Abstract
The intestinal glycosylceramidase of the mouse which we reported previously as a taurodeoxycholate-activated galactosylceramidase (Kobayashi, T., and Suzuki, K. (1981) J. Biol. Chem. 256, 1133-1137) has been purified to homogeneity. The enzyme gave a single band of a molecular weight of 130,000 in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight estimated by Sepharose 4B or Sephadex G-200 gel filtration under nondenaturing conditions was 290,000 to 300,000. In the double immunodiffusion test, rabbit antiserum raised against the purified enzyme gave a single precipitin band against the enzyme, but no cross-reactivity was observed against the brain or kidney galactosylceramidase (EC 3.2.1.46). The purified enzyme was active toward 4-methylumbelliferyl beta-D-galactoside, beta-D-glucoside, beta-D-xyloside, beta-D-fucoside, and alpha-L-arabinoside. Among potential glycolipid substrates, the enzyme was active, in the presence of sodium taurodeoxycholate, toward galactosylceramide, glucosylceramide, lactosylceramide, galactosylsphingosine, and glucosylsphingosine. It was inactive toward GM1 ganglioside, asialo-GM1 ganglioside, desialylated fetuin, and desialylated transferrin. Among disaccharides, the enzyme showed the highest catalytic activity toward lactose (18.9 mumol/min/mg of protein) and the lowest toward galactose beta (1 leads to 4)-N-acetylglucosamine (0.06 mumol/min/mg of protein). Galactose beta (1 leads to 6)-N-acetylglucosamine was not hydrolyzed. Phlorizin was also a substrate for the enzyme.
Highlights
The intestinal glycosylceramidaseotfhe mouse glucoside) hydrolase described by Leese and Semenza [3]
Tritium label was introduced into the C-6 of the terminal galactose of galactosylceramide, lactosylceramide, and asialo-GM, gangliosideby the galactose oxidase-sodium In a previous report, we described a highly active galacto- [’Hlborohydride procedure [5].The labeling of GMIganglioside was sylceramidase in the mouse intestine thatis activated b y by the same principle hut with minor modification of the original sodium taurodeoxycholate and is distinct from the two well method necessitated by its hydrophilic property [9]
Hydroxyapatite Chromatography-The dialyzed enzyme sample was applied to a hydroxyapatitecolumn (1X 6.5 cm) which had been pre-equilibrated with 2 m~ sodium phosphate buffer, pH 7.2
Summary
Commercial Materials conditions was 290,000 to 300,000.In thedouble im- The sources of some of the commercial materials were as described munodiffusiontest, rabbit antiserum raiseadgainst the in the previous report [1]. Fractions containing the enzyme activity were pooled and dialyzed against 2 mM sodium phosphate buffer, pH 7.2, for 12 h. Hydroxyapatite Chromatography-The dialyzed enzyme sample was applied to a hydroxyapatitecolumn (1X 6.5 cm) which had been pre-equilibrated with 2 m~ sodium phosphate buffer, pH 7.2. Sepharose 4B Gel Filtration-A Sepharose 4B column (1.6 X 86 cm) was pre-equilibrated with 10 mM sodium phosphate buffer, pH 7.2, and the concentrated enzyme preparation after the octyl-Sepharose step was applied and eluted with the same buffer at a flow rate of 3.6 ml/h. Sephadex G-200Gel Filtration-The fraction from the decyl-agarose chromatography was applied to a Sephadex G-200 column (1.6 X 86 cm), pre-equilibrated with 10 m~ sodium phosphate buffer, pH 7.2, containing 0.1% Triton X-100. The enzyme was stable for a t least 6 months
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