Reconstitution of CMP-N-acetylneuraminic acid hydroxylation activity using a mouse liver cytosol fraction and soluble cytochrome b5 purified from horse erythrocytes.
The hydroxylation of CMP-N-acetylneuraminic acid (CMP-NeuAc) in the formation of CMP-N-glycolylneuraminic acid requires several components which comprise an electron transport system. A protein, which replaces one of the components, was purified to homogeneity from a horse erythrocyte lysate. Based on its partial amino acid sequence and immunological cross-reactivity, this protein was identified as soluble cytochrome b5 lacking the membrane domain of microsomal cytochrome b5. The electron transport system involved in CMP-NeuAc hydroxylation was reconstituted, and then characterized using the purified horse soluble cytochrome b5 and a fraction from mouse liver cytosol. The hydroxylation reaction requires a reducing reagent, DTT being the most effective. Either NADH or NADPH was used as an electron donor, but the activity with NADPH amounted to about 74% of that with NADH. The hydroxylation was inhibited by salts and azide due to interruption of the electron transport from NAD(P)H to cytochrome b5 and in the terminal enzyme reaction, respectively.
Kozutsumi
Y
Y
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science.
Kawano
T
T
Kawasaki
H
H
Suzuki
K
K
Yamakawa
T
T
Suzuki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D004912:Erythrocytes / Q000378:metabolism*; D006736:Horses; D006900:Hydroxylation; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D015786:Cytochromes b5; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase