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 adrenodoxin, the total proteins of the bovine adrenal cortex gave two bands, suggesting the presence of two forms of adrenodoxin in vivo: full-length and carboxy-terminal deleted adrenodoxins. To examine the effect of the carboxy-terminal deletion of adrenodoxin on its activity, cDNAs for Arg115stop mutant adrenodoxin and for Asp113stop mutant adrenodoxin were constructed. The wild type [Ad(2-128)] and carboxy-terminal deleted [Ad(2-114) and Ad(2-112)] recombinant adrenodoxins expressed in Escherichia coli were purified to give a single band on SDS-PAGE. They showed an A414/A276 value of 0.92. In an NADPH-cytochrome c reduction assay, the Km values for cytochrome c in the reconstituted system with AD(2-128), Ad(2-114) and Ad(2-112) were 39, 235 and 618 mM, respectively. The Vmax values were 638, 700 and 898 mol/min/mol flavin, respectively. In an NADPH-acetylated cytochrome c reduction assay, the maximum activity of Ad(2-128) was obtained at 50 mM NaCl, while the maximum activities of Ad(2-114) and Ad(2-112) were obtained at 100 mM NaCl; the latter values were 4-times higher than that of Ad(2-128). In the presence of 100 mM NaCl, the Km 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 microM, respectively. The Vmax values were 352, 305 and 382 mol/min/mol flavin, respectively. These results indicate that the effects of the carboxy-terminal deletion of adrenodoxin on NADPH-cytochrome 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 acetylated cytochrome c.
Sagara
Y
Y
Department of Medical Biology, Kochi Medical School, Japan.
Hara
T
T
Ariyasu
Y
Y
Kajiyama
A
A
Yasukochi
T
T
Horiuchi
T
T
D000107:Acetylation; D000325:Adrenodoxin / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D000325:Adrenodoxin; D009249:NADP