Mandel M L, Moorthy B, Swartz S J, Garancis J C, Ghazarian J G
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
J Clin Lab Immunol. 1990 Jan;31(1):1-10.
The kidney mitochondrial monooxygenases known as 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases are two analogous enzymes which utilize the vitamin as a common substrate for the catalytic production of 1 alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. These two enzymes are complexes of NADPH-ferredoxin reductase, and an (Fe-S)-cluster containing ferredoxin with a redox potential that allows the ultimate transfer of reducing equivalents to the terminal oxidases distinctly known as cytochromes P-450(1) alpha and P-450(24). We have used in vitro immunizations of splenocytes obtained from mice sensitized with the purified cytochrome P-450(1) alpha to generate three hybridoma clones from fusion with p3 x 63.Ag8.653 myeloma ATCC cells which selectively secrete monoclonal antibodies (MAbs) of the IgM class. The MAbs have been partially purified by ammonium sulfate fractionation followed by size separation chromatography on Sephacryl S-200-HR. We have compared the structural similarities and differences between the two kidney enzymes in Western Blot analyses using horseradish peroxidase conjugated goat anti-mouse Igs specific for the heavy and light chains of mouse IgA, IgG and IgM. The MAbs from all three clones recognized and interacted with apparent common epitopes of the two hydroxylases but selectively discriminated against liver microsomal P-450LM2 type and adrenal mitochondrial P-450SCC cytochromes. The cytochromes P-450(1) alpha and P-450(24) were detected as two separate bands with approximate molecular weights of 57 and 55 KDa, respectively. In reconstitution of hydroxylase activities in vitro, the MAbs were equally effective in inhibiting the 1 alpha-hydroxylation and 24R-hydroxylation reactions. The ratio of micrograms of Igs to pmol cytochrome P-450 for a 50% inhibition of either activity was approximately 25. These results, collectively, seem to suggest the existence of a precursor-product relationship between the kidney mitochondrial 1 alpha- and the 24R-hydroxylases, or perhaps, a common ancestral origin. Immunochemical peroxidase anti-peroxidase staining of kidney tissue first exposed to the MAbs revealed that only the proximal tubular segment of the nephron was specifically enriched with the cytochromes.
被称为25-羟基维生素D3 1α-羟化酶和24R-羟化酶的肾脏线粒体单加氧酶是两种类似的酶，它们利用维生素作为共同底物，催化生成1α,25-二羟基维生素D3和24R,25-二羟基维生素D3。这两种酶是NADPH-铁氧化还原蛋白还原酶的复合物，以及一种含有铁氧化还原蛋白的（Fe-S）簇，其氧化还原电位允许将还原当量最终转移到被明确称为细胞色素P-450(1)α和P-450(24)的末端氧化酶上。我们使用从小鼠脾脏细胞中获得的脾细胞进行体外免疫，这些小鼠用纯化的细胞色素P-450(1)α致敏，以与p3 x 63.Ag8.653骨髓瘤ATCC细胞融合，产生三个杂交瘤克隆，这些克隆选择性分泌IgM类单克隆抗体（MAb）。通过硫酸铵分级分离，然后在Sephacryl S-200-HR上进行尺寸分离色谱法，对MAb进行了部分纯化。我们在蛋白质印迹分析中使用辣根过氧化物酶偶联的山羊抗小鼠Ig，该Ig对小鼠IgA、IgG和IgM的重链和轻链具有特异性，比较了两种肾脏酶之间的结构异同。来自所有三个克隆的MAb识别并与两种羟化酶的明显共同表位相互作用，但选择性地区分肝微粒体P-450LM2型和肾上腺线粒体P-450SCC细胞色素。细胞色素P-450(1)α和P-450(24)被检测为两条单独的条带，分子量分别约为57和55 kDa。在体外重建羟化酶活性时，MAb在抑制1α-羟化和24R-羟化反应方面同样有效。对于50%抑制任何一种活性，Igs微克与细胞色素P-450 pmol的比率约为25。总体而言，这些结果似乎表明肾脏线粒体1α-羟化酶和24R-羟化酶之间存在前体-产物关系，或者可能存在共同的祖先起源。首先用MAb处理肾脏组织的免疫化学过氧化物酶抗过氧化物酶染色显示，只有肾单位的近端小管段特异性富含细胞色素。
