Huang T J, Maines M D
University of Rochester School of Medicine, Department of Biophysics, New York 14642.
Mol Pharmacol. 1990 Jan;37(1):25-9.
Here we report on the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat kidney and describe selective changes in the tissue profile of the variants after bromobenzene treatment (2 mmol/kg, subcutaneously, 24 hr). Using two-dimensional electrophoresis and isoelectric focusing, two major molecular mass species, Mr 30,400 and 30,700, a minor form of Mr 31,400, and five net-charge groups of pI = 6.23, 5.91, 5.77, 5.61, and 5.48 were detected; the net-charge variants with pI = 5.61 and 5.77 were the most abundant forms. The Mr 30,400 form was the main component of two isoelectric focusing bands with pI = 6.23 and 5.91, and the relative amounts of these net-charge variants was severely decreased in the kidneys of bromobenzene-treated rats. The effect of bromobenzene in vivo could not be duplicated by in vitro experiments involving the direct treatment of purified enzyme with bromobenzene, or incubation of the purified preparation with bromobenzene in the presence of a NADPH-dependent microsomal drug-metabolizing system. Bromobenzene treatment did not alter the immunochemical properties of biliverdin reductase variants, as judged by the similarity of isoelectric focusing patterns of preparations on a Western blot using antibody raised against a rat liver total biliverdin reductase preparation. The treatment, however, caused an alteration in the kinetic properties of the enzyme, and the activity with NADH appeared to be selectively decreased. The possible mechanisms involved in the expression of multiple forms of the reductase and the biological significance of the multiplicity, as well as the change in composition caused by bromobenzene, are discussed.
在此，我们报告了在大鼠肾脏中检测到胆绿素还原酶的多种净电荷和分子量变体，并描述了溴苯处理（2 mmol/kg，皮下注射，24小时）后这些变体组织分布的选择性变化。使用二维电泳和等电聚焦，检测到两种主要的分子量形式，Mr 30,400和30,700，一种次要形式Mr 31,400，以及五个净电荷组，其pI分别为6.23、5.91、5.77、5.61和5.48；pI为5.61和5.77的净电荷变体是最丰富的形式。Mr 30,400形式是pI为6.23和5.91的两条等电聚焦带的主要成分，在溴苯处理的大鼠肾脏中，这些净电荷变体的相对含量大幅下降。溴苯在体内的作用不能通过体外实验重现，体外实验包括用溴苯直接处理纯化的酶，或在NADPH依赖的微粒体药物代谢系统存在的情况下将纯化制剂与溴苯一起孵育。根据使用针对大鼠肝脏总胆绿素还原酶制剂产生的抗体在蛋白质印迹上制备物的等电聚焦模式的相似性判断，溴苯处理并未改变胆绿素还原酶变体的免疫化学性质。然而，该处理导致了酶动力学性质的改变，并且与NADH的活性似乎选择性降低。文中讨论了还原酶多种形式表达所涉及的可能机制、多样性的生物学意义以及溴苯引起的组成变化。
