Purchase this article with an account.
C. Grayson, R.S. Molday; Functional Characterisation of Enzymes Involved in the Elongation of Very–Long Chain Fatty Acids . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2070.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
ELOVL4 is a member of the ELO family of proteins responsible for the elongation of very–long chain fatty acids. Protein truncating mutations in ELOVL4 have been identified in patients with autosomal dominant macular degeneration, implying a role for fatty acid biosynthesis in the pathogenesis of macular degeneration. The purpose of this study was to establish an assay to investigate the putative enzymatic activity ELOVL4 and related elongase enzymes, to elucidate their role in fatty acid biosynthesis and the development of macular degeneration.
Human elongase enzymes were amplified from cDNA libraries and immunoaffinity tags were added to the constructs. The heterologously expressed proteins were examined in a mammalian cell culture model by immunofluorescence and western blotting. Microsomal membranes were isolated from the transfected cells and analysed for enzyme activity using an in vitro assay to detect the elongation of fatty acid substrates.
Microsomal membranes from cells expressing the heterologous elongase enzymes were compared with microsomal membranes isolated from mock–transfected cells in the in vitro assay. Very–long chain fatty acids involved in docosahexaenoic acid (DHA) biosynthesis were elongated in the presence of microsomes containing heterologously expressed enzymes in a substrate–specific manner.
The in vitro assay described in this study is a valuable tool to study the fatty acid specificity of human elongase enzymes in mammalian cells. These data agree with the hypothesis that different enzymes are involved in the elongation of substrates in the DHA biosynthetic pathway. The study of these enzymes will help to gain insight into the role of fatty acid biosynthesis in the retina, particularly the functional role of ELOVL4 in the pathogenesis of macular degeneration.
This PDF is available to Subscribers Only