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 DEAEcellulose 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 K , of 0.66 mM for I-0-hexadecylglycerol. The microsomal alkylglycerol monooxygenase was stimulated by Triton X-1 00, 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. NEthylmaleimide and p-chloromercuribenzoate significantly inhibited the enzyme activity, suggesting that alkylglycerol monooxygenase is a -SH enzyme. Thus the membrane-bound form of this enzyme has been well characterized in microsomal preparations from rat liver [i ,4,5] and Tetrahymena pyrqormis [6,7].Thereafter Pfleger et al. found a significant amount of cleavage activity in the soluble fraction of rat liver using zonal centrifugation procedures, and they characterized it by electron microscopy [8]. However, they did not carry out recovery studies to determine what proportion of the total activity had been solubilized. Actual attempts to remove effectively the enzyme from its membrane environment have been unsuccessful.In this paper we describe for the first time an effective procedure for solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes.Abbreviations. Pte.H,, 2-amino-6,7-dimethyl-4-hydroxy-5,6,7,8-tetrahydropteridine hydrochloride; GSH, reduced glutathione.
MATERIALS AND METHODSThe following materials were obtained from commerical sources: 2-amino-6,7-dimethyl-4-hydroxy-5, 6, 7, 8-tetrahydropteridine hydrochloride (Pte. H4) from Aldrich; GSH from Yamanouchi; Triton X-100 and Phospholipids test kit from Wako; DEAE-cellulose (DE 52) from Whatman; 6-aminohexyl-Sepharose and Sepharose 6B from Pharmacia; precoated silica gel F254 plate from E. Merck; 1-0-hexadecylglycerol (chimyl alcohol) from Sigma; catalase and human immuno globulin from Boehringer, Mannheim; dye reagent for protein determination from Bio-Rad; and asolectin and 1-O-[l-14C]hexadecylglycerol (1 .I Ci/mol) were kindly donated by Drs T. Takenawa and H. Mangold respectively. All of the other chemicals were of analytical grade.
Preparation o j MicrosomesMicrosomal fractions were prepared as described previously [9], with the following small modification. The livers, excised from slightly anesthesized Wister rats weighing 200 -300 g, were perfused with ice-cold 0.25 M sucrose and homogenized with a Potter-Elvehjem homogenizer in 3 vol. of 0.25 M sucrose. The homogenate was cen...