The cerebroside-sulfate activator protein~CSAct or Saposin B! is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct~ϩ16 Da!, separated from nonoxidized CSAct by reversedphase high-performance liquid chromatography~RP-HPLC!, showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. Highresolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass~ϩ16 Da!. The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61~26% of control! with other residues in the Q 60 MMMHMQ 66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.

Membrane fractions from a lon strain of Escherichia coli but not a wild-type strain catalyze the incorporation of fucose from guanosine 5'-diphosphate-fucose into a lipid and into polymeric material. Both incorporation reactions specifically require only uridine 5'-diphosphate (UDP)-glucose. The sugar lipid was shown to be an intermediate in the synthesis of the polymer which was related to colanic acid. The sugar lipid had the structure (fucose3, glucose2)-glucose P-P-lipid. Its behavior on column and thin-layer chromatography, the rates of its hydrolysis in acid and base, and the response of its synthesis to inhibitors are all identical to the other sugar-lipid intermediates which have been shown to contain sugars attached to the C55-polyisoprenol, undecaprenol, by a pyrophosphate linkage. The membrane fractions from both the lon strain and the wild-type strain also catalyzed the incorporation of either glucose from UDP-glucose or galactose from UDP-galactose into a lipid fraction which was shown to contain the free sugar attached by a monophosphate linkage to an undecaprenol-like lipid. This lipid was isolated and its nuclear magnetic resonance spectra was identical to undecaprenol. The membrane fractions from both strains also incorporated glucose from UDP-glucose into glycogen and into a polymer that behaved like Escherichia coli lipopolysaccharide. Conditions were found where the incorporation of glucose could be directed specifically into each compound by adding the appropriate inhibitors.