PubMed · 403653
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S P Merinov, V A Shirokov. 1977. [Transamination processes in Brucella].. https://pubmed.ncbi.nlm.nih.gov/403653/
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A comparative study was made on the glycoform of O-glycan from human myeloma immunoglobulin A1. By gas-phase hydrazinolysis, O-glycan was released from its hinge portion. The released oligosaccharide was pyridylaminated and separated by a two-dimensional analytical method of gel filtration and reverse-phase HPLC. Four major pyridylamino derivatives (P1-P4) were obtained. The neutral component (P4) among them was identified as Gal beta 1,3GalNAc-PA by cochromatography with an authentic standard pyridylamino sugar. The desialylation of the other components indicated the largest P1 and middle size P2 components possibly corresponded to a disialylated structure, NeuAc alpha 2,3Gal beta 1,3(NeuAc alpha 2,6)GalNAc-PA, and a monosialylated component, NeuAc alpha 2,3Gal beta 1,3GalNAc-PA, respectively. The structural assignment of P3 is still incomplete. Four similar components were also detected in bovine fetuin whose relative content (P1:P2: P3:P:4) was 16:43:19:22. The relative content (%) of P1-P4 (glycoform) in IgA1 from the healthy control was 10.1 +/- 3.3, 48.2 +/- 4.6, 7.0 +/- 2.6, and 34.7 +/- 4.5. The glycoform of O-glycan on IgA1 thus appears the same for any individual. Analysis of IgA1 myeloma protein indicated glycoforms distinct from those of the healthy controls. The relative content of these component could be classed as 2:8:0:90 (Type I, only one case designated as Kita), 5:24:3:68 (Type II, seven cases), and 9:41:5:45 (Type III, four cases). Thus, the results for IgA1 myeloma protein indicate that at least three glycoforms of O-glycan are possible for the IgA1 hinge structure. However, only one glycoform was found in the healthy controls.
A highly effective method for the synthesis of neoglycoconjugates has been developed. The technique is based on two steps. First, a sulfhydryl group is introduced into the carbohydrate by reductive amination with cysteamine, and secondly the sulfhydryl group carrying carbohydrate is coupled to a protein or carrier via a disulfide bridge. The method was used to couple sialic acid-containing carbohydrates such as N-acetylneuraminyllactose and the carbohydrate moieties of gangliosides (GM1, GM3 and GD1b) to bovine serum albumin. Biological functionality of the generated GM1- and GD1b-neoglycoprotein was demonstrated by a choleratoxin-binding assay and an immunoassay, respectively. The applicability of neoglycoconjugates for the quantification of gangliosides in solution by a competitive assay is shown.
The pH dependence of the kinetic parameters of the L-aspartase-catalyzed reaction have been examined in both the amination and the deamination directions. The enzyme isolated from Escherichia coli exists in a pH-dependent equilibrium between a higher pH form that has an absolute requirement for a divalent metal ion and for substrate activation, and a low pH form that does not require activation by either substrate or metal ions. The interconversion between these enzyme forms is observed near neutral pH in the profiles examined for the reaction in either direction. This pH-dependent activation has not been observed for other bacterial aspartases. Loss of activity is observed at high pH with a pK value of 9. The pH profiles of competitive inhibitors such as 3-nitropropionic acid and succinic acid have shown that the enzyme group responsible for this activity loss must be protonated for substrate binding at the active site. An enzymatic group has also been identified that must be protonated in the amination reaction, with a pK value near 6.5, and deprotonated in the deamination reaction. This group, tentatively assigned as a histidyl residue, fulfills the criteria for the acid-base catalyst at the active site of L-aspartase.