Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.
1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.
Güray
T
T
Department of Biology, Middle East Technical University, Ankara, Turkey.
Arinç
E
E
D000818:Animals; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D003902:Detergents; D005292:Ferricyanides / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008954:Models, Biological; D009243:NAD / Q000378:metabolism; D012756:Sheep
D003902:Detergents; D005292:Ferricyanides; D009243:NAD; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; C028033:potassium ferricyanide