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PubMed1991

Light-dependent de-activation/re-activation of Anabaena variabilis ferredoxin: NADP+ reductase.

The activity of ferredoxin: NADP+ reductase (FNR) was found to decline to approximately 20% maximal levels with little or no loss in enzyme levels when cultures of the cyanobacterium Anabaena variabilis were maintained in the stationary phase of growth. Re-activation of enzyme activity occurred when cells were diluted into either fresh or re-utilized media and illuminated. This reversible de-activation/re-activation process was found, in vivo, to be dependent on the intensity of light illuminating the cells. The de-activated form of FNR was purified to homogeneity and exhibited the same molecular mass, isoelectric-focusing pattern and N-terminal amino acid sequence as the native form. Both de-activated and native FNR preparations each exhibited three reactive thiol groups on denaturation in urea; however, the rate of reaction with Ellman's reagent was much faster with the de-activated form than with the native form. Both preparations contain a single disulphide bond. Upon reduction of the disulphide bond in either form of the enzyme, the five reactive thiol groups exhibited identical reactivities in the presence of urea. Steady-state kinetic analysis of the de-activated form showed a marked increase in Km values for NADPH in diaphorase assays and an increase in Km for ferredoxin in the ferredoxin-mediated reduction of cytochrome c. No significant difference in kcat. was observed in comparison of the de-activated with the native form in any of the above assays; however, the de-activated form did exhibit a lower kcat. value in the transhydrogenase assay. The de-activated form of FNR bound ferredoxin with a 16-fold lower affinity than the native enzyme. These data suggest that the de-activation of FNR in vivo in response to low light intensity involves an alteration in protein structure, possibly via an intramolecular thiol disulphide interchange, which influences the interaction of the enzyme with its substrates.

Binding, Competitive↗
PubMed1992

cAMP post-transcriptionally diminishes the abundance of adrenodoxin reductase mRNA.

Adrenodoxin reductase (AR; ferridoxin: NADP+ oxidoreductase, EC 1.18.1.2) is a flavoprotein that mediates electron transport from NADPH to all known mitochondrial forms of cytochrome P450. AR mRNA was found in all human adult and fetal tissues examined; however, it was vastly more abundant in tissues that synthesize steroid hormones. The ratio of the 18- form of mRNA lacking 18 alternately spliced bases to the 18+ form was approximately 100:1 and remained constant irrespective of the tissue or hormonal manipulation, indicating that the alternate splicing is a passive nonregulated event. AR protein was unchanged by forskolin treatment of human JEG-3 cytotrophoblast cells for 24 h, but the mRNA diminished. Phorbol 12-myristate 13-acetate and cycloheximide had no effect, even though these agents had the expected effects on P450scc and adrenodoxin mRNAs. cAMP decreased the abundance of AR mRNA expressed from both transfected plasmids and the endogenous gene, indicating the effect was post-transcriptional. AR gene transcription in JEG-3 cells and promoter-chloramphenicol acetyltransferase constructs transfected into JEG-3 cells were unresponsive to forskolin. Powerful basal transcription elements were identified between -46 and -214 bases from the principal transcriptional initiation site, a region containing six elements closely resembling the binding site for transcription factor SP1.

Cells, Cultured↗
PubMed1992

Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase.

The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of the glpFKX regions of Escherichia coli and Shigella flexneri.

We have identified a new gene, glpX, belonging to the glp regulon of Escherichia coli, located directly downstream of the glpK gene. The transcription of glpX is inducible with glycerol and sn-glycerol-3-phosphate and is constitutive in a glpR mutant. glpX is the third gene in the glpFKX operon. The function of GlpX remains unknown. GlpX has an apparent molecular weight of 40,000 on sodium dodecyl sulfate-polyacrylamide gels. In addition to determining the E. coli glpX sequence, we also sequenced the corresponding glpFKX region originating from Shigella flexneri, which after transfer into E. coli was instrumental in elucidating the function of glpF in glycerol transport (D. P. Richey and E. C. C. Lin, J. Bacteriol. 112:784-790, 1972). Sequencing of the glpFKX region of this hybrid strain revealed an amber mutation instead of the tryptophan 215 codon in glpF. The most striking difference between the E. coli and S. flexneri DNA was found directly behind glpK, where two repetitive (REP) sequences were present in S. flexneri, but not in the E. coli sequence. The presence or absence of these REP sequences had no effect on transport or on growth on glycerol. Not including the REP sequence-containing region, only 1.1% of a total of 2,167 bp sequenced was different in the two sequences. Comparison of the sequence with those in the EMBL data library revealed a 99% identity between the last third of glpX and the first part of a gene called mvrA. We show that the cloned mvrA gene (M. Morimyo, J. Bacteriol. 170:2136-2142, 1988) originated from the 88-min region of the Escherichia coli chromosome and not, as reported, from the 7-min region and that the gene product identified as MvrA is in fact encoded by a gene distal to glpX.

