Molecular cloning and immunological characterization of porcine kidney ferredoxin

ARCHIVES

OF BIOCHEMISTRY

AND BIOPHYSICS

Vol. 293, No. 2, March, pp. 213-218,1992

Molecular Cloning and Immunological of Porcine Kidney Ferredoxin John L. Omdahl,l Department

Characterization

Keith Wilson,2 Harold Swerdlow,3 and William

of Biochemistry,

University

of New Mexico School of Medicine,

J. Driscol14

Albuquerque,

New Mexico 87131

Received June 28, 1991, and in revised form October 28, 1991

Porcine renodoxon is a kidney mitochondrial ironsulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A fulllength cDNA clone to porcine renodoxin was isolated in the current investigation and used to study the protein’s primary structure and immunological properties. The cysteine ligands for the iron-sulfur center, and the surface protein-binding and phosphorylation sites occupied identical positions in both porcine renodoxin and bovine adrenodoxin. Furthermore, porcine renodoxin was functionally indistinguishable from bovine adrenodoxin and the mature forms of both proteins had the same encoded length and shared -91% sequence similarity. A synthetic peptide to the surface protein-binding region was used to demonstrate the antigenicity of the domain in both the porcine and the bovine ISPs. However, porcine renodoxin displayed only limited immunological identity to other regions of bovine adrenodoxin as measured by competitive enzyme-linked immunosorbent assay. Part of this immunological distinction was attributed to the COOH-terminal processing of porcine renodoxin, an action which negated expression of a COOH-terminal antigenic site that is present in bovine adrenodoxin. Other antigenic differences were linked to charged-residue substitutions that were located in predicted surface domains. The highest frequency of surface-residue substitutions in ferredoxin proteins was predicted for porcine renodoxin, which could provide a basis for understanding why the pig protein appears more antigenically divergent than other ferredoxins. o 1992Academic press, hc.

i To whom correspondence should be addressed. * Current address: Department of Biochemistry & Molecular Biology, Oregon Health Sciences University, Portland, OR 97201. 3 Currant address: Department of Bioangineering, University of Utah, Salt Lake City, UT 84112. 4 Current address: National Institutes of Health, Bldg. 10, Room BlL466, Bethesda, MD 20892. ow3-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Tissue-specific expression of mitochondrial steroid hydroxylases is dependent upon the presence of an ironsulfur protein (BP or ferredoxin), which functions to shuttle electrons between NADPH-ferredoxin reductase and the monooxygenase cytochromes P450.5 These obligate ferredoxins are nuclear encoded, have -90% sequence similarity across species (l-4), and display a high degree of immunological cross-reactivity when measured by Ouchterlony double-diffusion analysis against bovine adrenodoxin antibody (5). As part of our molecular regulatory study of the vitamin D cytochromes P450, we recently isolated the iron-sulfur protein from pig kidney mitochondria, i.e., renodoxin,6 and demonstrated its biochemical similarity to the bovineadrenodoxin paradigm (6). However, during the immunoblot analysis of porcine renodoxin only a partial immunological cross-reactivity was observed with bovineadrenodoxin antibody (7). The extent of the antigenic differences was analyzed by competitive ELBA in the current report and found to be more divergent than would have been predicted from earlier work comparing the immunological reactivity of bovine adrenodoxin with ferredoxins from several species, which had not included the porcine protein (5). Using computer examination of cDNA7-derived primary structures and ELISA analysis, we found the antigenic distinctions between porcine and bovine ferredoxin to be associated with the COOH-terminal processing of porcine ferredoxin and charged-res’ Abbreviations used: P450,, cholesterol side-chain-cleavage enzyme (EC 1.14.15.6); P450,, 25-hydroxyvitamin D 1-monooxygenase (EC 1.14.13.13); ELISA, enzyme-linked immunosorbent assay; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium; PMSF, phenylmethylsulfonyl fluoride; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis. ’ In accordance with the recommendations of the IUPACOOH-IUB Commission on Biochemical Nomenclature (38), the terms renal ferredoxin and renodoxin will denote the iron-sulfur protein from kidney mitochondria and the terms adrenal ferredoxin and adrenodoxin will denote the iron-sulfur protein from adrenal mitochondria. r The nucleotide sequence for pig kidney ferredoxin has been reported to the GenBank/EMBL data base, accession number 57674. 213

