DIFFERENT EFFECTS OF CARBOXY-TERMINAL DELETION IN THE ADRENODOXIN MOLECULE ON CYTOCHROME-C AND ACETYLATED CYTOCHROME-C REDUCTIONS

In immunoblotting analysis using a rabbit antibody to bovine adrenodox in, the total proteins of the bovine adrenal cortex gave two bands, su ggesting the presence of two forms of adrenodoxin in vivo: full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of t he carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrenodoxin w ere constructed. The mild type [Ad(2-128)] and carboxy-terminal delete d [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Esch erichia coli mere purified to give a single band on SDS-PAGE. They sho wed an A(414)/A(276) value of 0.92. In an NADPH-cytochrome c reduction assay, the K-m values for cytochrome c in the reconstituted system wi th Ad(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 nM, respect ively. The V-max values were 638, 700 and 898 mol/min/mol flavin, resp ectively. In an NADPH-acetylated cytochrome c reduction assay, the max imum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maxim um activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values mere 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the K-m values for acetylated cytochrome c in the system reconstituted with Ad(2-128), Ad(2-114) and Ad(2-112) were 220, 33 and 22 mu M, respectively. The V values were 352, 305 and 382 mol/min/mol flavin, respectively. These results indicate that the eff ects of the carboxy-terminal deletion of adrenodoxin on NADPH-cytochro me c and acetylated cytochrome c reductions are different; the carboxy -terminal region (residues 113-128) of adrenodoxin largely contributes to the binding with cytochrome c but disturbs the binding with acetyl ated cytochrome c.