S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose-and time-dependent manner. The IC 50 of SNAP for GPx was 2 M at 1 h of incubation and was 20% of the IC 50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 M SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N -methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NOinduced apoptotic cell death.

GDP-L-Fuc:N-acetyl-␤-D-glucosaminide ␣136fucosyl-transferase (␣1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAc␤1-2Man␣1-6(GlcNAc␤1-2Man␣1-2)-Man␤1-4GlcNAc␤1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440,000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The ␣1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. ␣1-6FucT activity was found to be high in the transfected cells, as compared with non-or mocktransfected cells. Northern blotting analyses of rat adult tissues showed that ␣1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.It has been reported that the structures of glycopeptides change during the development and differentiation of embryos (1-4). Detailed analysis of specific antigens on the surface of various carcinoma cells revealed that carcinoma-specific sugar chains are expressed on the cell surface. A well documented phenotypic alteration of these specific sugar chains is the increase in the molecular weight of cell surface complex type N-linked glycan in transformed cells. This change has been observed regardless of the nature of the transforming agent: oncogenic viruses (5-9), chemical mutagens (10 -11), or DNA from unrelated tumor cells (12)(13)(14). This phenomenon was thought to reflect the deviation of carcinoma cells from the ordinary differentiation processes. ␣-Fucose residue attached to asparagine-linked GlcNAc also have some relationship with carcinogenesis. A difference in the binding pattern of serum ␣-fetoprotein with lentil lectin between hepatocellular carcinomas and benign liver diseases has been reported (15-17). Analyses of the carbohydrate structure of ␣-fetoprotein from hepatocellular carcinoma cell lines have indicated that almost all of the carbohydrates of ␣-fetoprotein are ␣1-6-fucosylated (18). ␣-Fetoprotein produced by germ cell tumors, such as yolk sac tumors, is also highly fucosylated (19). The activity of ␣1-6FucT 1 was higher in hepatocellular carcinoma tissue than in non-tumor tissue (20) and was induced by the transfection of the ras protooncogene into 3T3 fibroblast cells (21). Schachter et al. (22,23) first characterized ␣1-6FucT in porcine liver using a partially purified enzyme extract. The special release of ␣1-6FucT from platelets during blood clotting has been reported (24, 25), alteration of fucosylation has been reported in cystic fibrosis glycoproteins from different s...

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with l-serine was determined at 2.3 A resolution. The electron density map showed the Schiff base formation between l-serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the Calpha-H bond of the PLP-l-serine aldimine was not perpendicular to the PLP-Schiff base plane. This conformation is unfavourable for the alpha-proton abstraction by Lys244 and the reaction is expected to stop at the PLP-l-serine aldimine. Structural modelling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of l-serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the l-serine Calpha-H bond perpendicular to the PLP-Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the alpha-deprotonation of l-serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.

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