Basic Research
Copyright ©The Author(s) 2002. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 15, 2002; 8(6): 1123-1128
Published online Dec 15, 2002. doi: 10.3748/wjg.v8.i6.1123
Analysis of Heme oxygenase isomers in rat
Zhen-Wei Xia, Wen-Jun Cui, Xue-Hong Zhang, Qing-Xiang Shen, Jian Wang, Yun-Zhu Li, Shen-Nian Chen, Shan-Chang Yu
Zhen-Wei Xia, Yun-Zhu Li, Shan-Chang Yu, Shun-Nian Chen, Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, Shanghai 200025, China
Wen-Jun Cui, Xue-Hong Zhang, College of Life Science and Biotechnology, Shanghai Jiaotong University & The Chinese Academy of Science, Shanghai Branch, Shanghai 200030, China
Qing-Xiang Shen, Jian Wang, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China
Author contributions: All authors contributed equally to the work.
Supported by National Natural Scientific Foundation of China, No. 39600159
Correspondence to: Dr. Zhen-Wei Xia, Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, 197 Rui Jin Er Road, Shanghai 200025, China. xzw63@hotmail.com
Telephone: +86-21-64333414 Fax: +86-21-64333414
Received: June 11, 2002
Revised: July 15, 2002
Accepted: July 26, 2002
Published online: December 15, 2002
Abstract

AIM: To purify and identify heme oxygenase (HO) isomers which exist in rat liver, spleen and brain treated with hematin and phenylhydrazine and in untreated rat liver and to investigate the characteristics of HO isomers, to isolate and confirm the rat HO-1 cDNA that actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells), to prepare the rat heme oxygenase-1 (HO-1) mutant and to detect inhibition of HO-1 mutated enzyme.

METHODS: First, rat liver, spleen and brain microsomal fractions were purified by DEAE-Sephacel and hydroxylapatite. The characteristics including activity, immunity and inducibility of two isomers (HO-1 and HO-2), and their apparent molecular weight were measured by detecting enzymatic activities, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis, respectively. Second, plasmid pcDNA3HO1 containing native rat HO-1 cDNA and pcDNA3HO1D25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed by site-directed mutagenesis. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1D25 were collected and disrupted by sonication, the microsomes were prepared by ultracentrifugation. Third, the inhibition of rat HO-1 mutant was analyzed.

RESULTS: Two isomers were purified and identified in treated rat liver, spleen, brain and untreated rat liver. HO-1 was the predominant form with a ratio of 2.0:1 and 3.2:1 of HO-1 and HO-2 in liver and spleen, respectively, but only the activity of HO-2 in the brain and untreated liver could be detected. The apparent molecular weights of HO-1 and HO-2 were about Mr 30000 and Mr 36000 under reducing conditions, respectively. The antiserum against liver HO-2 was employed in Western blotting analysis, the reactivity of HO-1 in the liver was not observed. The plasmid pcDNA3HO1 was highly expressed in endoplasmic reticulum of transfected COS-1 cells. The specific activity was ≈5-fold higher than that of the control. However, the enzyme activity of mutated HO-1 declined. While an equal amount of mutant was added to the enzyme reaction system, the levels of bilirubin decreased 42%.

CONCLUSION: The studies suggest that HO-1 and HO-2 exist in the hematin and phenylhydrazine treated rat liver and spleen, but only HO-2 in the brain and untreated liver. Two constitutive forms are different in molecular weight, inducibility and immunochemical properties. The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It suggests that this clone has an insert of 1030 base-pairs encodes HO-1. His25Ala mutant reduced the formation of bilirubin and it suggests that the mutant could competely bind the heme with native enzyme.

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