Abstract
Ethanolamine kinase catalyzes the first step in the CDP-ethanolamine pathway for the formation of the major membrane phospholipid phosphatidylethanolamine (PtdEtn). In this work, the predicted Etnk2 cDNA was established as a soluble protein with ethanolamine-specific kinase activity that was most highly expressed in liver. Mice with an inactivated Etnk2 gene were derived, and its absence reduced the rate of PtdEtn synthesis from exogenous ethanolamine in hepatocytes. PtdEtn is a major precursor to phosphatidylcholine in liver; however, Etnk2(-/-) mice did not have reduced amounts of either PtdEtn or phosphatidylcholine or an altered phospholipid molecular species distribution. The knock-out animals were able to adapt to a choline-deficient diet. The Etnk2(-/-) mice exhibited a maternal-specific intrauterine growth retardation phenotype that resulted in a 33% reduction in litter size and frequent perinatal death. Histological analysis of pregnant Etnk2(-/-) females showed that fetal development failed at the late stage of pregnancy in a significant percentage of embryos because of the appearance of extensive placental thrombosis. These results illustrate a non-redundant role for EtnK2 expression in regulating placental hemostasis.
Highlights
Our work is the first step in the CDP-Etn pathway, EtnK
Sequence and Distribution of the Murine Etnk2 cDNA—In our previous work with ETNK1, a second human gene called ETNK2 was detected by a bioinformatic analysis [12]
The mouse Etnk2 gene is located on chromosome 1 and consists of eight exons that span 16.7 kb
Summary
Cloning of the Mouse Etnk cDNA and Construction of the Expression Vector—The expressed sequence tag data base was searched using the human ETNK1 sequence [12]. EtnK and ChoK Assays—Frozen 293T cell pellets transfected with plasmid pPJ184 or pPJ96 [12] or with a control empty vector were thawed on ice and incubated for 1 h in lysis buffer (20 mM Tris-HCl (pH 8.0), 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 2 g/ml aprotinin) on ice. The cells were disrupted by sonication (6 ϫ 30 s) in a cup horn (Microsonix Sonicator 3000), and the particulate fraction was removed by centrifugation at 5000 ϫ g for 5 min at 4 °C. The cell pellet was resuspended in wash buffer (each liter contained 8 g of NaCl, 0.35 g of KCl, 0.16 g of MgSO4, 0.18 g of CaCl2, 2.4 g of HEPES, and 15 g of bovine serum albumin, with the pH adjusted to 7.4 with NaOH) containing 10% (v/v) erythrocyte lysis buffer and centrifuged down three times. Sections (0.5 cm) were scraped from the plate, and the radioactivity was quantitated in 3 ml of scintillation fluid using a liquid scintillation counter
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