The purification and identification of heme oxygenase isoforms from spleen tissue of rat and the expression of heme oxygenase-1 cDNA in COS-1 cells
- Author:
Zhenwei XIA
1
;
Yunzhu LI
;
Shunnian CHEN
;
Qingxiang SHEN
;
Changlin LI
;
Hui SHEN
;
Shanchang YU
Author Information
1. 上海医科大学附属瑞金医院
- From:
Chinese Medical Journal
1998;111(9):842-846
- CountryChina
- Language:Chinese
-
Abstract:
Objective 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.Methods 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.Conclusion 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.
