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The enzyme alkylglycerol monooxygenase was solubilized with 2% of Triton X-100 and partially purified approximately to 83-fold with a 36% yield after a procedure which included 6-aminohexyl-Sepharose and DEAE-cellulose column chromatography, followed by a sucrose density gradient centrifugation. The molecular weight of the native form was estimated to be approximately 400000 as judged by Sepharose 6B column chromatography in the presence of Triton X-100. The enzyme had a pH optimum near 8.5 and a Km of 0.66 mM for 1-O-hexadecylglycerol. The microsomal alkylglycerol monooxygenase was stimulated by Triton X-100, but did not affect kinetically the initial velocity except to maintain it for a much longer period of time. The purified enzyme required absolutely both molecular oxygen and reduced pteridine as an electron donor. Furthermore, reduced glutathione and phospholipids were necessary for expression of full enzyme activity. N-Ethylmaleimide and p-chloromercuribenzoate significantly inhibited the enzyme activity, suggesting that alkylglycerol monooxygenase is a -SH enzyme.

The alkylglycerol monooxygenase catalyzing the cleavage of the ether bond in alkylglycerol resides in rat liver microsomes. The enzyme preparation was freed of phospholipids by sodium deoxycholate treatment followed by gel filtration in the presence of deoxycholate. The removal of phospholipids markedly decreased the alkylglycerol monooxygenase activity. The activity of the delipidated enzyme, however, could be completely restored by the addition of phospholipid vesicles without detergent. When individual phospholipids were added, anionic phospholipids such as phosphatidylglycerol and diphosphatidyl-glycerol were the most effective. These findings, along with our previous observation of a similar effect of liposomes on the purified enzyme, indicate that the amphipathic nature of the protein is responsible for the lipid dependence of enzymatic activity.

Synthetic alkyl lysophospholipids (ALP) show antineoplastic activity. In the present discussion, the cytotoxicity of ALP is attributed to their accumulation in tumor cells. Some neoplastic cell species, in contrast to normal cells, cannot metabolize ALP because of a lack of O-alkylglycerol monooxygenase (AGMO) activity. To understand the metabolic fate of ether lipids and ether-linked phospholipids, AGMO substrate specificity studies were undertaken. Thirty-five different natural and synthetic ether lipids and their metabolites (including a thioether) were tested as substrates for AGMO. The study revealed that the potent cytostatic substance, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is not a substrate for AGMO. Therefore, its selective toxicity to tumor cells cannot be based on the differences in direct detoxification of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine by AGMO in normal and malignant cells. However, 1-O-octadecyl-2-O-methyl-rac-glycerol, which can be formed by phospholipase C hydrolysis, is a good substrate for AGMO.

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.