214

OMDAHL

idue substitutions mains. MATERIALS

in predicted

surface (antigenic)

do-

AND METHODS

Reagents and biochemicals. Restriction enzymes were obtained from Bethesda Research Laboratories (BRL), Pharmacia LKB Biotechnology, Inc., and New England Biolabs. Nitrocellulose was obtained from Schleicher & Schuell, Inc. The pIBI76 plasmid was purchased from International Biotechnologies Inc., Sequenase was obtained from United States Biochemical Corp., T1 kinase was purchased from Pharmacia LKB Biotechnology, Inc., and the Bethesda Research Laboratories kit was used for nick-translation. [cy-32P]dATP, [-Y-~*P]ATP, [a-32P]dCTP, and [cy-36S]dATP were purchased from DuPont-New England Nuclear. Goat anti-rabbit IgG alkaline phosphatase conjugate andp-nitrophenyl phosphate were obtained from Sigma and the phosphatase substrate BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium) was purchased from Kirkegaard & Perry Laboratories, Inc. Other chemicals were of reagent grade or higher. The bovine adrenodoxin clone (pBAdx4) and antibody ta a COOH-terminal peptide for bovine adrenodoxin were kindly provided by M. Waterman (Dallas, TX). Porcine renodoxin and bovine adrenodoxin were isolated as described (6,7) with fluoride (PMSF) in all soinclusion of 0.2 mM phenylmethylsulfonyl lutions. Antibodies were raised in New Zealand white rabbits (7). The synthetic peptide LEAITDEENDMLDLA (porcine renodoxin, Leu-67 through Ala-81) was obtained from the Protein Resource Laboratory in the Department of Biochemistry at the University of New Mexico School of Medicine. Analytical procedures. PAGE was run using 20% SDS gels (6) and ELISA as described (8). Proteins for immunoblot analysis were run on 20% SDS-PAGE, electroblottad to nitrocellulose, and stained according to the manufacturer’s procedure using alkaline phosphatase IgG conjugate with BCIP-NBT as substrate. COOH-terminal residues were released from denatured protein (4 M guanidium HCl, 30 min) by treatment with carboxypeptidase Y (1:40) (a kind gift from B. McMullen, University of Washington, Seattle) at pH 5.25 (20 mM acetate). Amino acids were separated on a Gilson Dionex system using o-phthaldehyde detection. DNA-oligomer synthesis. A mixture of DNA oligomers (14-mers) was synthesized using &cyanoethyl phosphoramidites (Applied Biosysterns DNA Synthesizer, Model 308A), and purified by PAGE and gelexclusion chromatography before end-labeling. The oligomer contained 24 different 14-mers [GT(A/G/T)AT(T/C)TT(G/A)TC(T/C)TC] that were complementary to a pig-renodoxin mRNA segment encoding for the NH&rminal sequence Glu-Asp-Lys-Ile-Thr (7). Construction of cDNA library. Total RNA was isolated from pig kidney cortical-tissue induced for cytochrome P450D1 (kindly provided by R. Horst, Ames, IA) using the guanidinium isothiocyanate and cesium chloride gradient method (9). Poly(A)+-enriched mRNA was prepared using oligo(dT) chromatography (10). Double-stranded cDNA was synthesized (11) (BRL one-tube procedure), inserted into the PstI site of pBR322 DNA, and used to transform DH5 Escherichiu coli (12), resulting in a cDNA library with 2 X lo* recombinants. Screening of cDNA library. The cDNA library was screened by colony hybridization (10) using replicate filters. The library was plated onto nitrocellulose filters at high density (-150,000 colonies per 150-mm plate) and DNA fixed to replicate filters was hybridized with ?P-labeled heterologous bovine-adrenodoxin DNA (pBAdx4) (1) or a ll-mer mixture encoding pig renodoxin. Pig-renodoxin clones were isolated and restriction mapped following a second screening at low-density plating. The and bovine-adrenodoxin DNA was 3zP labeled by nick-translation the mixed DNA ll-mers were end-labeled using Td polynucleotide kinase (10). Subcloning and DNA sequence analysis. Positive pBR322 recombinants were subcloned into PstI cut pIBI76, which contains a reversesequence primer 5’ to the multiple cloning polylinker and the phage Fl origin of replication. Single-stranded DNA was sequenced by the dideoxy

ET AL. chain-termination method (13) using [~v~‘S]~ATP and Sequenase. Regions of gel compression were sequenced using both dITP and dGTP. The reverse-sequence primer in pIBI76 and three internal primer sites were used to walk through the renodoxin sequence in both directions. Nucleotide and protein sequence data were analyzed by DNASTAR (Madison, WI).

