The Arabidopsis Iron-Sulfur (Fe-S) Cluster Gene MFDX1 Plays a Role in Host and Nonhost Disease Resistance by Accumulation of Defense-Related Metabolites.

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.

Analysis of the effect of temperature on protein abundance in Demodex-associated Bacillus oleronius.

A potential role for bacteria in the induction of rosacea has been suggested. The aim of this work was to characterise the effect of temperature on the production of immunostimulatory proteins by Bacillus oleronius-a bacterium to which rosacea patients show sera reactivity and which was originally isolated from a Demodex mite from a rosacea patient. The affected skin of rosacea patients is at a higher temperature than unaffected skin, and it was postulated that this might alter the protein expression pattern of B. oleronius. B. oleronius growth was reduced at 37°C compared to 30°C but resulted in increased expression of the immune-reactive 62kDa protein (1.65 fold [P < 0.05]). Proteomic analysis revealed increased abundance of a wide range of proteins involved in the stress response (e.g. stress proteins [21.7-fold increase], phosphocarrier protein HPr [438.5-fold increase], 60 kDa chaperonin [12.6-fold increase]). Proteins decreased in abundance after growth at 37°C included ferredoxin (325-fold decrease) and peptidase (244-fold decrease). This work indicates that the increased skin temperature of rosacea patients may alter the growth and protein production pattern of B. oleronius and lead to the greater production of immuo-stimulatory proteins.

Bioinformatics analysis to assess potential risks of allergenicity and toxicity of HRAP and PFLP proteins in genetically modified bananas resistant to Xanthomonas wilt disease.

Banana Xanthomonas wilt (BXW) disease threatens banana production and food security throughout East Africa. Natural resistance is lacking among common cultivars. Genetically modified (GM) bananas resistant to BXW disease were developed by inserting the hypersensitive response-assisting protein (Hrap) or/and the plant ferredoxin-like protein (Pflp) gene(s) from sweet pepper (Capsicum annuum). Several of these GM banana events showed 100% resistance to BXW disease under field conditions in Uganda. The current study evaluated the potential allergenicity and toxicity of the expressed proteins HRAP and PFLP based on evaluation of published information on the history of safe use of the natural source of the proteins as well as established bioinformatics sequence comparison methods to known allergens (www.AllergenOnline.org and NCBI Protein) and toxins (NCBI Protein). The results did not identify potential risks of allergy and toxicity to either HRAP or PFLP proteins expressed in the GM bananas that might suggest potential health risks to humans. We recognize that additional tests including stability of these proteins in pepsin assay, nutrient analysis and possibly an acute rodent toxicity assay may be required by national regulatory authorities.

Roles of the host oxidative immune response and bacterial antioxidant rubrerythrin during Porphyromonas gingivalis infection.

The efficient clearance of microbes by neutrophils requires the concerted action of reactive oxygen species and microbicidal components within leukocyte secretory granules. Rubrerythrin (Rbr) is a nonheme iron protein that protects many air-sensitive bacteria against oxidative stress. Using oxidative burst-knockout (NADPH oxidase-null) mice and an rbr gene knockout bacterial strain, we investigated the interplay between the phagocytic oxidative burst of the host and the oxidative stress response of the anaerobic periodontal pathogen Porphyromonas gingivalis. Rbr ensured the proliferation of P. gingivalis in mice that possessed a fully functional oxidative burst response, but not in NADPH oxidase-null mice. Furthermore, the in vivo protection afforded by Rbr was not associated with the oxidative burst responses of isolated neutrophils in vitro. Although the phagocyte-derived oxidative burst response was largely ineffective against P. gingivalis infection, the corresponding oxidative response to the Rbr-positive microbe contributed to host-induced pathology via potent mobilization and systemic activation of neutrophils. It appeared that Rbr also provided protection against reactive nitrogen species, thereby ensuring the survival of P. gingivalis in the infected host. The presence of the rbr gene in P. gingivalis also led to greater oral bone loss upon infection. Collectively, these results indicate that the host oxidative burst paradoxically enhances the survival of P. gingivalis by exacerbating local and systemic inflammation, thereby contributing to the morbidity and mortality associated with infection.

