclonal antibody followed by a spectrophotometric assay for AIDH activity. Next we demonstrated the unique properties of the purified BCP 54 as AIDH. The major isoelectric point is 6.41. BCP 54 preferentially oxidizes aromatic aldehyde such as benzaldehyde with NAD as coenzyme, but cannot oxidize phenylacetaldehyde. After heat treatment the AIDH activity is more stable with propionaldehyde-NAD than with benzaldehyde-NADP. With propionaldehyde-NAD the pH profile shows a broad plateau from pH 6-9 followed by a sharp rise up to pH 10. In contrast, with benzaldehyde-NADP there is a sharp optimum at pH 9.0. The activity with only benzaldehyde-NADP is inhibited by p-hydroxymercuribenzoate, but is not affected by disulfiram and diethylstilbestrol. So we suggested that BCP 54 is an AIDH with kinetic properties different from the rat tumor-associated AIDH.
牛角膜的主要可溶性蛋白（BCP 54：牛角膜蛋白54千道尔顿）先后通过凝胶过滤、阴离子交换色谱和色谱聚焦法进行分离。通过赖氨酰内肽酶消化获得的纯化BCP 54片段的氨基酸序列显示出与肿瘤相关的以及2,3,7,8 - 四氯二苯并 - p - 二恶英诱导型醛脱氢酶（AIDH）有显著同源性。从BCP 54与肿瘤相关AIDH在结构形式上的高度相似性来看，提示BCP 54具有AIDH活性。我们通过使用小鼠抗BCP 54单克隆抗体进行免疫沉淀，随后进行AIDH活性的分光光度测定，证实了BCP 54具有高AIDH活性。接下来我们展示了纯化的BCP 54作为AIDH的独特性质。主要等电点为6.41。BCP 54以NAD作为辅酶时优先氧化芳香醛如苯甲醛，但不能氧化苯乙醛。热处理后，AIDH活性对于丙醛 - NAD比对于苯甲醛 - NADP更稳定。对于丙醛 - NAD，pH曲线在pH 6 - 9显示出一个宽平台，随后在pH 10急剧上升。相比之下，对于苯甲醛 - NADP，在pH 9.0有一个尖锐的最佳值。仅苯甲醛 - NADP的活性受到对羟基汞苯甲酸的抑制，但不受双硫仑和己烯雌酚的影响。因此我们认为BCP 54是一种具有与大鼠肿瘤相关AIDH不同动力学性质的AIDH。