RESULTS Isolation and characterization of renodoxin cDNA clone. A full length -2.4-kb renodoxin clone was identified by screening 2 X lo6 colonies with both a bovine adrenodoxin cDNA clone and an oligomer mixture that encodes an amino-terminal sequence of pig renodoxin. The renodoxin clone (pPRdx2) had seven nucleotides in the 5’-untranslated region, which included the optimal eucaryotic translation-initiation sequence (A/GNNATGG) (14) that was used to identify the initiator methionine (Fig. 1). The renodoxin open reading frame encoded for 186 amino acids and was followed by an -1.9-kb noncoding 3’-region. Derived primary structure for renodoxin. The cDNAderived sequences for the porcine presequence (58 residues) and mature protein (128 residues) were the same length as their bovine counterparts. The porcine presequence contained a high level of glycine, serine, threonine (-30% of total), and positively charged arginine, and shared 69% sequence similarity with bovine adrenodoxin. The mature porcine and bovine ferredoxins displayed a high degree of sequence similarity (-91% identity) with amino acid differences clustered into two distinct regions (Fig. 2). Substitutions occurred in the NHz-terminal (residues 17-35) and COOH-terminal (residues 99-127) domains with a single substitution present in the midmolecule region (residue 61) (Fig. 2). No substitutions occurred in: (i) the cysteine domains of the iron-sulfur center (residues 46,52,55, and 92) (15), (ii) the highly acidic proteinbinding region that is involved with the binding of ferredoxin reductase and cytochromes P450 (residues 7286) (16), or (iii) the Ser-88 residue that has been identified as a phosphorylation site (17) (Fig. 2). The primary structure for mature porcine ferredoxins was the same for both kidney renodoxin and adrenal adrenodoxin except for the presence of an 11-residue COOHterminal peptide that was not detected during the original Edman degradation analysis (18) (Fig. 2). Sequence identity between the mature pig kidney and adrenal ferredoxins is consistent with the expression of a single ferredoxin gene within species (19). Secondary structural analysis. Predicted secondary structural features of the porcine and bovine ferredoxins were determined by Kyte-Doolittle (20), Hopp-WOO& (21) and Chou-Fasman (22) analyses. The reliability of these programs with small iron-sulfur proteins was verified (personal data) with crystallographic data for Spirulinu platensis ferredoxin (23).

PORCINE -7 actggct

KIDNEY

215

FERREDOXIN

ATG GCC GTC CGGCTC CTG CGCGTC GCC TCC GCA GCC CTG CCC GACACG GCA +51 Met Ala Val Arg Leu Leu Arg Val Ala Ser Ala Ala Leu Gly Asp Thr Ala 10 1

GTT CGGTGC CAG Ccc CX GTC GGA CCC CCC GCGGGAMC CGGCGGCCG GGCGGCAGC +lOg Val Arg Trp Gln Pro Leu Val Gly Pro Arg Ala Gly Asn Arg Gly Pro Cly Gly Ser 30 20 ATC TGG CTG GGT CTG GGT GGCCGT GCCGCC GCA GCGCGGACG CTG AGC TTG TCG GCG+165 Ile Trp Leu Gly Leu Gly Gly Arg Ala Ala Ala Ala Arg Thr Leu Ser Leu Ser Ala 50 40 . TCA CAA GACAAA ATA ACA GTC GAGTTT ATA MC CGT GAT GGT +222 CGCGCGTGGI AGC AGC Arg Ala Trp Ser Ser Ser Glu Asp Lys Ile Thr Val His Phe Ile Am Arg Asp Gly 70 60 GTT GTGATT GAA +279 AAG ACG TTA ACA ACC CAA GGAAAA GTT -4TCT GCGC Lys Thr Leu Thr Thr Gin Gly Lys Val Gly Asp Ser Leu Leu Asp Val Val Ile Glu 90 80 MT AAT CTA CAT ATT GAT GGT TTT GGT GCA TGT GAGGGGACC TTG GCT TGC TCT ACC +336 Am Am Leu Asp Ile Asp Gly Phe Gly Ala Cys Glu Gly Thr Leu Ala Cys Ser Thr 110 100 TGT &AC CTT ATC TTT GM GAGCAC ATA TTT GAGAAA TTA GAA GCA ATC ACC GAT GAG +393 Cys His Leu Ile Phe Glu Asp His Ile Phe Glu Lys Leu Glu Ala Ile Thr Asp Glu 130 120 GAGMT GAC ATG CTG GAT CTG GCA TAT GGACTA ACA GAT AGA TCA CGGCTG GGCTGC +450 Glu Asn Asp Het Leu Asp Leu Ala Tyr Gly Leu Thr Asp Ax-g Ser Arg Leu Gly Cys 150 140 CM ATC TGT TTG ACA AAG GCT ATG GACAA; ATG ACC GTT CGA CTG CCT GAGGCT GTG +507 Gin Ile Cys Leu Thr Lys Ala Her Asp Am Met Thr Val Arg Val Pro Glu Ala Val 160 CCC GAT CCC AGA GAGTCC ATT GAT TTG GGCAAG MC TCC TCT MA CTA GM TAA ata +564 Ala Asp Ala Arg Glu Ser Ile Asp Leu Gly Lys Am Ser Ser Lys Leu Glu stop 170 186 180

