Abraham N G, Mitrione S M, Hodgson W J, Levere R D, Shibahara S
Department of Medicine, New York Medical College Valhalla 10595.
Adv Exp Med Biol. 1988;241:97-116. doi: 10.1007/978-1-4684-5571-7_13.
Heme oxygenase has been purified to electrophoretic homogeneity from detergent solubilized adult human liver microsomes. Treatment of microsomes with Triton X-100, sodium cholate and subsequent batchwise DEAE-cellulose, 2', 5' ADP-sepharose 4B, Sepharose CLB and hydroxylapatite column resulted in 17% yield of the purified heme oxygenase. The reconsituted system of heme oxygenase, composed of heme oxygenase, NADPH cytochrome c (P450) reductase and biliverdin reductase was equiactive with 1 mM NADPH and 4 nM NADH and showed complete dependence on added heme for catalytic activity. The Km values for NADPH and NADH were .046 and .526 mM, respectively. While NADPH concentration was held constant, the Km value for heme was 1.01 microM with a specific activity of 583 unit/mg protein. The activity of the reconstituted heme oxygenase system was not affected by preincubation with heavy metals despite their inhibitory effect of NADPH cytochrome c (P450) reductase and biliverdin reductase. However, the metalloporphyrins of these heavy metals were found to be strong inhibitors of the reconsituted system with Ki values of 0.015, 0.6, 2.3 and 5 microM for Sn-, Co-, Zn- and Mg- protoporphyrins, respectively. Similarly, the sulfhydryl inactivating reagents, HgCl2, iodoacetamide and p-chloromercurylbenzoate, inhibited the reconstituted heme oxygenase activity. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from rat and human liver as well as HepG2 cells were identified on dot and Western blots by their reaction with the anti-heme oxygenase similar to the purified enzyme protein. Anti-heme oxygenase precipitated quantitatively, the entire heme oxygenase of rat liver microsomes obtained from animals maintained on standard diet. The human bone marrow microsomal heme oxygenase activity was also quantitatively precipitated by this antibody. Antibody inhibition of rat and human heme xoygenase demonstrated a degree of conservation of both enzyme proteins between the species. As judged by Western blotting, the anti-heme oxygenase recognized only a single protein in spleen, liver, kidney, brain, heart, bone marrow, integtine and corneal epithelium. The human heme oxygenase cDNA was isolated by screening a cDNA library in the Okayama-Berg vector with a rat liver cDNA and was subjected to nucleotide sequence analysis. The deducted human heme oxygenase is also composed of 288 amino acids with a molecular mass of 32,800 Da.(ABSTRACT TRUNCATED AT 400 WORDS)
血红素加氧酶已从经去污剂溶解的成人肝脏微粒体中纯化至电泳纯。用 Triton X - 100、胆酸钠处理微粒体，随后经分批离子交换层析（DEAE - 纤维素柱）、亲和层析（2', 5' ADP - 琼脂糖 4B 柱）、凝胶过滤层析（琼脂糖 CLB 柱）和羟基磷灰石柱层析，得到了产率为 17%的纯化血红素加氧酶。由血红素加氧酶、NADPH - 细胞色素 c（P450）还原酶和胆绿素还原酶组成的重组血红素加氧酶系统，在 1 mM NADPH 和 4 nM NADH 时具有同等活性，且其催化活性完全依赖于添加的血红素。NADPH 和 NADH 的 Km 值分别为 0.046 mM 和 0.526 mM。当 NADPH 浓度保持恒定时，血红素的 Km 值为 1.01 μM，比活性为 583 单位/毫克蛋白。尽管重金属对 NADPH - 细胞色素 c（P450）还原酶和胆绿素还原酶有抑制作用，但重组血红素加氧酶系统的活性不受与重金属预孵育的影响。然而，发现这些重金属的金属卟啉是重组系统的强抑制剂，对于锡 - 、钴 - 、锌 - 和镁 - 原卟啉，其 Ki 值分别为 0.015 μM、0.6 μM、2.3 μM 和 5 μM。同样，巯基灭活试剂 HgCl2、碘乙酰胺和对氯汞苯甲酸也抑制重组血红素加氧酶活性。用纯化的人肝脏血红素加氧酶免疫兔子，所得抗体制剂用于检测该酶的种属特异性。通过与抗血红素加氧酶的反应，在斑点印迹和 Western 印迹上鉴定出大鼠和人肝脏以及 HepG2 细胞中分子量为 32,000 的微粒体蛋白，类似于纯化的酶蛋白。抗血红素加氧酶能定量沉淀从标准饮食饲养的动物获得的大鼠肝脏微粒体中的全部血红素加氧酶。该抗体也能定量沉淀人骨髓微粒体血红素加氧酶活性。抗体对大鼠和人血红素加氧酶的抑制表明这两种酶蛋白在种属间有一定程度的保守性。通过 Western 印迹判断，抗血红素加氧酶在脾脏、肝脏、肾脏、大脑、心脏、骨髓、肠道和角膜上皮中仅识别单一蛋白。通过用大鼠肝脏 cDNA 筛选冈山 - 伯格载体中的 cDNA 文库，分离出了人血红素加氧酶 cDNA，并进行了核苷酸序列分析。推导的人血红素加氧酶也由 288 个氨基酸组成，分子量为 32,800 Da。（摘要截短至 400 字）
