Purification and characterization of leukotriene A4 hydrolase from human epidermis

FEBS Letters

Iversen, Lars¹  Kragballe, Knud¹  Nissen, Judith B.¹  Kristensen, Peter³  Merrick, William C.² 

[1] Department of Dermatology, Marselisborg Hospital, University of Aarhus, DK-8000 Aarhus C, Denmark;Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, USA;Department of Chemistry, University of Aarhus, Aarhus, Denmark

关键词: Leukotriene A4 hydrolase;    Human epidermis;    Purification;    Eicosanoid;    DTT;    dithiothreitol;    FCS;    fetal calf serum;    HSA;    human serum albumin;    KGM;    keratinocyte growth medium;    5-LO;    5-lipoxygenase;    LT;    leukotriene;    PAGE;    polyacrylamide gel-electrophoresis;    PBS;    phosphate buffered saline;    RP-HPLC;    reversed-phase high-performance liquid chromatography;    SDS;    sodium dodecyl sulfate;    Tris-HC1;    Trizma hydrochloride;   

DOI  :  10.1016/0014-5793(94)01459-E

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The leukotriene A₄ hydrolase is a central enzyme in leukotriene B₄, formation. Unlike 5-lipoxygenase, leukotriene A₄ hydrolase activity is present in normal human epidermis, where it is likely to be involved in transcellular leukotriene formation. In this study the leukotriene A₄ hydrolase was purified from human epidermis and human cultured keratinocytes and compared with leukotriene A₄ hydrolase from human neutrophils. To purify leukotriene A₄ hydrolase from human epidermis a new non-specific affinity chromatography column, with the leukotriene A₄ hydrolase inhibitor bestatin coupled to AH-Sepharose, was introduced. The epidermal leukotriene A₄ hydrolase was purified to apparent homogeneity and the molecular weight was determined to be approximately 70,000 Da by SDS-PAGE. The pI was 5.1–5.4 for the epidermal as well as the keratinocyte and neutrophil leukotriene A₄ hydrolase, as determined by chromatofocusing. Only minor differences in the amino acid composition were seen between the three enzyme sources. The optimal pH for the hydrolase activity was 7.5–8.5 for the epidermal and neutrophil leukotriene A₄ hydrolases. Finally, it was also shown that the epidermal leukotriene A₄ hydrolase undergoes suicide inactivation when transforming leukotriene A₄ into leukotriene B₄. It was concluded that there is a close resemblance between the epidermal leukotriene A₄, hydrolase and the hydrolase found in other cell types. Therefore, the human epidermis may be a good model for the in vivo study of transcellular leukotriene formation.

【 授权许可】

Unknown