Purification, Identification, and Cloning of Lysoplasmalogenase, the Enzyme That Catalyzes Hydrolysis of the Vinyl Ether Bond of Lysoplasmalogen*
| العنوان: | Purification, Identification, and Cloning of Lysoplasmalogenase, the Enzyme That Catalyzes Hydrolysis of the Vinyl Ether Bond of Lysoplasmalogen* |
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| المؤلفون: | Marianne S. Jurkowitz, Guido Marcucci, Douglas R. Pfeiffer, Elisabeth A. Calhoon, Shujun Liu, Lai-Chu Wu, Francesca Madiai |
| المصدر: | The Journal of Biological Chemistry |
| بيانات النشر: | American Society for Biochemistry and Molecular Biology, 2011. |
| سنة النشر: | 2011 |
| مصطلحات موضوعية: | Male, Plasmalogen, Lysoplasmalogen, Hydrolases, Plasmalogens, Enzyme Purification, Biochemistry, Mass Spectrometry, Rats, Sprague-Dawley, 03 medical and health sciences, Fatty aldehyde, chemistry.chemical_compound, Membrane Lipids, 0302 clinical medicine, Western blot, Complementary DNA, medicine, Animals, Humans, Northern blot, Gene Identification, Molecular Biology, Lysoplasmalogenase, 030304 developmental biology, chemistry.chemical_classification, Octyl glucoside, 0303 health sciences, medicine.diagnostic_test, Chemistry, HEK 293 cells, Membrane Proteins, Cell Biology, Liver Metabolism, Hydrogen-Ion Concentration, Intestinal Metabolism, Molecular biology, Lipids, Phospholipid Metabolism, Rats, Enzyme, HEK293 Cells, Liver, 030220 oncology & carcinogenesis, Microsomes, Liver, Lysophospholipids |
| الوصف: | Lysoplasmalogenase (EC 3.3.2.2 and EC 3.3.2.5) is an enzyme that catalyzes hydrolytic cleavage of the vinyl ether bond of lysoplasmalogen, forming fatty aldehyde and glycerophosphoethanolamine or glycerophosphocholine and is specific for the sn-2-deacylated form of plasmalogen. Here we report the purification, characterization, identification, and cloning of lysoplasmalogenase. Rat liver microsomal lysoplasmalogenase was solubilized with octyl glucoside and purified 500-fold to near homogeneity using four chromatography steps. The purified enzyme has apparent K(m) values of ∼50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent V(m) values of 24.5 and 17.5 μmol/min/mg protein for the two substrates, respectively. The pH optimum was 7.0. Lysoplasmalogenase was competitively inhibited by lysophosphatidic acid (K(i) ∼20 μm). The predominant band on a gel at ∼19 kDa was subjected to trypsinolysis, and the peptides were identified by mass spectrometry as Tmem86b, a protein of unknown function. Transient transfection of human embryonic kidney (HEK) 293T cells showed that TMEM86b cDNA yielded lysoplasmalogenase activity, and Western blot analyses confirmed the synthesis of TMEM86b protein. The protein was localized in the membrane fractions. The TMEM86b gene was also transformed into Escherichia coli, and its expression was verified by Western blot and activity analyses. Tmem86b is a hydrophobic transmembrane protein of the YhhN family. Northern blot analyses demonstrated that liver expressed the highest level of Tmem86b, which agreed with tissue distribution of activity. Overexpression of TMEM86b in HEK 293T cells resulted in decreased levels of plasmalogens, suggesting that the enzyme may be important in regulating plasmalogen levels in animal cells. |
| اللغة: | English |
| تدمد: | 1083-351X 0021-9258 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0717866b365afce90c3a69d46b8df701Test http://europepmc.org/articles/PMC3137066Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....0717866b365afce90c3a69d46b8df701 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 1083351X 00219258 |
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