Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells.
A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.
del Castillo-Olivares
A
A
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencas, Universidad de Málaga, Spain.
Medina
M A
MA
Núñez de Castro
I
I
Márquez
J
J
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005292:Ferricyanides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
D005292:Ferricyanides; C007931:hexacyanoferrate III; D009245:NADH Dehydrogenase