Cytochrome b5-like hemoprotein/cytochrome b5 reductase complex in rat liver mitochondria has NADH-linked aquacobalamin reductase activity.
Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.
Department of Agricultural Chemistry, University of Osaka Prefecture, Japan.
H
H
Saido
F
F
Watanabe
Y
Y
Tamura
K
K
Miyatake
A
A
Ito
T
T
Yubisui
Y
Y
Nakano
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003579:Cytochrome Reductases / Q000302:isolation & purification / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000378:metabolism*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D013053:Spectrophotometry; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000378:metabolism
C043960:aquacobalamin; D015786:Cytochromes b5; C068157:aquacobalamin reductase; D009247:NADH, NADPH Oxidoreductases; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D014805:Vitamin B 12