Purification, Identification, and Cloning of Lysoplasmalogenase, the Enzyme That Catalyzes Hydrolysis of the Vinyl Ether Bond of Lysoplasmalogen*

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 Km values of ∼50 μm for both lysoplasmenylcholine and lysoplasmenylethanolamine and apparent Vm 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 (Ki ∼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

Pharmacological evidence that the activation of the Na(+)-Ca(2+) exchanger protects C6 glioma cells during chemical hypoxia

1. In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium-iodide positive dead cells. 2. Extracellular Na(+) removal, which activates the Na(+)-Ca(2+) exchanger as a Na(+) efflux pathway and prevents Na(+) entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. 3. During chemical hypoxia, in the presence of extracellular Na(+) ions, a progressive increase of [Ca(2+)](i) occurred; in the absence of extracellular Na(+) ions [Ca(2+)](i) was enhanced to a greater extent. 4. The blockade of the Na(+)-Ca(2+) exchanger by the amiloride derivative 5-(N-4-chlorobenzyl)-2′,4′-dimethylbenzamil (CB-DMB), lanthanum (La(3+)) and the Ca(2+) chelator EGTA, completely reverted the protective effect exerted by the removal of Na(+) ions on C6 glioma cells exposed to chemical hypoxia. 5. The inhibition of the Na(+)-Ca(2+) antiporter enhanced chemical hypoxia-induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na(+) ions (145 mM), suggesting that the blockade of the Na(+)-Ca(2+) antiporter during chemical hypoxia can lead to increased cell damage. 6. Collectively, these results suggest that activation of the Na(+)-Ca(2+) exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury