{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T18:14:59Z","timestamp":1788372899061,"version":"build-2803163510"},"reference-count":21,"publisher":"American Chemical Society (ACS)","issue":"9","license":[{"start":{"date-parts":[[2017,2,24]],"date-time":"2017-02-24T00:00:00Z","timestamp":1487894400000},"content-version":"vor","delay-in-days":9,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_termsofuse.html"}],"funder":[{"DOI":"10.13039\/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","doi-asserted-by":"publisher","award":["9848"],"award-info":[{"award-number":["9848"]}],"id":[{"id":"10.13039\/501100000038","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2017,3,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Diacylglycerol kinase \u03b5 (DGK\u03b5) catalyzes the phosphorylation of diacylglycerol, producing phosphatidic acid. DGK\u03b5 demonstrates exquisite specificity for the acyl chains of diacylglycerol. This contributes to the enrichment of particular acyl chains within the lipids of the phosphatidylinositol cycle. Phosphatidylinositol is highly enriched with 1-stearoyl-2-arachidonoyl, which is important for maintaining cellular health. Dysregulation of DGK\u03b5 perturbs lipid signaling and biosynthesis, which has been linked to epilepsy, Huntington\u2019s disease, and heart disease. Recessive loss-of-function mutations in the DGK\u03b5 gene cause atypical hemolytic uremic syndrome. Because DGK\u03b5 has never been purified, little is known about its molecular properties. We expressed human DGK\u03b5 and a truncated version lacking the first 40 residues (DGK\u03b5\u039440) and purified both proteins to near homogeneity using nickel affinity chromatography. Kinase activity measurements showed that both purified constructs retained their acyl chain specificity for diacylglycerol with an activity level comparable to that of N-terminally FLAG epitope-tagged forms of these proteins expressed in COS7 cells. Both constructs lost activity upon being stored, particularly upon freezing and thawing, which was minimized by the addition of glycerol. Circular dichroism revealed that DGK\u03b5 and DGK\u03b5\u039440 both contain significant amounts of \u03b1-helical and \u03b2 structure and exhibit biphasic thermal denaturations. The loss of secondary structure upon heating was irreversible for both constructs, with relatively little effect of added dioleoylphosphatidylcholine. The addition of 50% glycerol stabilized both constructs and facilitated refolding of their secondary structures after heating. This is the first successful purification and characterization of DGK\u03b5\u2019s enzymatic and conformational properties. The purification of DGK\u03b5 permits detailed analyses of this unique enzyme and will improve our understanding of DGK\u03b5-related diseases.<\/jats:p>","DOI":"10.1021\/acs.biochem.6b01193","type":"journal-article","created":{"date-parts":[[2017,2,15]],"date-time":"2017-02-15T14:55:03Z","timestamp":1487170503000},"page":"1337-1347","source":"Crossref","is-referenced-by-count":13,"title":["Expression, Purification, and Properties of a Human\nArachidonoyl-Specific Isoform of Diacylglycerol Kinase"],"prefix":"10.1021","volume":"56","author":[{"given":"William","family":"Jennings","sequence":"first","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sejal","family":"Doshi","sequence":"additional","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Prasanta Kumar","family":"Hota","sequence":"additional","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aaron","family":"Prodeus","sequence":"additional","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie","family":"Black","sequence":"additional","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9602-9558","authenticated-orcid":true,"given":"Richard M.","family":"Epand","sequence":"additional","affiliation":[{"name":"McMaster University , , 1280 Main Street West , , ,","place":["Hamilton, Ontario, Canada, L8S 4K1"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2017,2,15]]},"reference":[{"key":"2026090213322369600_cit1","doi-asserted-by":"publisher","first-page":"6186","DOI":"10.1021\/cr1004106","article-title":"Regulation\nand functions of diacylglycerol\nkinases","volume":"111","author":"Shulga","year":"2011","journal-title":"Chem. 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