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A continuous assay forO-alkylglycerol monooxygenase (E.C. 1.14.16.5)

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Lipids

Abstract

The antitumor activity of alkyl lysophospholipids has raised some questions concerning the degradation ofO-alkyl bonds in naturally occurring ether lipids. In this report, we describe the first continuous assay forO-alkylglycerol monooxygenase (AGMO), the only enzyme known to cleave theO-alkyl bond in saturated ether lipids and ether phospholipids. AGMO activity was monitored at 340 nm by coupling the NADH redox reaction to the tetrahydropteridine cofactor of the rat liver microsomal enzyme. Turnover rates as low as 0.6 nmol/min could be measured. Using radiolabeled substrates, the products were identified with a TLC-Linear-Analyzer. The only interference with this assay can arise from other reducing agents, e.g. dithiothreitol. The assay was used to develop protocols for the solubilization of AGMO from membrane preparations in the presence of detergents.

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Abbreviations

AGMO:

O-alkylglycerol monooxygenase

ALP:

alkyl lysophospholipids

BSA:

bovine serum albumine

DTT:

dithiothreitol

1-[3H]H-G:

1-O-[3H]hexadecylglycerol

3H-sn-G-1PC:

3-O-hexadecyl-sn-glycero-1-phosphocholine

1H-sn-G-3PC:

1-O-hexadecyl-sn-glycero-3-phosphocholine

PtH4 :

D,L-5,6,7,8-tetrahydropterine

DMPtH4 :

D,L-6,7-dimethyl-PtH4

MPtH4 :

D,L-6-methyl-PtH4

PtH2 :

dihydropteridine, q-, quinoid isomer or 7,8-isomere, see Fig. 4

DMPtH2 :

MPtH2, 6,7-dimethyl- or 6-methyl-derivate, respectively of PtH2

TLC:

thin layer chromatography

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Authors and Affiliations

  1. Abt. Haematologie/Onkologie, Zentrum Innere Medizin, Robert-Koch-Str. 40, D-3400, Goettingen, Federal Republic of Germany

    Clemens Unger

  2. Dept. 140, Max-Planck-Institut f. biophysikal. Chemie, Am Fassberg 11, Postfach 2841, D-3400, Göttingen, Federal Republic of Germany

    Jochem Koetting & Hansjoerg Eibl

Authors
  1. Jochem Koetting
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  2. Clemens Unger
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  3. Hansjoerg Eibl
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Koetting, J., Unger, C. & Eibl, H. A continuous assay forO-alkylglycerol monooxygenase (E.C. 1.14.16.5). Lipids 22, 824–830 (1987). https://doi.org/10.1007/BF02535538

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  • DOI: https://doi.org/10.1007/BF02535538

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