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

Abstract

The antitumor activity of alkyl lysophospholipids has raised some questions concerning the degradation of O-alkyl bonds in naturally occurring ether lipids. In this report, we describe the first continuous assay for O-alkylglycerol monooxygenase (AGMO), the only enzyme known to cleave the O-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.

Glyceryl-ether Monooxygenase [EC 1.14.16.5J Part V: Some Aspects of the Stoichiometry

“…The rapid formation of aliphatic aldehyde, alcohol and acid from batyl alcohol by the monooxygenase reaction had been reported before (1,(3)(4)(5)(6), together with only small amounts of unidentified products. Under our conditions a substantial amount of radioactive material remains at the origin on the TLC plates, and this amount increased as the reaction proceeded (equal to ca 50010 of batyl alcohol consumed).…”

“…(5) (6) We have examined in detail the stoichiometry of the oxidation of 6-MePH 4 and the consumption of batyl alcohol in the monooxygenase reaction as well as the formation of products that followed enzymic hydroxylation. Repeated spectral scans of a solution set for the direct spectrophotometric assay of monooxygenase activity (Materials and Methods) between 260 and 450 nm revealed the presence of 6-MePH4 (maximum at 300 nm with tail absorption between 340 and 400 nm) which slowly changed to the spectrum of q-6-MePH 2 with little, if any, evidence of the characteristic spectrum of 6-methyl-4a-hydroxy-5, 6.7,8-tetrahydropterin (5).…”

“…Inhibition of the DHPR reaction was observed at higher concentrations of 6-MePH4 with ca 20% decrease at 0.210 mM excess of 6-MePH4 which accounts for all the decoupling observed in the coupled reaction at 0.24 mM of 6-MePH4 (Table 1). Attempts to carry out similar experiments with a limited amount of potassium ferricyanide (0.108 mM, one electron oxidant) were unsuccessful because unlike dichloroindophenol which oxidised 6-MePH 4 within a few seconds, ferricyanide required more than seven minutes during which time q-6-MePH 2 had started to rearrange to the DHPR-inactive dihydropterin (6). When the errors in the measurement of the reaction extinction coefficient for the monooxygenase (ca± 5%) and the effects of product inhibition are taken into account, then all the ratios of NADH/q-6-MePH 2 in Table I may not be significantly different from one within experimental error.…”

“…This work was confirmed and extended by Snyder and coworkers (3,4,5) who used similar assay procedures with radio labeled eH and 14(:) glyceryl ethers. Kotting, Unger and Eibl, (6,7) developed a continuous spectrophotometric assay whereby they followed the monooxygenase reaction in a coupled system with dihydropteridine reductase (DHPR) …”

Glyceryl-ether monooxygenase [EC 1.14.16.5]. A microsomal enzyme of ether lipid metabolism

“…III-I). [89][90][91] This enzyme is thus capable of regulating the levels of a variety of glyceryl ethers either directly or indirectly.…”