atacatttattattatg

+658

FIG. 1. DNA and deduced amino acid sequence for porcine renodoxin. The arrow before Ser-59 denotes the processing site between the signal peptide and mature protein. Underlined sequences represent the primer-extension sites used in the sequence analysis. Lower case DNA sequences represent untranslated flanking regions and the capitol letters in the 3’ flanking region denote a putative polyadenylation signal. Dots above the DNA sequence are spaced at 20-base intervals.

The presequence for renodoxin contained a high concentration of arginine and hydrophobic residues that were asymmetrically distributed into positively charged and hydrophobic regions within a predicted NHz-terminal (Yhelical domain (24, 25) (Figs. 1 and 3), which is charac-

teristic of imported mitochondrial proteins (24-28). Secondary structural analysis (32,33) of the COOH-terminal region of the presequence predicted a hydrophilic-surface domain with an ampliphilic P-strand containing a consensus two-step cleavage motif (28) consisting of Arg-49,

20 BOVINE/liver' BOVINEjadrennl PORCINE/kidney

40

t ----------------E----K--------Vo-----"Q--.--------.--~----.--. ---------.--..--E----K--I--------Vd-----------------------..

60

t

.f

SSSEDKITVHFINRDGKTLTTQGKVCDSLLDVVIENNLDIDGFGACEGT~CSTCHLIFE PORCINE/adrenal'--------------------------------------.---~------------.----

61

80

t t Q----------------------------------------------~--D--S---~-~.

100

120

128

t

t

t

~---.-------------------------------------~-----D--S-------M-M~---I-. DHIFEKLEAITDEENDMLDLAYGLTDRSRLGCQICLTKAMDNMTVRVPEAVADARESIDLGKNSSKLEb ---------------------------------------------~-.---------. FIG. 2. Comparison of porcine and bovine mature ferredoxin sequences. Dashed lines signify that the residue is the same as porcine renodoxin. Sequences denoted by an asterisk were determined by Edman-degradation analysis [bovine liver (39), porcine adrenal (l&3)] and other sequences were derived from cDNA data [bovine adrenal (l), porcine kidney (Fig. l)].

216

OMDAHL 2 Porcine 1

antigenic

-3

0

",':"":I"""'4 50

100

150

190

RESIDUE

FIG. 3. Hydropathy profiles of mammalian ferredoxins. The profiles were calculated according to Kyte and Doolittle (20) using a window of nine residues. The arrow notation for the mature proteins (4) marks the processing site between presequence and mature protein. Other notations include: (V) approximate locations of residue substitutions in bovine adrenodoxin relative to porcine renodoxin; (+) internal cysteine residues; (-), protein-binding site; (m), serine-phosphorylation site.

ET AL.

munological interest, since they involved changes in acidic or basic residues (i.e., Glu-17 to Lys, Lys-22 to Gln, Gln35 to Glu, Gln-61 to Asp, and Met-122 to Lys) (Fig. 2). In addition to predicted changes in surface residues, it was also noted that several functionally important surface regions were conserved between the proteins. Of particular immunochemical interest was the acidic region that participates in protein-protein interactions during electron transfer (i.e., residues 72 to 86). A synthetic peptide that contained five of the six acidic residues in this region reacted with both porcine renodoxin and bovine adrenodoxin antibodies (Fig. 4B) and, thereby, established the antigenicity of the protein-binding site. COOH-terminal analysis. The antigenic map for mammalian ferredoxins has not been determined, however, one antigenic site has been identified within the COOH-terminal 14 residues of bovine adrenodoxin (29). Porcine and bovine ferredoxins differ by three residues in this part of their COOH-terminal regions (Fig. 2), which could contribute to their distinct antigenic activities. To test this possibility, we compared the immunochemical reactivity of the two proteins to antibody made against

