Human Leukotriene C$ sb4$ synthase is a membrane-bound glutathione S-transferase that catalyzes the first committed step leading to the biosynthesis of the pro-inflammatory mediators, the cysteinyl leukotrienes (LTC$ sb4,$ LTD$ sb4$ and LTE$ sb4$). Using chromatographic separation and photoaffinity labelling techniques, LTC$ sb4$ synthase was found to be a unique enzyme distinct from all other known glutathione S-transferases. An 18 kDa membrane polypeptide was specifically labelled in the microsomal membranes of myelocytic cell lines containing LTC$ sb4$ synthase activity (U937 and THP-1), with a photoaffinity derivative of LTC$ sb4$ (azido (I$ sp{125}$) -LTC$ sb4$) and was identified as a candidate for being LTC$ sb4$ synthase or a subunit thereof. LTC$ sb4$ synthase was subsequently purified to homogeneity from THP-1 cells and the purified preparation contained only one polypeptide which had a molecular mass of 18 kDa. By gel filtration chromatography, the native molecular mass of LTC$ sb4$ synthase was determined to be approximately $39 pm3$ kDa. It was therefore concluded that LTC$ sb4$ synthase is enzymatically active as a homodimer. The sequence of the N-terminal 35 amino acids of purified human LTC$ sb4$ synthase was determined and was found to be unique, composed primarily of hydrophobic amino acids and containing a protein kinase C (PKC) consensus sequence. Further analysis of the potential phosphoregulation of LTC$ sb4$ synthase in neutrophilic and eosinophilic HL-60 cells demonstrated that cysteinyl leukotriene biosynthesis, but not non-cysteinyl leukotriene biosynthesis, was specifically attenuated by phorbol ester-mediated activation of PKC. In eosinophilic HL-60 and THP-1 cells this decrease in cysteinyl leukotriene production was demonstrated to be due to non-competitive inhibition of LTC$ sb4$ synthase activity. Concomitant with the PKC-mediated decrease in cysteinyl leukotriene biosynthesis, an increase in prostanoid biosynthesis occurred. Based
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.41520 |
| Date | January 1994 |
| Creators | Ali, Ambereen |
| Contributors | Ford-Hutchinson, A. W. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Pharmacology & Therapeutics.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001397716, proquestno: NN94573, Theses scanned by UMI/ProQuest. |
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