The accurate crystal structure of nicotinamide, 3-pyridinecarboxamide, was determined from X-ray and neutron diffraction experiments: C(6)H(6)N(2)O, M(r) = 122.13, monoclinic, P2(1)/c, Z = 4. The electron distribution at 150 K was determined by the maximum entropy method and the electrostatic potential in the crystal was calculated by Fourier convolution of the electron distribution. The electrostatic properties of the nicotinamide molecule depend on the molecular conformation. The asymmetric electrostatic potential field observed above and below the pyridine-ring plane is related to the rotation of the carboxamide group with respect to the pyridine plane. The positive potential peak at the C4 atom of the pyridine ring extends to the C=O-group side of the plane. The asymmetry of the potential on the C4 atom is consistent with the stereospecificity of hydride transfer in NAD(+)/NADH oxidoreduction.

Human ␤1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal␤1-3Gal␤1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 Å in the presence of the donor substrate product UDP, the catalytic Mn

Chondroitin sulfate (CS)-D and CS-E, which are characterized by oversulfated disaccharide units, have been shown to regulate neuronal adhesion, cell migration, and neurite outgrowth. CS proteoglycans (CSPGs) consist of a core protein to which one or more CS chains are attached via a serine residue. Although several brain CSPGs, including mouse DSD-1-PG/phosphacan, have been found to contain the oversulfated D disaccharide motif, no brain CSPG has been reported to contain the oversulfated E motif. Here we analyzed the CS chain of appican, the CSPG form of the Alzheimer's amyloid precursor protein. Appican is expressed almost exclusively by astrocytes and has been reported to have brain-and astrocyte-specific functions including stimulation of both neural cell adhesion and neurite outgrowth. The present findings show that the CS chain of appican has a molecular mass of 25-50 kDa. This chain contains a significant fraction (14.3%) of the oversulfated E motif GlcUA␤1-3GalNAc(4,6-O-disulfate). The rest of the chain consists of GlcUA␤1-3GalNAc(4-O-sulfate) (81.2%) and minor fractions of GlcUA␤1-3GalNAc and GlcUA␤1-3GalNAc(6-O-sulfate). We also show that the CS chain of appican contains in its linkage region the 4-O-sulfated Gal structure. Thus, appican is the first example of a specific brain CSPG that contains the E disaccharide unit in its sugar backbone and the 4-O-sulfated Gal residue in its linkage region. The presence of the E unit is consistent with and may explain the neurotrophic activities of appican.

The Drosophila melanogaster genome contains three putative glucuronyltransferases homologous to human GlcAT-I and GlcAT-P. These enzymes are predicted to be ␤1,3-glucuronyltransferases involved in the synthesis of the glycosaminoglycan (GAG)-protein linkage region of proteoglycans and the HNK-1 carbohydrate epitope of glycoproteins, respectively. The genes encode active enzymes, which we have designated DmGlcAT-I, DmGlcAT-BSI, and DmGlcAT-BSII (where BS stands for "broad specificity"). Protein A-tagged truncated soluble forms of all three enzymes efficiently transfer GlcUA from UDP-GlcUA to the linkage region trisaccharide Gal␤1-3Gal␤1-4Xyl. Strikingly, DmGlcAT-I has specificity for Gal␤1-3Gal␤1-4Xyl, whereas DmGlcAT-BSI and DmGlcAT-BSII act on a wide array of substrates with non-reducing terminal ␤1,3-and ␤1,4-linked Gal residues. Their highest activities are obtained with asialoorosomucoid with a terminal Gal␤1-4GlcNAc sequence, indicating their possible involvement in the synthesis of the HNK-1 epitope in addition to the GAG-protein linkage region. Gal␤1-3GlcNAc and Gal␤1-3GalNAc, disaccharide structures widely found in N-and O-glycans of glycoproteins and glycolipids, also serve as acceptors for DmGlcAT-BSI and -BSII. Transcripts of all three enzymes are ubiquitously expressed throughout the developmental stages and in adult tissues of Drosophila. Thus, all three glucuronyltransferases are likely involved in the synthesis of the GAG-protein linkage region in Drosophila, and DmGlcAT-BSI and -BSII appear to be involved in various GlcUA transfer reactions for the synthesis of proteoglycans, glycoproteins, and glycolipids. This activity distinguishes these glucuronyltransferases from their mammalian homologs GlcAT-P and GlcAT-D (or -S). Sequence alignment of the Drosophila glucuronyltransferases with homologs in human, rat, and Caenorhabditis elegans demonstrates the conservation of a majority of the critical amino acid residues in the active sites of the three Drosophila enzymes. Proteoglycans (PGs)1 play an essential role in a variety of biological processes such as cell-cell adhesion, cell proliferation, and tissue morphogenesis (1, 2). PGs consist of a core protein and sulfated glycosaminoglycan (GAG) side chains. PGs can be classified into three groups based on the nature of their GAGs: heparan sulfate (HS)-type PGs, chondroitin sulfate (CS)-type PGs, and keratan sulfate PGs. There is increasing evidence that uniquely sulfated domain structures of GAG side chains are critically involved in various functions of these PGs (3-5), and defective synthesis of GAGs causes aberrant morphology and even embryonic lethality during development (6).In biosynthesis, HS or CS linear chains are differentially assembled on the common linkage region tetrasaccharide Glc-UA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-, which takes place on a specific serine residue in a given core protein (7). Transfer of either ␣1,4-GlcNAc or ␤1,4-GalNAc to the tetrasaccharide linkage region terminus mediated by the GAG-specific hexosaminyltransferases determin...