Antibody profiles characteristic of Mycobacterium tuberculosis infection state.

The relationship between specific antibody profiles and tuberculosis (TB) state was investigated by measuring serum antibody levels to six Mycobacterium tuberculosis antigens in human subjects grouped into four diagnostic categories: active disease, inactive (past) tuberculosis, latent infection without radiographic chest abnormalities, and infection free. Statistical data analyses showed that the latter two groups were serologically indistinguishable and that active tuberculosis and inactive tuberculosis were characterized by different antibody profiles. Antibodies to the 38-kDa antigen, alanine dehydrogenase, and Rv2626c were associated with active TB, while antibodies to the 16-kDa antigen, ferredoxin A, and ESAT-6 were associated with inactive TB. Thus, the targets of the immune response vary with tuberculosis state. The correlation between bacterial antigen production and infection stage was investigated in mice infected with M. tuberculosis by bacterial transcription profiling. It was found that levels of transcripts encoding the six M. tuberculosis antigens varied during infection. Together, the data indicate that antigen composition of tubercle bacilli varies with stage of infection and that immunoprofiling can distinguish between tuberculosis states.

The sweet pepper ferredoxin-like protein (pflp) conferred resistance against soft rot disease in Oncidium orchid.

Genetic engineering to date has not been used to introduce disease resistance genes into the orchid gene pool. The ferredoxin-like protein gene originally isolated from sweet pepper is thought to function as a natural defense against infection due to its antimicrobial properties. Hence it was reasoned that introduction of this gene might produce Oncidium plants resistant to Erwinia carotovora, the causal agent for the soft rot disease. An expression vector containing sweet pepper ferredoxin-like protein (pflp) cDNA, hph and gusA coding sequence was successfully transformed into protocorm-like bodies (PLBs) of Oncidium orchid, using Agrobacterium tumefaciens strain EHA105. A total of 17 independent transgenic orchid lines was obtained, out of which six transgenic lines (beta-glucuronidase (GUS) positive) were randomly selected and confirmed by Southern, northern and western blot analyses. A bioassay was conducted on the transgenic lines. Transgenic plants showed enhanced resistance to E. carotovora, even when the entire plant was challenged with the pathogen. Our results suggest that pflp may be an extremely useful gene for genetic engineering strategies in orchids to confer resistance against soft rot disease.

On the two iron centers of desulfoferrodoxin.

Desulfoferrodoxin from Desulfovibrio vulgaris, strain Hildenborough, is a homodimer of 28 kDa; it contains two Fe atoms per 14.0 kDa subunit. The N-terminal amino-acid sequence is homogeneous and corresponds to the previously described Rho gene, which encodes a highly charged 14 kDa polypeptide without a leader sequence. Although one of the two iron centers, FeA, has previously been described as a 'strained rubredoxin-like' site, EPR of the ferric form proves very similar to that of the pentagonal bipyramidally coordinated iron in ferric complexes of DTPA, diethylenetriaminepentaacetic acid: both systems have spin S = 5/2 and rhombicity E/D = 0.08. Unlike the Fe site in rubredoxin the FeA site in desulfoferrodoxin has a pH dependent midpoint potential with pKox = 9.2 and pKred = 5.3. Upon reduction (Em,7.5 = +2 mV) FeA exhibits an unusually sharp S = 2 resonance in parallel-mode EPR. The second iron, FeB, has S = 5/2 and E/D = 0.33; upon reduction (Em,7.5 = +90 mV) FeB turns EPR-silent.

The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate stimulates the dephosphorylation of mitochondrial ferredoxin in cultured chick kidney cells.

The tumor promoting phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), presumably through activation of protein kinase C, decreases the production of 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] and increases that of 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] by primary cultures of chick kidney cells. We have previously shown that the regulation of the cellular output of 1,25(OH)2D3 and 24,25(OH)2D3 by PTH and 1,25(OH)2D3 can be quantitatively accounted for by altered hydroxylase activities within isolated mitochondria. In the present paper, we examined the effects of TPA and 1-oleoyl-2-acetyl-glycerol (OAG) on the state of mitochondrial protein phosphorylation and on 25-hydroxyvitamin D3 [25(OH)D3] metabolism. There was a good correlation between 25(OH)D3- 1 alpha- and 24-hydroxylase activities in mitochondria isolated from cells pretreated with either TPA or OAG and the pattern of 1- and 24-hydroxylation of 25(OH)D3. The most notable change in protein phosphorylation in the molecular mass range of 10-60 kilodaltons (kDa) was a dramatic decrease in the phosphorylation of a 12.5-kDa mitochondrial matrix protein after treatment of kidney cells with TPA or OAG. The amino acid composition of the 12.5-kDa protein was similar to bovine and human ferredoxins and it comigrated with bovine and human ferredoxins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 12.5-kDa phosphoprotein was immunoprecipitated specifically by an antipeptide polyclonal antibody for chick ferredoxin. The dephosphorylation of ferredoxin in response to TPA was both rapid and transient, with the phosphate content of the 12.5-kDa protein reduced by 70% after a 5-min exposure and returning to control levels by 20 min. A similar transience was observed with regard to the rapid effects of TPA on 1 alpha-hydroxylase activity, again showing maximal inhibition at 5 min. The results of our studies are consistent with the idea that ferredoxin phosphorylation plays a role in the regulation of steroid hydroxylation.

Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413.

Three soluble ferredoxins were purified to homogeneity from nitrogen-fixing cultures of Anabaena variabilis (ATCC 29413) and characterized. The purified proteins have different absorption spectra, molecular mass, iron content, amino-acid composition and resistance to O2 inactivation. Two were plant-type ferredoxins FdI and FdxH, corresponding to the previously reported ferredoxins II and I (Böhme, H. and Schrautemeier, B. (1987) Biochim. Biophys. Acta 891, 1-7). The third ferredoxin (ferredoxin III) (previously not described in cyanobacteria) was a bacterial-type ferredoxin. Ferredoxin III has a molecular mass of about 6 kDa and contains 3-4 atoms Fe/mol. Native (oxidized) ferredoxin III shows an EPR-signal at g = 2.014 that disappears after reduction by dithionite, characteristic of ferredoxins containing three-iron clusters. Ferredoxin III, like ferredoxin FdxH, is inactivated by oxygen. Ferredoxin III supports higher rates of C2H2 reduction by Rhodobacter capsulatus nitrogenase than FdI and higher rates of H2 evolution by clostridial hydrogenase than FdI and FdxH. Combined nitrogen suppresses the synthesis of both nitrogenase and ferredoxin III. These data suggest a possible role of ferredoxin III (bacterial-type) in nitrogen fixation by A. variabilis.

Nigerythrin and rubrerythrin from Desulfovibrio vulgaris each contain two mononuclear iron centers and two dinuclear iron clusters.

The trivial name 'rubr-erythrin' is a contraction of two other trivial names: rubredoxin (ruber, red) and hemerythrin. It names a protein of undetermined biological function which putatively carries rubredoxin-like mononuclear iron and hemerythrin-like dinuclear iron. The name 'nigerythrin' (niger, black) is an analogy of rubrerythrin. It identifies a second protein of undetermined function which has prosthetic groups similar to rubrerythrin. Rubrerythrin was initially described [LeGall, J., Prickril, B. C., Moura, I., Xavier, A. V., Moura, J. J. G. & Huynh, B.-H. (1988) Biochemistry 27, 1636-1642] as a homodimer with four iron ions arranged into two rubredoxin sites and one inter-subunit dinuclear cluster. Nigerythrin is a novel protein. Here, we report that both proteins are homodimers, each dimer carrying not four but six iron ions in two mononuclear centers and two dinuclear clusters. Rubrerythrin and nigerythrin are probably both located in the cytoplasm; they are differentially characterized with respect to molecular mass, pI, N-terminal sequence, antibody cross-reactivity, optical absorption, EPR spectroscopy, and reduction potentials. All three reduction potentials in both proteins are > +200 mV. These appear too high to be of practical relevance in the cytoplasm of the sulfate reducer Desulfovibrio vulgaris (Hildenborough). We suggest the possibility of a non-redox role for both proteins with all six iron ions in the ferrous state.

Molecular cloning and immunological characterization of porcine kidney ferredoxin.

