Ennis O, Maytum R, Mantle T J
Department of Biochemistry, Trinity College, Dublin, Ireland.
Biochem J. 1997 Nov 15;328 ( Pt 1)(Pt 1):33-6. doi: 10.1042/bj3280033.
Native biliverdin IX alpha reductase (BVR-A) is a monomer of molecular mass 34 kDa. We have developed an expression vector that allows the isolation of 40 mg of a glutathione S-transferase (GST)-BVR-A fusion protein from 1 litre of culture. The fusion protein (60 kDa) behaves as a dimer on gel filtration (120 kDa), so that we have artificially created a BVR-A dimer. The recombinant rat kidney enzyme exhibits pre-steady-state 'burst' kinetics that show a pH dependence similar to that already described for ox kidney BVR-A. Similar behaviour was obtained in the presence and absence of the GST domain both for the burst kinetics and during initial-rate studies in the presence and absence of albumin. The stereospecificity of the BVR-A-catalysed oxidation of [4-3H]NADH, labelled at the A and B faces, was shown to occur exclusively via the B face.
天然胆红素原IXα还原酶（BVR-A）是一种分子量为34 kDa的单体。我们构建了一种表达载体，可从1升培养物中分离出40毫克谷胱甘肽S-转移酶（GST）-BVR-A融合蛋白。该融合蛋白（60 kDa）在凝胶过滤时表现为二聚体（120 kDa），因此我们人工构建了一个BVR-A二聚体。重组大鼠肾脏酶表现出前稳态“爆发”动力学，其pH依赖性与已报道的牛肾BVR-A相似。在有无GST结构域的情况下，对于爆发动力学以及在有无白蛋白存在的初始速率研究中，均获得了类似的行为。BVR-A催化的[4-³H]NADH氧化反应（在A面和B面标记）的立体特异性显示，氧化仅通过B面发生。
