PURIFICATION AND CHARACTERIZATION OF A PLASMA-MEMBRANE FERRICYANIDE-UTILIZING NADH DEHYDROGENASE FROM EHRLICH TUMOR-CELLS

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidore ductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comp rises the isolation of an enriched plasma membrane fraction, solubiliz ation with Triton X-100, ion-exchange chromatography, ammonium sulphat e precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific a ctivity 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 su ggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximu m activity at pH values from 7.5 to 8.5. The purified enzyme showed Mi chaelis-Menten kinetics for the substrates, with apparent K-m values o f 4.3 x 10(-5) M and 6.7 x 10(-5) M for NADH and ferricyanide respecti vely. The isolated protein was strongly inhibited by Zn2+ and the thio l-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.