CMP-N-acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands. Interaction with membrane-bound and soluble cytochrome b5-dependent electron transport chains.
In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5 Ac) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5 and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane-associated, forms of cytochrome b5 and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b5 and cytochrome b5 reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b5. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b5 interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b5. Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycolylneuraminic acid.
Shaw
L
L
Biochemisches Institut, Christian-Albrechts Universität, Kiel, Germany.
Schneckenburger
P
P
Schlenzka
W
W
Carlsen
J
J
Christiansen
K
K
Jürgensen
D
D
Schauer
R
R
D000818:Animals; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D003579:Cytochrome Reductases / Q000378:metabolism; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism; D003600:Cytosol; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D012995:Solubility; D013363:Submandibular Gland / Q000201:enzymology*; D013552:Swine
D015786:Cytochromes b5; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase