Toxoplasma gondii: analysis of the active site insertion of its ferredoxin-NADP(+)-reductase by peptide-specific antibodies and homology-based modeling.

Apicomplexan parasites possess an apicoplast-localized redox system consisting of a plant-type ferredoxin-NADP(+)-reductase (FNR) and its redox partner ferredoxin, a small [2Fe-2S] protein. We show here that several apicomplexan FNRs contain unique amino acid insertions of various lengths which are located in close proximity to the enzymatically important FAD and ferredoxin-binding sites of these proteins. Using the insertion of the Toxoplasma gondii reductase as an example we raised epitope-specific antibodies against an 11 amino acids long peptide predicted to be surface-exposed within this insertion. This peptide was found to be immunogenic when presented to the immune system as part of a carrier protein, but also in its natural structural context in the whole recombinant protein, implying that the epitope is surface-exposed. Three-dimensional modeling of T. gondii FNR based on the known 3D-structure of maize root FNR predicts that the overall structure of plant and apicomplexan FNRs are very similar and that the 11 amino acids are part of an alpha-helix, looping out of the molecule. Collectively, these data suggest that the insertion in T. gondii FNR does not affect the overall structure of the protein but may have an effect on the binding dynamics of FAD, NADP(+), and/or ferredoxin to FNR.

Immunological analysis of developmental changes in ecdysone 20-mono-oxygenase expression in the cotton leafworm, Spodoptera littoralis.

The developmental changes in ecdysone 20-mono-oxygenase during the sixth larval instar of the cotton leafworm, Spodoptera littoralis, were investigated. The specific activity of mitochondrial ecdysone 20-mono-oxygenase in the fat-body exhibited a distinct peak at 72 h, at which time the larvae stop feeding. Immunoblot analyses, using antibodies raised against components of vertebrate mitochondrial steroidogenic enzyme systems [anti-(cytochrome P-450scc), anti-(cytochrome P-450(11) beta), anti-adrenodoxin and anti-(adrenodoxin reductase) antibodies], revealed the presence of specific immunoreactive polypeptides in fat-body mitochondrial extracts. In addition, these antibodies effectively inhibited fat-body mitochondrial ecdysone 20-mono-oxygenase activity. This suggests that the S. littoralis steroid-hydroxylating system(s) may contain polypeptide components analogous to those present in vertebrates. A close correlation between developmental changes in mitochondrial ecdysone 20-mono-oxygenase activity and the abundance of polypeptides (approx. 66 kDa and 50 kDa) recognized by the anti-(cytochrome P-450(11) beta) antibody and a polypeptide (approx. 52 kDa) recognized by the anti-(adrenodoxin reductase) antibody were observed in both fat-body and midgut. These results suggest that developmental changes in the abundance of components of the ecdysone 20-mono-oxygenase system may play an important role in the developmental regulation of the enzyme expression and, hence, of 20-hydroxyecdysone titre.

Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides.

Antiserum to a native protein may cross-react with the corresponding denatured protein or with peptides. The cross-reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation-sensitive immunoassays must be employed to distinguish a genuine from an apparent cross-reaction. In the present study, we have analyzed critically the cross-reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross-reaction with the help of the protein A antibody-capture ELISA, a new conformation-sensitive ELISA format. Three systems were analyzed: cross-reaction of antisera to native yeast and horse cytochrome c with unfolded apo-cytochrome c; cross-reaction of antisera to a coiled-coil leucine-zipper peptide with a homologous random-coil peptide obtained by introducing two proline residues into the leucine-zipper sequence; cross-reaction of antisera to two peptides that correspond to the N-terminal and an internal sequence of ferredoxin: NADP+ reductase (FNR), with the native enzyme. The reaction of the anti-(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross-reaction. Furthermore, the apparently cross-reactive antibodies to horse cytochrome c did not discriminate against sequence-related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine-zipper peptide did cross-react in a genuine way with the homologous random-coil peptide, that is, the cross-reactive antibodies do not seem to have been produced against the unfolded form of the leucine-zipper peptide. Of the two anti-peptide sera the one against the unstructured and highly accessible N-terminal segment reacted strongly with the native protein. The second serum against a solvent-accessible turn-like sequence of FNR showed apparent cross-reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide. The generation of cross-reactive antibodies depends on the conformational stability and integrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier-bound. The results clarify the different nature of cross-reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation-specific probes.

The purification and properties of ferredoxin-NADP(+)-oxidoreductase from roots of Pisum sativum L.

A ferredoxin-NADP(+)-oxidoreductase (FNR) was purified to homogeneity from pea root plastids to a specific activity of 200 nkat.mg protein-1, following acetone precipitation and ferredoxin affinity chromatography. The molecular weight of the enzyme was estimated to be 36,000 and 33,800 by SDS-polyacrylamide gel electrophoresis and molecular exclusion chromatography, respectively. The absorption spectrum of the enzyme suggests it contains flavin as a prosthetic group. The enzyme requires NADPH and did not use NADH as an electron donor. The Km values for NADPH and ferredoxin were calculated to be 28 and 5 microM, respectively. The enzyme exhibited optimal activity at pH 8.0. Although resembling the leaf enzyme in most properties, amino terminal sequencing demonstrates clear differences between the leaf and root proteins and suggests closer homology of the pea root enzyme with the enzyme from spinach roots. A polyclonal antibody against the pea root plastid enzyme was raised by the immunization of rabbits. Judging by immunodiffusion only partial identity was observed between the root plastid and chloroplast FNR. The root plastid FNR enzyme activity was precipitated with increasing concentrations of the antibody, in contrast to the chloroplast enzyme which was not inhibited. The potential usefulness of these antibodies is discussed.

Monoclonal antibody studies of ferredoxin:NADP+ oxidoreductase.

Eleven independent monoclonal antibodies, all IgG's, have been raised against the ferredoxin:NADP+ oxidoreductase of spinach leaves. All 11 monoclonal antibodies were able to produce substantial inhibition of the NADPH to 2,6-dichlorophenol indophenol (DCPIP) diaphorase activity of the enzyme, but none of the antibodies produced any significant inhibition of electron flow from NADPH to ferredoxin catalyzed by the enzyme. Spectral perturbation assays were used to demonstrate that antibody interaction with NADP+ reductase did not interfere significantly with the binding of either ferredoxin or NADP+ to the enzyme. Ultrafiltration binding assays were used to confirm that the monoclonal antibodies did not interfere with complex formation between ferredoxin and the enzyme. These results have been interpreted in terms of the likely presence of one or more highly antigenic epitopes at the site where the nonphysiological electron acceptor, DCPIP, binds to the enzyme. Furthermore, the results suggest that the site where DCPIP is reduced differs from both of the two separate sites at which the two physiological substrates, ferredoxin and NADP+/NADPH, are bound.

Crystallization and comparative characterization of reduced nicotinamide adenine dinucleotide phosphate-ferredoxin reductase from sheep adrenocortical mitochondria.

1. Sheep NADPH-ferredoxin reductase (E.C. 1.18.1.2) was purified from the adrenocortical mitochondria. The reductase was typical flavoenzyme and crystallized in ammonium sulfate solution. 2. The properties of the reductase were investigated physicochemically and immunochemically. The minimum molecular weight of the reductase was 52,000 and the reductase has one FAD per mole as a coenzyme. 3. The sheep NADPH-ferredoxin reductase showed a precipitate line against antibody to bovine NADPH-ferredoxin reductase. 4. The compositions and sequences of amino acid residues of this reductase and porcine, bovine, and human enzymes were compared. In spite of differences of mammalian species, the sequence of amino acid residues in the amino-terminal regions were highly homologous. 5. It is suggested that the amino-terminal region may be essential for the function of the NADPH-ferredoxin reductase.

Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex.

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.

On the specificity of pig adrenal ferredoxin (adrenodoxin) and spinach ferredoxin in electron-transfer reactions.

Spinach leaf ferredoxin and ferredoxin:NADP oxidoreductase as well as pig adrenodoxin and adrenodoxin reductase have been purified to homogeneity. Ferredoxin-NADP reductase and adrenodoxin-NADP reductase can perform the same diaphorase reactions (dichloroindophenol, ferricyanide and cytochrome c reduction) albeit not with the same efficiency. Despite the differences in their redox potentials, animal and plant ferredoxins can be used as heterologous substrates by the ferredoxin-NADP reductases from both sources. In heterologous systems, however, the ferredoxin/adrenodoxin concentrations must be increased approximately 100-fold in order to reach rates similar to those obtained in homologous systems. Ferredoxin and adrenodoxin can form complexes with the heterologous reductases as demonstrated by binding experiments on ferredoxin-Sepharose or ferredoxin-NADP-reductase-Sepharose and by the realization of difference spectra. Adrenodoxin also weakly substitutes for ferredoxin in NADP photoreduction, and can be used as an electron carrier in the light activation of the chloroplastic enzyme NADP-dependent malate dehydrogenase. In addition adrenodoxin is a good catalyst of pseudocyclic photophosphorylation, but not of cyclic phosphorylation and can serve as a substrate of glutamate synthase. These results are discussed with respect to the known structures of plant and animals ferredoxins and their respective reductases.

Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase.

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplast completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.

Immunological studies of the binding protein for chloroplast ferredoxin-NADP+ reductase.

Monospecific rabbit antibodies against the ferredoxin-NADP+ reductase binding protein of spinach thylakoids were obtained and characterized. The immunoglobulin G (IgG) fraction gave single precipitation arcs with the purified antigen or with Triton X-100 extracts of thylakoids or the reductase binding protein complex. Antibodies against the flavoprotein behave similarly. Both antibodies agglutinated thylakoids and precipitated the diaphorase activity of a Triton X-100 extract of these membranes. Isolated Fab fragments of the IgG anti-binding protein inhibited NADP+ photoreduction in a time- and Fab concentration-dependent manner. The presence of ferredoxin diminished the rate of inhibition. In the light, the inactivation rate was higher than in dark and this effect was abolished in the presence of uncouplers. These results suggest that the binding protein is protruding from the thylakoids and could be sensing the proton gradient.

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase.

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.

Stoichiometry of mitochondrial cytochromes P-450, adrenodoxin and adrenodoxin reductase in adrenal cortex and corpus luteum. Implications for membrane organization and gene regulation.

We have estimated the concentrations of cytochromes P-450scc and P-45011 beta and the electron-transfer proteins adrenodoxin reductase and adrenodoxin in the adrenal cortex and corpus luteum using specific antibodies against these enzymes. While in the adrenal cortex the concentrations of these enzymes are relatively constant in different animals and show no significant sex differences, in corpora lutea they vary considerably and can increase at least up to fifty-fold over the levels found in the ovary. The average relative concentrations of adrenodoxin reductase, adrenodoxin and P-450 are 1:3:8 in the adrenal cortex (which has two cytochromes P-450, P-450scc and P-450(11) beta, in equal concentrations) and 1:2.5:3 in the corpus luteum (which has only P-450scc). We further present evidence that the levels of cytochrome c oxidase also show a degree of correlation with the levels of the mitochondrial steroidogenic enzymes.

[Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria]. / Immunokhimicheskaia kharakteristika kholesteringidroksiliruiushchego tsitokhroma P-450 mitokhondrii kory nadpochechnikov.

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).

The involvement of ferredoxin-NADP+ reductase in cyclic electron transport in chloroplasts.

The sites of action, in spinach thylakoid, of known inhibitors of electron transport at the reducing end of photosystem I have been more accurately located by parallel investigation of effects on three partial reactions: photo-reduction (from water) of added NADP+, photoreduction of added cytochrome c, and dark reduction of cyto-chrome c by added NADPH. Comparison with inhibitory effects on cyclic electron flow (registered by the slow phase of the electrochromic response during repetitive flash excitation) permitted assessment of the role of ferredoxin and ferredoxin-NADP+ reductase (ferredoxin: NADP+ oxidoreductase, EC 1.18.1.3) in the cyclic electron transport pathway around photosystem I. Disulfodisalicylidenepropane-1,1-diamine inhibited all the above partial reactions except the ferredoxin-dependent photoreduction of cytochrome C. thereby indicating its interference with the reductase or the complexation between reductase and ferredoxin. Studies with purified ferredoxin-NADP+ reductase established it as the sensitive component. Cyclic flow is also sensitive to the above inhibitor and thus presumably involves the reductase. Supporting evidence for this came from studies of inhibition by substituted maleimides, which are inhibitors of electron transfer through the isolated reductase; these also inhibited the slow phase of the electrochromic response and all partial reactions except the photoreduction of cytochrome c. In contrast, an antiserum against the reductase affected only reactions involving NADP. The conclusion is drawn that the pathway of cyclic electron transport includes both ferredoxin and ferredoxin-NADP+ reductase, but not the NADP-binding site on the reductase.

