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.
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.


RESULTS
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.


CONCLUSIONS
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.
Xia
Z
Z
Department of Pediatrics, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Li
Y
Y
Chen
S
S
Shen
Q
Q
Li
C
C
Shen
H
H
Yu
S
S
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis; D006419:Heme Oxygenase (Decyclizing) / Q000096:biosynthesis* / Q000302:isolation & purification*; D051547:Heme Oxygenase-1; D007527:Isoenzymes; D051381:Rats; D013154:Spleen / Q000201:enzymology*
D018076:DNA, Complementary; D007527:Isoenzymes; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1