Okamoto Y, DeLuca H F
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Proc Soc Exp Biol Med. 1994 Jan;205(1):52-5. doi: 10.3181/00379727-205-43676.
We previously reported the development of monoclonal antibodies against chick kidney mitochondrial 1,25-dihydroxyvitamin D3 24-hydroxylase. However, these monoclonal antibodies were able to immunoprecipitate only approximately 25% of total 24-hydroxylase activity present in solubilized preparations, suggesting the existence of different forms of the enzyme. Chromatography of the solubilized preparations on a mono-Q column resolved the 24-hydroxylase activity into two peaks. The protein found in the first peak of enzyme activity (peak 1) did not immunoblot with the monoclonal antibody (IVC2F10) that was raised against the partially purified 24-hydroxylase. However, the protein in the second peak (peak 2) reacted on immunoblot with the same antibody. The enzyme for both peaks had similar kinetics, required adrenodoxin and adrenodoxin reductase, and acted on both 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3. It is not yet clear whether these are two distinct enzymes or whether it is the same enzyme complexed with different proteins.
我们之前报道了针对鸡肾线粒体1,25 - 二羟基维生素D3 24 - 羟化酶的单克隆抗体的研发情况。然而，这些单克隆抗体仅能免疫沉淀溶解制剂中约25%的总24 - 羟化酶活性，这表明存在该酶的不同形式。将溶解制剂在单Q柱上进行色谱分析，可将24 - 羟化酶活性分离为两个峰。在酶活性的第一个峰（峰1）中发现的蛋白质不能与针对部分纯化的24 - 羟化酶产生的单克隆抗体（IVC2F10）发生免疫印迹反应。然而，第二个峰（峰2）中的蛋白质在免疫印迹上能与同一抗体发生反应。两个峰的酶具有相似的动力学特性，需要肾上腺皮质铁氧化还原蛋白和肾上腺皮质铁氧化还原蛋白还原酶，并且作用于1,25 - 二羟基维生素D3和25 - 羟基维生素D3。目前尚不清楚这是两种不同的酶，还是与不同蛋白质复合的同一种酶。
