Purification, characterization and reconstitution of CMP-N-acetylneuraminate hydroxylase from mouse liver

Abstract

CMP-N-acetylneuraminate hydroxylase was isolated from mouse liver high speed supernatant with a yield of 0.4% and an apparent 1000-fold purification. The enzyme is a monomeric protein with a molecular weight of 66 kDa, as determined by gel filtration and SDS-PAGE. The hydroxylase system was reconstituted with Triton X-100-solubilized mouse liver microsomes and purified soluble or microsomal forms of cytochrome b5 reductase and cytochrome b5. The systems were characterized in detail and kinetic parameters for each system were determined.

A structural difference between the cell surfaces of humans and the great apes

“…1). This oxygen atom is added by the enzymatic action of a specific hydroxylase which converts the nucleotide sugar donor CMP-Neu5Ac to CMP-Neu5Gc (Shaw and Schauer, 1988;Bouhours and Bouhours, 1989;Muchmore et al, 1989;Kozutsumi et al, 1990;Kozutsumi et al, 1991;Shaw et al, 1992;Kawano et al, 1993;Schneckenburger et al, 1994;Shaw et al, 1994;Takematsu et al, 1994;Kawano et al, 1995). The expression of Neu5Gc is widespread in mammalian cells (it can be the major sialic acid in some cell types), and shows tissue-specific and developmentally regulated expression in a variety of systems (Schauer, 1982;Bouhours and Bouhours, 1983;Muchmore et al, 1987;Muchmore, 1992;Varki, 1992).…”

CMP‐N‐acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron‐sulphur protein to be described in Eukarya

“…Although the N-terminal primary structure of the pig enzyme could not be determined by cDNA sequencing, the 92% identity of the obtained sequence with the analogous region of the mouse enzyme suggests that the remaining 5' sequence of the pig enzyme is also homologous with the mouse hydroxylase. Indeed, the identical molecular weight and catalytic properties of both enzymes [12][13][14] underscore their structural similarity.…”

“…Since (2Fe-2S) groups do not usually incorporate exogenous iron in vitro, the activatory effect of Fe 2+ ions on the hydroxylase [13,14] may be due to the formation of a mononuclear iron centre. A combination of a Rieske (2Fe-2S) centre and mononuclear iron was proposed to exist in the benzoate and phthalate dioxygenases of bacterial origin [19,26] and also in the O2-activating component of 4-methoxybenzoate monooxygenase from Pseudomonas putida [27].…”

“…The purified enzyme is monomeric and has a molecular weight of 65 kDa [12][13][14]. The fact that CMP-Neu5Ac hydroxylase is dependent on cytochrome b5 and is inhibited by the addition of iron-binding agents [14] suggests that this enzyme may possess an ironcontaining prosthetic group.…”

“…CMP-Neu5Ac hydroxylase has been purified from mouse liver and pig submandibular gland and found to be a cytosolic monooxygenase, which requires NADH, cytochrome b5 re-ductase and cytochrome b5 for activity and can be stimulated by the addition of iron ions [9][10][11]. The purified enzyme is monomeric and has a molecular weight of 65 kDa [12][13][14]. The fact that CMP-Neu5Ac hydroxylase is dependent on cytochrome b5 and is inhibited by the addition of iron-binding agents [14] suggests that this enzyme may possess an ironcontaining prosthetic group.…”