Lipid alkyl radicals generated from polyunsaturated fatty acids via chemical or enzymatic H-abstraction have been a pathologically important target to quantify. In the present study, we established a novel method for the quantification of lipid alkyl radicals via nitroxyl radical spintrapping. These labile lipid alkyl radicals were converted into nitroxyl radical-lipid alkyl radical adducts using 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-N -oxyl (Cm ⌬ P) (a partition coefficient between octanol and water is approximately 3) as a spin-trapping agent. The resulting Cm ⌬ P-lipid alkyl radical adducts were determined by HPLC with postcolumn online thermal decomposition, in which the adducts were degraded into nitroxyl radicals by heating at 100 ؇ C for 2 min. The resulting nitroxyl radicals were selectively and sensitively detected by electrochemical detection. With the present method, we, for the first time, determined the lipid alkyl radicals generated from linoleic acid, linolenic acid, and arachidonic acid via soybean lipoxygenase-1 or the radical initiator 2,2 -azobis(2,4-dimethyl-valeronitrile). Peroxidation of polyunsaturated fatty acids, which are components of cellular membrane and lipoproteins, leads to vital damage to several types of cells. Reactive oxygen/nitrogen species (1), such as hydroxyl radical, hypochlorous acid (2-4), peroxynitrite (5, 6), and singlet oxygen molecule (7-9) are involved in the peroxidation of polyunsaturated fatty acids in the inflammatory lesions. On the other hand, lipoxygenases are speculated to be involved in the peroxidation of polyunsaturated fatty acids in the atheromatous plaque (10). Commonly, lipid alkyl radicals seem to be necessarily produced as an intermediate in these reactions.To detect labile free radicals via ESR, spin-trapping techniques have been established, in which unstable radicals react with spin-trapping agents to form relatively stable radical adducts (11). To date, nitrone spin-trapping agents, including ␣ -[4-pyridyl-1-oxide] N -tert-butyl nitrone and 5,5 Ј -dimethyl-1-pyroline-N -oxide, have been used to detect these lipid-derived radicals (12-17). Furthermore, Mason and coworkers (13-15) established an HPLC/ESR system to identify the resulting radical adducts. However, the trapping efficiency of these nitrone compounds toward lipid alkyl radicals is comparatively low. Consequently, these spin-trapping agents are not applicable to the quantification of lipid alkyl radicals. Five-or six-membered cyclic nitroxyl radicals are relatively stable. Nitroxyl radicals appeared to possess an ability to scavenge carbon-centered radicals (18). This special character of nitroxyl radicals makes it possible to quantitatively trap carbon-centered radicals. Johnson, Caron, and Blough (19) used hydrophilic 3-aminomethyl-2,2,5,5-tetramethylpyrrolidine-N -oxyl to evaluate carbon-centered radical generation: in this method, the adducts were fluorometrically detected after derivatization of amino groups by fluorescamine.The nitroxyl radical-carbon-centered radi...

During the course of a study to elucidate the role of modification of the common polysaccharide-protein linkage structure, GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser, in biosynthetic sorting mechanisms of the different sulfated glycosaminoglycan chains, a novel N-acetylgalactosamine (GalNAc) transferase was discovered in fetal bovine serum. The enzyme catalyzed the transfer of

We report the occurrence, in bovine serum of a soluble form, of N-acetylgalactosaminyl-transferase activity, which is involved in chondroitin sulfate biosynthesis, and we describe a simple assay method for the enzyme. Chondroitin with the general structure [GlcA-GalNAc], was found to serve as acceptor substrates for the enzyme. To develop a routine assay, bovine serum was incubated with polymeric chondroitin and UDP-[3H]GalNAc. After the removal of labeled endogenous acceptors derived from serum by trichloroacetic acid treatment, the labeled chondroitin sulfate chains were separated from residual unincorporated label by centrifugation using syringe columns packed with Sephadex G-25, and the effluent fractions were monitored by scintillation counting. This simple method allowed us to perform multiple assays and to establish assay conditions for the serum N-acetylgalactosaminyltransferase involved in chondroitin polymerization.

Lipoxygenases have been proposed to be a possible factor that is responsible for the pathology of certain diseases, including ischaemic injury. In the peroxidation process of linoleic acid by lipoxygenase, the E,Z-linoleate allyl radical-lipoxygenase complex seems to be generated as an intermediate. In the present study, we evaluated whether E,Z-linoleate allyl radicals on the enzyme are scavenged by radical scavengers. Linoleic acid, the content of which was greater than the dissolved oxygen content, was treated with soya bean lipoxygenase-1 (ferric form) in the presence of radical scavenger, CmP (3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl). The reaction rate between oxygen and lipid allyl radical is comparatively faster than that between CmP and lipid allyl radical. Therefore a reaction between linoleate allyl radical and CmP was not observed while the dioxygenation of linoleic acid was ongoing. After the dissolved oxygen was depleted, CmP stoichiometrically trapped linoleate-allyl radicals. Accompanied by this one-electron redox reaction, the resulting ferrous lipoxygenase was re-oxidized to the ferric form by hydroperoxylinoleate. Through the adduct assay via LC (liquid chromatography)-MS/MS (tandem MS), four E,Z-linoleate allyl radical-CmP adducts corresponding to regio- and diastereo-isomers were detected in the linoleate/lipoxygenase system, whereas E,E-linoleate allyl radical-CmP adducts were not detected at all. If E,Z-linoleate allyl radical is liberated from the enzyme, the E/Z-isomer has to reach equilibrium with the thermodynamically favoured E/E-isomer. These data suggested that the E,Z-linoleate allyl radicals were not liberated from the active site of lipoxygenase before being trapped by CmP. Consequently, we concluded that the lipid allyl radicals complexed with lipoxygenase could be scavenged by radical scavengers at lower oxygen content.