Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Gunawan, Johannes; Debuch, Hildegard

doi:10.1111/j.1471-4159.1982.tb07948.x

Cited by 24 publications

(14 citation statements)

References 19 publications

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“…The jejunum is an important site of lipid absorption, metabolism, and distribution. Collectively, the high levels of Tmem86b transcripts in the liver and small intestines correlated with empirical findings of high contents of lysoplasmalogenase activity in those tissues (23, 25, 26). …”

Section: Resultssupporting

confidence: 74%

“…The enzyme properties are also similar to those of rat brain microsomes with the exception that activity levels are much lower in the brain (24, 25). A highly sensitive assay for detecting hydrolysis of the vinyl ether bond was developed in the laboratory of Debuch (24, 25), in which the plasmenylethanolamine and lysoplasmenylethanolamine substrates were radiolabeled with 14 C at the C-1 of the alkenyl group (19). These workers found no evidence for an enzyme that catalyzed hydrolysis of the vinyl ether group of the intact plasmalogen molecule in any subcellular fraction of rat brain (25).…”

Section: Discussionmentioning

confidence: 52%

“…The enzyme has been identified and studied in microsomes from the liver (21–23) and small intestinal mucosa (26), where its specific activities are high. The enzyme has also been found in brain microsomes, where its activity is low, only

\frac{1}{700}

of liver activity (24, 25). Thus, in these tissues, brain, liver, and small intestinal mucosa, there exist reciprocal relationships between content of plasmalogens and the activity of lysoplasmalogenase.…”

Section: Introductionmentioning

confidence: 99%

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

Pfeiffer

Calhoon

et al. 2011

Journal of Biological Chemistry

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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.

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Section: Resultssupporting

confidence: 74%

Section: Discussionmentioning

confidence: 52%

\frac{1}{700}

Section: Introductionmentioning

confidence: 99%

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

Pfeiffer

Calhoon

et al. 2011

Journal of Biological Chemistry

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“…Enzymes capable of specifically cleaving the ethanolamine and choline substituents of the phosphate moiety of plasmalogens have also been identified in the brain (52) and other tissues (53). A lysoplasmalogenase enzyme (EC 3.3.2.2 and 3.3.2.5), specifically catalyzing the hydrolysis of the vinyl-ether bond of plasmalogens, has also been described in the brain (54). However, preliminary data indicate that alkenyl-GP is not cleaved by this enzyme 2 ; the possibility that it could have a sufficiently long half-life in biological fluids is supported by its ϳ5 M in vivo steady-state concentration in the anterior chamber fluid of the eye (19).…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Growth Factor-like Lipid, 1-O-cis-Alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (Alkenyl-GP) That Is Present in Commercial Sphingolipid Preparations

Liliom¹,

Fischer²,

Virág³

et al. 1998

Journal of Biological Chemistry

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Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1 H and 13 C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 M was only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065-C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.

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“…1A ). The involved enzymes are fatty aldehyde dehydrogenase (ALDH3A2) ( 1, 2 ), prenylcysteine oxidase 1 (PCYOX1) ( 3, 4 ), 2-hydroxyacylCoA lyase (HACL1) ( 5, 6 ), sphingosine-1-phosphate lyase (SGPL1) ( 7-9 ), MPO ( 10 ), and lysoplasmalogenase ( 11,12 ). In particular, two of these enzymes, HACL1 and SGPL1, have intensively been studied in our laboratory.…”

mentioning

confidence: 99%

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

Mezzar

Schryver

Veldhoven

2014

Journal of Lipid Research

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Long-chain aliphatic aldehydes are produced during various metabolic processes, such as microsomal oxidation of long-chain alcohols, lysosomal degradation of prenylated proteins, peroxisomal ␣ -oxidation of 3-methylbranched fatty acids and 2-hydroxy long-chain fatty acids, microsomal breakdown of phosphorylated sphingoid bases, attack of plasmalogens by myeloperoxidase (MPO)-derived hypochlorous acid, and microsomal degradation of (lyso)plasmalogens (see Fig. 1A ). The involved enzymes are fatty aldehyde dehydrogenase (ALDH3A2) ( 1, 2 ), prenylcysteine oxidase 1 (PCYOX1) ( 3, 4 ), 2-hydroxyacylCoA lyase (HACL1) ( 5, 6 ), sphingosine-1-phosphate lyase (SGPL1) ( 7-9 ), MPO ( 10 ), and lysoplasmalogenase ( 11,12 ). In particular, two of these enzymes, HACL1 and SGPL1, have intensively been studied in our laboratory. Their activity measurements are complicated by their low specifi c activity [ ‫ف‬ 115 ( 5 ) and ‫ف‬ 15 mU/g rat liver ( 13 ), respectively], low K m (10-15 M), and the considerable interference of enzymes acting on their substrates (acylCoA hydrolases for HACL1; phosphatases for SGPL1) or on their cofactors (phosphatases acting on TPP, cofactor for HACL1, and on pyridoxal-phosphate, cofactor for SGPL1). In addition, the generated aldehyde is chemically not stable and subject to further metabolism, and compounds normally used to trap the aldehyde or block its metabolism interfere with the cofactor (pyridoxalphosphate). Moreover, the presence of plasmalogens in mammalian tissues, which give rise to aldehydes under acidic conditions ( 14 ), can cause a high background when using nonradioactive substrates, especially in nervous tissue.To measure HACL1 and SGPL1 activities in tissue or cell lysates, various protocols were developed by our group.Abstract Long-chain aldehydes are commonly produced in various processes, such as peroxisomal ␣ -oxidation of longchain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fl uorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantifi ed in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or defi ciency in tissue homogenates and fi broblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 ac...

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Lysoplasmalogenase–A Microsomal Enzyme from Rat Brain

Cited by 24 publications

References 19 publications

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

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

Identification of a Novel Growth Factor-like Lipid, 1-O-cis-Alk-1′-enyl-2-lyso-sn-glycero-3-phosphate (Alkenyl-GP) That Is Present in Commercial Sphingolipid Preparations

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1

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