N-linked oligosaccharide structures in the diamine oxidase from porcine kidney.
ABSTRACT Structures of the N-linked glycans released from porcine kidney diamine oxidase (DAO) were characterized utilizing various analytical techniques, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), high-performance capillary electrophoresis (HPCE), and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The oligosaccharide sequences present in DAO were conclusively determined using specific exoglycosidases in conjunction with MALDI/TOF-MS. The structures found in the glycoprotein are primarily linear, di-, or tribranched fucosylated complex type. MS analysis of the esterified N-glycan pool derived from DAO indicated the presence of several di- and trisialylated structures.
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ABSTRACT: Copper amine oxidases (EC 184.108.40.206) exhibit atypical stereochemical patterns in the reactions they catalyze. Dopamine and tyramine are oxidized with abstraction of the pro-R hydrogen by the porcine plasma amine oxidase, the pro-S hydrogen by pea seedling amine oxidase and a net nonstereospecific proton abstraction by the bovine plasma enzyme. This provides the first example in which a reaction catalyzed by enzymes in the same formal class occurs by all three possible stereochemical routes. To assess the underlying mechanistic significance of this heterogeneity, we have established the stereochemical course of the oxidation of tyramine by five additional copper amine oxidases using 1H NMR spectroscopy. Reactions catalyzed by rabbit and sheep serum amine oxidases are nonstereospecific. These enzymes exhibit rare mirror image binding with differential flux through two opposite and stereospecific reaction pathways. Differential primary kinetic isotope effects are observed for each mode, 8 and 4.6 for pro-S abstraction and 2.6 and 2.7 for pro-R abstraction by the sheep and rabbit amine oxidases, respectively. Tyramine oxidations catalyzed by the soybean and chick pea amine oxidases and porcine kidney diamine oxidase, however, are all stereospecific, occurring with loss of the pro-S hydrogen at C-1. Solvent exchange profiles are consistent within each stereochemical class of enzyme; the pro-R and nonstereospecific enzymes exchange solvent into C-2 of product aldehydes, the pro-S enzymes do not.Journal of Biological Chemistry 05/1991; 266(11):6795-800. · 4.65 Impact Factor
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ABSTRACT: The heterogeneous nature of most polysaccharides found in nature includes distribution in molecular weight, primary sequence, and branching. The analytical methodology used in the characterization of these structural aspects must ensure high separation efficiency and selectivity. This paper reports on the high-performance capillary electrophoresis (HPCE) separation of branched forms of oligosaccharides as well as some variants in the primary structure. Oligosaccharide maps were obtained after selective debranching using isoamylase, laminarinase, and cellulase enzymes. The samples investigated were alpha-D-glucans (amylose, amylopectin, and pullulan) and beta-D-glucans (exemplified by lichenan). The solutes were separated as fluorescent derivatives with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and detected by laser-induced fluorescence at 514 nm using a He/Cd laser (excitation at 325 nm). The number of theoretical plates was in excess of one million per meter. Baseline resolution of oligosaccharides with a degree of polymerization approximately 70 was obtained within 15 min using borate buffer as the electrolyte.Carbohydrate Research 06/1994; 258:1-9. · 2.04 Impact Factor
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ABSTRACT: A new way of combining exoglycosidase digestion with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS) is described which permits the structural characterization of underivatized oligosaccharides on low picomole amounts of starting material. The key feature of the new approach is that an oligosaccharide sample can be recovered after a MALDI experiment and a series of sequential exoglycosidase digestions can be carried out on that sample within a single working day. The following steps are involved: (i) recording a molecular mass profile of the starting material by MALDI/TOF-MS using a mixture of 2,5-dihydroxybenzoic acid and 1-hydroxyisoquinoline as the matrix; (ii) recovery of the sample from the target and removal of the matrix by droplet dialysis (molecular mass cut-off 500 Da); (iii) exoglycosidase digestion in a volume of 1 microliter; (iv) removal of the incubation buffer by droplet dialysis; (v) removal of the enzyme by absorption on a Nafion membrane and (vi) start of the next cycle from (i). The method exhibits the following advantages over traditional oligosaccharide sequencing techniques: (i) analysis of digestion products by MALDI/TOF-MS is much faster than by chromatographic techniques; (ii) no derivatization of the analyte is required; (iii) exoglycosidase digestions work faster in small reaction volumes because substrate concentrations are closer to the Km of the enzyme; (iv) advanced sample handling techniques ensure reduced losses and (v) no sample splitting is needed for analysis and therefore the sensitivity of the overall method is increased. The method is illustrated by the analysis of isolated glycans and complex mixtures derived from chicken ovalbumin and human immunoglobulin G.Journal of Mass Spectrometry 11/1996; 31(10):1131-40. · 3.21 Impact Factor