1.6 1.4

Leu-51 and Ser-54 (Fig. 1). However, it remains to be determined whether the cleavage of renodoxin’s presequence occurs by a one- or two-step process. Hydropathy analysis (20) of the mature porcine and bovine proteins predicted extensive hydrophilic (surface) sequences in the NH2- and COOH-terminal regions, and in a more medial domain that contains a surface loop for protein-protein interactions and serine phosphorylation (Fig. 3). The predicted surface loop is bound by two highly conserved hydrophobic regions (Glu-35 to Glu-60 and Gly91 to Ala-99) that contain the four cysteine ligands of the iron-sulfur center (15). Porcine renodoxin and bovine adELBA analysis. renodoxin reacted differently in a competitive ELISA analysis using renodoxin as bound antigen and anti-renodoxin antibody. The adrenodoxin competition curve was shifted toward higher antigen concentration when compared to renodoxin competition with self (Fig. 4A). It was evident from the higher antigen requirement in the competition analysis that bovine adrenodoxin is an inefficient competitor for the binding of renodoxin to anti-renodoxin antibody. In the complementary experiment, porcine renodoxin functioned as a weak competitor in the binding of bovine adrenodoxin with anti-adrenodoxin antibody (data not shown). The lowered binding affinity of the bovine antigen for porcine antibody was associated with 11 residue substitutions (Fig. 2) that were confined to predicted surface (antigenic) domains (Fig. 3, see porcine profile). Five of these substitutions were of particular im-

1.2 1 .o

0.8 0.6 0.4 0.2 0.0

CJ. 8, +. 0. a. ” 27 Antigen

100 Antigen

9 3

1

.3

.l

Concentration

25

6.25 Concentration

a

8

3-.

.037.012.004 (pg/wsll)

1.6

0.4

0.01

(rig/well)

FIG. 4. (A) Competitive ELISA of bovine and porcine ferredoxins. Bovine serum albumin (O), bovine adrenodoxin (V), and porcine renodoxin (V) were used as competing antigens for the binding of porcine anti-renodoxin antibody to titer-plate-linked porcine renodoxin. (B) ELISA antigen-titration curve using bound peptide (Leu-67 through Ala-U) or BSA antigen and bovine-adrenodoxin (V) or porcine-renodoxin (V) antibody. The binding curve for bound BSA (0) was the same for both antibodies and is presented as the average binding by the porcine and bovine antibodies.

PORCINE

KIDNEY

the bovine COOH-terminal 14amino-acid peptide (29). Bovine adrenodoxin gave a positive immunoblot reaction to bovine COOH-peptide antibody, indicative of a functional COOH-terminal antigenic site, whereas, porcine renodoxin did not react with the antibody (Fig. 5). The porcine protein further distinguished itself from bovine adrenodoxin by migrating in SDS-PAGE as a smaller -13.4-kDa molecule (Fig. 5). Immunoblot and electrophoretic properties identical to those of porcine renodoxin were also observed for porcine adrenodoxin (data not shown), which is consistent with a species-specific processing event. Mature renodoxin and bovine adrenodoxin each had an intact NHz-terminus (7) and the same encoded length (Fig. 1); therefore, it was reasoned that renodoxin’s lack of an antigenic site for the bovine COOH-antibody could be due to the COOH-terminal processing of the porcine protein. Processing of the COOH-terminus was evaluated by sequence analysis using carboxypeptidase Y. The COOH-terminal residue for pig renodoxin was determined to be Leu-120 followed by Asp-119 and Ile-118. This processing between Gly-121 and Leu-120 is three residues beyond the terminal Ser-117 detected during the original chemical sequencing of porcine adrenodoxin (18).

