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| http://purl.uniprot.org/citations/17915953 | http://www.w3.org/2000/01/rdf-schema#comment | "Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes."xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.org/dc/terms/identifier | "doi:10.1021/bi701496z"xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/author | "Backes W.L."xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/author | "Huber W.J."xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/date | "2007"xsd:gYear |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/name | "Biochemistry"xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/pages | "12212-12219"xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/title | "Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase."xsd:string |
| http://purl.uniprot.org/citations/17915953 | http://purl.uniprot.org/core/volume | "46"xsd:string |
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