Porcine renodoxon is a kidney mitochondrial iron-sulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A full-length 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 approximately 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.

The interaction of ferredoxin and glutamate synthase: cross-linking and immunological studies.

The water-soluble carbodiimide, N-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) serves as an effective reagent for cross-linking spinach leaf ferredoxin and the ferredoxin-dependent spinach leaf enzyme, glutamate synthase. The cross-linked complex was functional in the absence of added ferredoxin, suggesting that ferredoxin is cross-linked to glutamate synthase at the physiological binding site on the enzyme for this iron-sulfur protein electron donor. The ferredoxin:glutamate synthase stoichiometry of the cross-linked complex was estimated to be 2:1. The absorbance spectrum of the oxidized, cross-linked complex was very similar to that of an electrostatically stabilized, noncovalent, 2:1 complex of the two proteins. An antibody raised against spinach NADP+ reductase, which recognizes a ferredoxin-binding site on glutamate synthase, does not recognize the cross-linked ferredoxin-glutamate synthase complex. This implies that the ferredoxin-binding sites on the two enzymes are structurally similar enough so that an antibody raised against one of these ferredoxin-dependent enzymes recognizes an epitope at the ferredoxin-binding site of the second enzyme. Cross-linking of ferredoxin to its binding site on glutamate synthase renders this epitope inaccessible to the antibody.

Characterization and N-terminal amino acid sequence of multiple ferredoxins in kidney and adrenal mitochondria.

Two separate ferredoxins that differ in molecular mass by about 1.5 kDa were isolated from both pig kidney and bovine adrenal mitochondria. The proteins had different biochemical and immunological properties and appeared to be distinct gene products. The smaller ferredoxin from pig kidney (renodoxin, Mr approximately equal to 13,500) was very similar to bovine adrenodoxin (Mr = 14,048). Both proteins had nearly identical N-terminal amino acid sequences and electron-transfer activities. However, renodoxin and adrenodoxin expressed distinct antigenic determinants, although they were immunologically cross-reactive. The larger kidney (approximately equal to 15-kDa) and adrenal (approximately equal to 15.3-kDa) ferredoxins were biochemically similar to each other but they had lower specific activities and their N-terminal sequences were different when compared to renodoxin and adrenodoxin. Each of the four ferredoxins had a visible absorption spectrum characteristic of a [2Fe-2S] chromophore. But in addition, the larger ferredoxins displayed a prominent A276 peak due to their higher tyrosine content and the presence of tryptophan, which is absent in adrenodoxin and renodoxin. Consequently, the larger ferredoxins were termed Trp-ferredoxin. Using antibody to pig kidney Trp-ferredoxin, the larger adrenal and kidney ferredoxins were found to be very similar immunologically; however, the Trp-containing and adrenodoxin-type ferredoxins did not cross-react in immunoblot analysis. Nevertheless, it was shown from competition ELISA and activity-inhibition analysis that the two ferredoxin types had limited common antigenic determinants. Trp-ferredoxin was the major iron-sulfur protein in kidney whereas adrenodoxin was the dominant molecular form in adrenal gland.

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.

The 18-kD protein that binds to the chloroplast DNA replicative origin is an iron-sulfur protein related to a subunit of NADH dehydrogenase.

From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.

Ferredoxin and cytochrome P-450scc concentrations in granulosa cells of porcine ovaries during follicular cell growth and luteinization.

The concentrations of cytochrome P-450scc and ferredoxin, two of the three proteins which comprise the mitochondrial steroidogenic electron transport chain, were measured in granulosa and luteal cells from porcine ovaries by an immunoblot procedure. During the follicular phase of the ovarian cycle the concentration of cytochrome P-450scc increased 5-fold and ferredoxin increased 3-fold. When the large follicles developed into corpora lutea the cytochrome P-450scc concentration increased a further 7-fold while ferredoxin increased only 3-fold. These changes were coincident with an overall 4-fold increase in the concentration of ferredoxin reductase during follicular cell development and luteinization. Analysis of the data revealed that the concentration of ferredoxin, which shuttles electrons from ferredoxin reductase to cytochrome P-450scc, was always adequate to saturate both the reductase and cytochrome P-450scc. This came about from a co-ordinate increase in the concentration of cytochrome P-450scc and the concentration of ferredoxin minus ferredoxin reductase.