Crystalline reduced nicotinamide adenine dinucleotide phosphate-adrenodoxin reductase from pig adrenocortical mitochondria. Essential histidyl and cysteinyl residues of the NADPH-binding site and environment of the adrenodoxin-binding site.

Pig NADPH-adrenodoxin reductase was crystallized from pig adrenocortical mitochondria and its physicochemical properties were investigated. Pig NADPH-adrenodoxin reductase is a typical flavoprotein. Its optical absorption spectrum showed peaks at 272, 377, and 450 nm in the oxidized form. The adrenodoxin reductase contained one FAD per mol. The molecular weight was 49,000. The isoelectric points of the adrenodoxin reductase and its complex with adrenodoxin were 5.3 and 4.6, respectively. Pig NADPH-adrenodoxin reductase, unlike bovine NADPH-adrenodoxin reductase, was found to be free of carbohydrate. The fluorescences of tryptophanyl residues and FAD of the adrenodoxin reductase were quenched by holo- and apo-adrenodoxins. The NADPH-binding site of the adrenodoxin reductase was examined by photooxidation and selective chemical modifications with diethyl pyrocarbonate and sulfhydryl reagents. The results indicate that a histidyl and a cysteinyl residue of the adrenodoxin reductase are essential for the NADPH-binding site. The circular dichroism spectrum of the adrenodoxin reductase showed negative ellipticity in the visible region. Spur formation was observed between pig and bovine NADPH-adrenodoxin reductases against the antibody to bovine NADPH-adrenodoxin reductase in Ouchterlony double-diffusion agar plates. The antibody did not interact with spinach ferredoxin-NADP+ reductase.

Reactions of antibodies against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin with spinach chloroplasts.

Purified antisera against ferredoxin, ferredoxin-NADP+ reductase and plastocyanin agglutinated osmotically shocked and washed spinach chloroplasts, prepared according to standard procedures. The monomeric antibody (immunoglobulin G fraction) of the reductase antiserum agglutinated chloroplasts specifically and directly, indicating that protruding structures (for example, the coupling factor) do not act as steric hindrances as has been suggested. With ferredoxin antiserum, the presence of a pentameric antibody (immunoglobulin M fraction) was obligatory to observe a positive agglutination reaction. Immunoglobulin G only inhibited ferredoxin-dependent reactions, like NADP+-photoreduction, but did not cause agglutination. Ferredoxin seems to be located in depressions of the membrane, possibly caused by a partial release of this protein in shocked chloroplasts. Similar results were obtained with purified immunoglobulins from a plastocyanin antiserum. Again the immunoglobulin G fraction inhibited electron transport reactions catalyzed by plastocyanin, whereas immunoglobulin M showed a positive agglutination, but had no influence on electron transport. It is concluded that ferredoxin, ferredoxin-NADP+ reductase and plastocyanin are peripheral electron transport components, located at the outer thylakoid membrane.

On the role of ferredoxin and ferredoxin-NADP+ reductase in cyclic electron transport of spinach chloroplasts.

Antibodies prepared against purified spinach ferredoxin and ferredoxin-NADP+ reductase were used as specific inhibitors of electron-transfer reactions dependent on either ferredoxin or ferredoxin-NADP+ reductase; The possible role of both electron carriers in cyclic electron transport was checked using cytochrome b6 photoreactions as indicator. It could be demonstrated that the ferredoxin antibody inhibits cytochrome b6 photoreduction. Ferredoxin-NADP+ reductase, however, appears not to be involved in this pathway: reductase antibody in concentrations sufficient to completely inhibit electron transport to NADP+ had no effect on cytochrome b6 photoreduction. Quantitative treatment of the immunoassay data showed that osmotically shocked chloroplasts contain both bound ferredoxin and ferredoxin-NADP+ reductase in concentration approximately equal to that of cytochrome b6.