Four forms of bovhrc admnodoxin with modified amino-tennini obtained by direct cxpmssion of cDNAs in E~cIrerichia roli arc Ad(Mct').Ad(Met-'k Ad(Met-r-1 and Ad(Met'k The shoulder numbers rcprcsent the site of translation initiator Met at the amino-&mini. T!tc adrcnodoxins, exazpt for Ad(Met-'k UIXE puri!i& from the cell lysate and the ratios of A&o-A,, of the purified proteins were over 0.92. NADPH-cytochrome c reducta~ activities of the three forms of adrenodoxin in the prcscncc of admnodoxin mductasc wet-c the same as that of purified bovine adrenocortica! adrenodoxin. HOHEXL as c)-iochromc P-4X& reduction catalyzed by Ad(Mct') was about 60% of that by Ad(Mct'). the contribution of the amino-terminal region for the electron transfer or binding to cytochromc P-450sCx-would need to bc considered. MATERIALS AND METHODSThe rull-size cDNA Tar bovine Ad was digcjtcd with cxonuclcasc Bal3l to obtain various lengths ofdclction of the S-region as fo!!sws: the Psrl Fragment of the cDNA which cowzrs the cntirc sequcncc of bovine Ad [9! was su&!oncd to the Pst! site or pUC9. The &JR! Xnwl fragment ol' the piasmid which includes the Ad L.)NA was cloned to the Es&-Stra! site of the expression vector pKK223-3.With Xntnl dig&on. the 3'untranslatcd region or the Ad cDNA was cleaved o!T IO bases downstream From the stop codon. hftcr a 5.5 kb &JR! fragment of DNA dcrivcd from R plasmid was inscrtcd into the EroRl site of the clone. the plasmid was digcstcd with Sntu! and then withcxonuclease Ral31. T!tcSmul sitcisdcrived from pUC9.Thc tnrncatcd cDNAs were flash-ended and ligated to the .&JR! site of the vector pKK223-3 using the EruR! linker, CATGAATTCATG. which was dcsigncd to include an initiation codon. During this step. excess EcoRl linker and remaining R pksmid DNA wcrc rcmovcd by EcoRI digestion. '!ltc final constructs wcm used for transformation of E. ndi DIZIO

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.