Isolation of an antigen-specific T suppressor factor that suppresses the in vivo response of DBA/2 mice to ferredoxin.

A T cell hybridoma (Fd11) has been produced from B10.D2 mice that secretes a putative antigen-specific T suppressor factor (TsF). The TsF is isolable from culture supernatants of Fd11 by affinity purification over columns containing either a monoclonal antibody (B16G) shown previously to be capable of binding murine TsF or ferredoxin (Fd), the nominal antigen to which the Fd11 TsF binds. Specificity of the Fd11 TsF for Fd was established by comparing it to another TsF isolated by us (A10 TsF) in a sandwich ELISA, and by demonstrating the specific reactivity to Fd of the hybridoma in calcium flux studies. The Fd11 affinity-purified TsF was shown to contain two major unique components with m.w. in the region of 80,000 and 35,000 when run on reducing polyacrylamide gels in the presence of sodium dodecyl sulphate. Specific immunosuppressive properties of Fd11 were demonstrated when Fd11 TsF (10 micrograms) was injected i.v. into Fd-primed syngeneic mice at the time of antigen boost. Fd11 TsF specifically and significantly diminished the secondary antibody response to Fd in DBA/2 mice.

Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria.

Hepatic ferredoxin (hepatoredoxin) was purified from bovine liver mitochondria. The monomeric molecular mass of the hepatoredoxin was larger than that of adrenocortical ferredoxin (adrenodoxin) from bovine adrenocortical mitochondria at 14 kDa. We studied the amino acid residues and NH2-terminal sequence of this protein. The hepatoredoxin was organ-specific protein. The optical absorption spectrum of oxidized hepatoredoxin had two peaks, at 414 and 455 nm in the visible region. Hepatoredoxin formed an immunoprecipitin line against anti-adrenodoxin immunoglobulin in Ouchterlony double diffusion, and an immunochemical staining band in Western blotting.

Abrogation and reconstitution of nonresponsiveness: a correlation with high network connectivity.

A monoclonal antibody (Fd-B2) to ferredoxin, which bears an idiotype scarcely expressed in any of a wide variety of mouse strains, is able to markedly enhance the response to ferredoxin of both high-responder and intermediate-responder strains. A rabbit anti-idiotype serum to Fd-B2 also specifically enhances the response to ferredoxin in such mice. Most remarkably, the treatment of nonresponder T cells by either the idiotype (Fd-B2) plus complement or anti-idiotype antiserum plus complement causes them to be responsive in adoptive transfer experiments. The two responding populations (idiotype-treated and anti-idiotype-treated) can then be combined to reconstitute the nonresponsive state. When the nonresponders are treated with either Fd-B2 idiotype plus complement or anti-idiotype plus complement and subsequently respond, the idiotype of the anti-ferredoxin antibody produced does not bear the Fd-B2 idiotype. We interpret the results as being consistent with a model in which the unresponsive state for ferredoxin is a state of high network connectivity of the ferredoxin-specific T cells.

Immune response to ferredoxin; nonresponder status is not due to suppression.

Ferredoxin (Fd), a small protein from Clostridium pasteurianum, has been selected for immunologic studies because of its limited number (two) of antigenic determinants. Functionally (as determined by antibody binding), monodeterminant fragments of Fd can be generated enzymatically, leaving molecules only a few amino acids smaller than the native protein, with unaltered solid phase binding properties. These fragments were used to assess the immune response to each of the two determinants. Clear differences in immunologic properties can be assigned to sequences within Fd: the amino terminal tripeptide is responsible for inducing a proliferative response and limited antibody production, whereas the carboxy terminal dipeptide accounts for most of the antibody activity, yet little, if any, T-proliferative activity. Studies with the enzyme-generated fragments of Fd have unmasked a sequence proximal to the amino terminal that represents a second determinant for T cell proliferation but does not have any demonstrable antibody-inducing activity. This third determinant is shown to induce responsiveness to Fd in nonresponder animals after the removal of the amino terminal tripeptide. The results indicate that nonresponsiveness to this molecule in H-2d mice is not a direct effect of suppression.