Xia Z, Li Y, Chen S, Shen Q, Li C, Shen H, Yu S
Department of Pediatrics, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Chin Med J (Engl). 1998 Sep;111(9):842-6.
To demonstrate that heme oxygenase (HO) isoforms exist in rat spleen treated with hematin and phenylhydrazine and to confirm that the isolated cDNA actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells) in order to prepare HO-1 mutant for inhibiting the natural enzyme.
The rat spleen microsomal fractions were first purified by diethylaminoethyl (DEAE)-Sephacel and hydroxylapatite. The activity of two isoforms (HO-1 and HO-2) of enzyme and their apparent molecular weight on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) were measured. Secondly, using the isolated HO-1 cDNA clone, the expression plasmid pcDNA3HO1 was constructed and transfected into cultured COS-1 cells. The transfected cells were collected and disrupted by sonication, and the microsomes were prepared by ultracentrifugation. The activity of HO-1 was measured.
Two isoforms were purified and identified in treated rat spleen and HO-1 was the predominant form. The ratio of HO-1 to HO-2 was 3.2:1. The apparent molecular weights of HO-1 and HO-2 were about 30 kD and 36 kD under reducing conditions, respectively. The HO-1 was highly expressed in endoplasmic reticulum of transfected cells. The specific band was located in molecular weight of 30 kD. The specific activity was five times higher than that of the control.
The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It is suggested that this clone having an insert of 1030 base-pairs encodes HO-1 and that we can prepare HO-1 mutant by site-directed mutagenesis of HO-1 cDNA to prevent and treat hyperbilirubinemia.
证明血红素加氧酶（HO）同工型存在于用氯高铁血红素和苯肼处理的大鼠脾脏中，并通过在猴肾细胞（COS-1细胞）中表达cDNA来确认分离出的cDNA确实编码HO-1，以便制备用于抑制天然酶的HO-1突变体。
首先通过二乙氨基乙基（DEAE）-葡聚糖凝胶和羟基磷灰石纯化大鼠脾脏微粒体部分。测量了该酶的两种同工型（HO-1和HO-2）的活性及其在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的表观分子量。其次，使用分离出的HO-1 cDNA克隆构建表达质粒pcDNA3HO1，并将其转染到培养的COS-1细胞中。收集转染的细胞并通过超声破碎，然后通过超速离心制备微粒体。测量HO-1的活性。
在处理过的大鼠脾脏中纯化并鉴定出两种同工型，且HO-1是主要形式。HO-1与HO-2的比例为3.2:1。在还原条件下，HO-1和HO-2的表观分子量分别约为30 kD和36 kD。HO-1在转染细胞的内质网中高表达。特异性条带位于30 kD的分子量处。比活性比对照高五倍。
COS-1细胞中表达的HO-1的活性高于从大鼠脾脏组织纯化的酶的活性。提示这个插入了1030个碱基对的克隆编码HO-1，并且我们可以通过对HO-1 cDNA进行定点诱变来制备HO-1突变体，以预防和治疗高胆红素血症。
