人类胆绿素IXalpha还原酶是zinc-metalloprotein。表征纯化和酶大肠杆菌表达。
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玛蒂博士Polevoda BV、黄TJ McCoubrey小周
人类胆绿素IXalpha还原酶是zinc-metalloprotein。表征纯化和酶大肠杆菌表达。
欧元。1996 1月15日,235 (2):372 - 81。
- PubMed ID
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8631357 (在PubMed]
- 文摘
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胆绿素IXalpha还原酶(BVR)催化转化的血红素降解产物,胆绿素,胆红素。迄今BVR酶中是独一无二的特征,它具有双重pH /辅因子(NADH、NADPH)特异性。cDNA克隆编码人类BVR隔绝伽马库使用探针通过逆转录聚合酶链反应生成从人类胎盘RNA。这种方法拍摄,因为更直接的方法使用先前孤立鼠BVR cDNA作为杂交探针没有成功。人类的cDNA克隆测序;它被证明有一个开放阅读框编码296 -氨基酸蛋白可确定四肽之前被micro-sequencing纯化蛋白。单个消息的互补脱氧核糖核酸杂交大约1.2 kb在人类肾脏保利(a)丰富的RNA,并出现了,被印迹分析,单份基因的产物。序列分析表明,人类还原酶显示大约83%的身份,在核苷酸和氨基酸水平,与老鼠BVR。在一些地区,包括羧基终点站,蛋白质序列的身份下降到45%。还值得注意的是另外两个半胱氨酸残基的存在在人类还原酶编码(五比三鼠)。 The protein produced by an expression plasmid in which the insert was cloned in frame with lacZ sequences was characterized, and demonstrated dual pH and cofactor dependence. However, as suggested by kinetic analysis, the human enzyme may also use NADH as cofactor, as opposed to the rat reductase, which most likely utilizes only NADPH under physiological conditions. Western blot analysis and isoelectric focusing demonstrate that, although migrating as a single band on SDS/PAGE, the expressed protein, like that purified from tissue, consists of several isoelectric charge variants. Atomic absorption spectroscopy indicates that the protein purified from human liver contains Zn at an approximately 1:1 molar ratio. That human BVR is a Zn metalloprotein was further substantiated by 65Zn exchange analysis of both the purified and the fusion protein expressed in Escherichia coli. Exogenous Zn also inhibits NADPH-dependent, but not NADH-dependent, activity. Hence, the NADH and NADPH binding regions are differentiated by their ability to interact with Zn; Fe-hematoporphyrin, however, inhibited both NADH- and NADPH-dependent activity.