Soluble and catalytically active endothelin converting enzyme-1 is present in cerebrospinal fluid of subarachnoid hemorrhage patients

Don Monath Sanjaya Kuruppu, Sherry H Y Chou, Steven K Feske, Sarah Suh, Iresha Hanchapola, Eng H Lo, Mingming Ning, Alexander Ian Smith

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Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET and BigET18-34 (6 micrograms/mL) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 (5 micromol/L). The ECE-1 activity (Mean/SEM) in SAH CSF samples was 0.127/0.037 micromols of substrate cleaved/microlitre of CSF/24 hrs. The C-terminal peptides generated upon the cleavage of BigET and BigET18-34 were detected 48 hrs after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.
Original languageEnglish
Pages (from-to)1091 - 1094
Number of pages4
JournalMolecular & Cellular Proteomics
Issue number4
Publication statusPublished - 2014

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