217

FERREDOXIN

46 -

17.6,

FIG. 5. PAGE and immunoblot analysis of bovine and porcine ferredoxins. Samples (0.2 Kg) of bovine adrenodoxin (lanes 1 and 3) and porcine renodoxin (lanes 2 and 4) were run in 20% SDS-PAGE and either silver stained (lanes 1 and 2) or immunoblot analyzed (lanes 3 and 4) using bovine COOH-terminal anti-peptide antibody.

acids longer than the mature-protein sequencedetermined by Edman degradation (30). Processing of the bovine proComparative immunological studies have shown a high tein can be prevented by the addition of protease inhibcross-reactivity between bovine adrenodoxin antibody and itors during the purification procedures and, therefore, the cleavage appears to be an isolation artifact (31, 32). several animal ferredoxins (5). This antigenic similarity is consistent with rapidly emerging sequence data that This action of a protease inhibitor to block the COOHshow -90% sequence identity between ferredoxins from terminal processing of bovine adrenodoxin is not shared different species (l-4). However, the potential for greater by the porcine protein. Rather, mature porcine renodoxin antigenic diversity between ferredoxins was suggested is COOH-terminal processed when purified in the presence of PMSF (current study). We have also observed from immunoblot analysis performed during the purifiprocessing of the porcine protein when isolated from socation and characterization of pig renodoxin (7). The lutions containing a protease-inhibitor cocktail’ or charlowered cross-reactivity between porcine renodoxin and acterized in freshly prepared mitochondria by denaturing bovine adrenodoxin was established clearly in the current immunoblot analysis (data not shown). These results study through competitive ELISA analysis. This change suggest that the COOH-terminal processing of the porcine in antigen-antibody affinity is characteristic of altered protein is a normal post-translational event, but verifiantigenic determinants due to post-translational modication requires additional protease-cleavage studies. fication or amino acid substitution. Computer analysis of The specific activity of the COOH-processed pig rencDNA-derived sequences revealed a linkage between preodoxin is the same as bovine adrenodoxin in reconstitudicted surface charged-residue substitutions in porcine renodoxin and the lowered binding affinity between bo- tion studies with cytochrome c or cytochrome P4501 of vine antigen and porcine antibody (Fig. 3, porcine pro- the vitamin D pathway (7). Interestingly, the COOH-terfile). Furthermore, upon reviewing ferredoxin sequences minal cleavage of 13 residues in bovine adrenodoxin is from across species, we noticed that porcine renodoxin associated with an increase in the protein’s affinity for contained the highest number of charged-residue sub- P450,,, (32). Whether a similar activity enhancement ocstitutions. This observation could provide a basis for curs in COOH-processed porcine ferredoxins is not known understanding why porcine ferredoxin appears more an- due to the present unavailability of full-length mature porcine protein. This issue is currently being addressed tigenically divergent than other ferredoxins. COOH-terminal cleavage of mitochondrial ferredoxins was discovered initially during the cDNA cloning of bo’ The protease-inhibitor cocktail consisted of EDTA (1 mM), PMSF vine adrenodoxin (1). In that study, the cDNA-predicted (0.2 mM), and leupeptin, antipain, and pepstatin A (0.5 mg/ml each) COOH-terminal sequence was observed to be 14 amino with final concentrations given in parentheses. DISCUSSION

218

OMDAHL

through the bacterial expression of pig renodoxin (study in progress). It has been determined that the highly acidic region in bovine adrenodoxin (residues 72 to 86) is involved with binding adrenodoxin reductase, cytochrome c and cytochrome P450,,, (16). The position of this acidic domain as well as the iron-sulfur center is identical in all known mitochondrial ferredoxin sequences and appears fundamental to the ferredoxins’ ability to indiscriminately transfer electrons to different mitochondrial cytochromes P450 (7, 33-37). Having demonstrated the antigenicity of this acidic domain (current study), it will be useful to prepare the attendant epitope-specific antibody for use in the atlinity isolation of ferredoxins and in the study of their complex protein-protein interactions. In summary, mature porcine and bovine ferredoxins each have encoded lengths of 128 residues and equivalent electron transfer activities. The porcine protein lacks eight COOH-terminal residues when isolated from renal tissue, and therefore it does not express antigenic activity with a bovine COOH-terminal antibody. Distinct antigenie domains present in other regions of the two ferredoxins are linked to differences in charged residues within predicted surface domains. A common antigenic site was determined for a highly acidic surface loop (residues 7281), which is expressed in all mitochondrial ferredoxins and involved with the binding of electron acceptor and donor proteins. REFERENCES 1. Okamura, T., John, M. E., Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1985) Proc. Natl. Acad. Sci. USA 82,5705-5709. 2. Mittal, S., Zhu, Y., and Vickery, L. E. (1988) Arch. B&hem. 264,383-391.

Biophys.

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