Kitajima S, Minakami S
J Biochem. 1983 Feb;93(2):615-20. doi: 10.1093/oxfordjournals.jbchem.a134217.
NADH-cytochrome b5 reductases purified from human red cell membranes and cytosol were compared with those prepared from human liver microsomes. Minimal molecular weights of the membrane and the cytosol enzymes as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were 36,000 and 32,000 daltons, respectively, which are comparable to those of the detergent-solubilized reductase (dfp) and the protease-solubilized one (tfp) of liver microsomes, respectively. All the enzymes contained FAD and had essentially the same turnover numbers and apparent Km values for NADH and protease-solubilized cytochrome b5. The membrane enzyme and liver dfp reduced cytochrome c in the presence of detergent-solubilized cytochrome b5 70-80 times faster than in the presence of trypsin-solubilized cytochrome b5, whereas the cytosol enzyme and liver tfp showed essentially the same low activities with both preparations of cytochrome b5. SDS-PAGE mapping of the limited proteolytic products of the reductases obtained by digestion with staphylococcal protease or a-chymotrypsin showed essentially the same patterns of peptides between the red cell membrane enzyme and liver dfp and between the red cell cytosol enzyme and liver tfp. These results suggest that the NADH-cytochrome b5 reductase of human red cell membranes is identical with that of liver microsomes and that the enzyme of red cell cytosol is a proteolytic product of the membrane enzyme.
将从人红细胞膜和胞质溶胶中纯化得到的NADH-细胞色素b5还原酶与从人肝微粒体中制备的还原酶进行了比较。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定，膜酶和胞质溶胶酶的最小分子量分别为36,000和32,000道尔顿，分别与人肝微粒体中去污剂增溶的还原酶（dfp）和蛋白酶增溶的还原酶（tfp）的分子量相当。所有酶都含有黄素腺嘌呤二核苷酸（FAD），并且对于NADH和蛋白酶增溶的细胞色素b5具有基本相同的周转数和表观米氏常数（Km值）。在去污剂增溶的细胞色素b5存在下，膜酶和肝dfp还原细胞色素c的速度比在胰蛋白酶增溶的细胞色素b5存在下快70-80倍，而胞质溶胶酶和肝tfp在两种细胞色素b5制剂中表现出基本相同的低活性。用葡萄球菌蛋白酶或α-胰凝乳蛋白酶消化得到的还原酶有限蛋白水解产物的SDS-PAGE图谱显示，红细胞膜酶与肝dfp之间以及红细胞胞质溶胶酶与肝tfp之间的肽谱基本相同。这些结果表明，人红细胞膜的NADH-细胞色素b5还原酶与肝微粒体的相同，并且红细胞胞质溶胶中的酶是膜酶的蛋白水解产物。