Aquaporins↗
PubMedDate not supplied

Ferredoxin-NADP+ oxidoreductase of C. paradoxa nucleus encoded, but cyanobacterial gene transfer from symbiont to host, an evolutionary mechanism originating new species.

The nucleus encoded cyanoplast ("cyanellar") ferredoxin-NADP + oxidoreductase (FNR) of Cyanophora paradoxa, characterized by an N-terminal amino acid sequence, is compared with homologous sequences of other photoautotrophic organisms. The high degree of similarity to the cyanobacterial sequences indicates a cyanobacterial origin. This could be a first direct demonstration of an intertaxonic combination: a gene transfer from an original endocytobiont (cyanobacterium) to the nucleus of its host, one of the most important demands of the Endosymbiosis Theory, an evolutionary mechanism leading to the origin of a new species.

Amino Acid Sequence↗
PubMed1976

Adrenodoxin reductase-adrenodexin complex.

Adrenodoxin reductase and adrenodoxin have been shown (Chu, J.-W., and Kimura, T. (1973) J. Biol. Chem. 248, 5183-5187) to form a low dissociation constant, 1:1 complex when both proteins are in the oxidized form. We have found that when adrenodoxin: adrenodoxin reductase ratios are varied by increasing the adrenodoxin concentration, with adrenodoxin reductase held constant, an increasing rate of cytochrome c reduction, with NADPH as reductant, is seen up to a ratio of 1:1, indicating that cytochrome c reduction occurs via the protein-protein complex. Spectra observed during titration of this protein-protein complex with NADH were resolved into components by the linear programming method, using a computer program written in Fortran IV. Analysis of the data has shown that the flavoprotein is reduced prior to the iron sulfur protein, and that the midpoint oxidation-reduction potentials (pH 7.5) of the two proteins are -295 and -331 mV, respectively, when both are present in the complex. Complex formation does not alter the potential of adrenodoxin reductase, but changes that of adrenodoxin by -40 mV. Equilibrium constants derived from potential measurements show that the strength of the protein-protein interaction in the complex is unaltered by reduction of adrenodoxin reductase, but is decreased by about 1 kcal due to reduction of adrenodoxin. The low dissociation constants for both oxidized reduced forms of the adrenodoxin reductase-adrenodoxin complex indicate that the complex must remain associated throughout its catalytic cycle. Titration of the adrenodoxin reductase-adrenodoxin complex with the physiologic reductant, NADPH, was followed by EPR and visible spectra, and yielded an order of reduction of the components identical with that seen when NADH was used as reductant. Reduction of the protein-protein complex with NADPH yielded a ternary complex between NADP+, flavoprotein, and iron sulfur protein, with the two electrons located in a "charge transfer" complex between flavoprotein and pyridine nucleotide.

Adrenal Cortex↗
PubMed1977

[Study of the NADH and NADPH-ferredoxin oxidoreductase activities in Clostridium acetobutylicum].

The NADH and NADPH-ferredoxin oxidoreductase have been studied in Clostridium acetobutylicum. Acetyl-CoA is an obligatory activator of NADH-ferredoxin reductase activity and NADH a competitive inhibitor of ferredoxin-NAD+ reductase activity. These regulations are the same when C. acetoburylicum moves from 'butylic-type metabolism' to 'butyric-type metabolism'; this demonstrates that NADH-ferredoxin oxidoreductase cna, through its reversible action, meet the very different cell needs imposed by these two types of culture. The physiological function of the clostridial NADPH-ferredoxin oxidoreductase was anabolic as it has been with other clostridia.

Acetyl Coenzyme A↗
PubMed1978

Effect of corn and sorghum diets in N1 methylnicotinamide excretion and hepatic enzymes in rats.

This study was carried out to compare the effects of corn (Zea mays) and sorghum (Sorghum vulgare) diets on urinary N1 methylnicotinamide (N'MN) excretion and on the activity of hepatic enzyme in young adult rats. Thirty rats, weighing an average of 174.3 g at the beginning of the experiments, were divided into three groups and studied for 13 weeks. The two experimental diets supplied 7% protein, and a casein diet was used as control. Niacin was excluded from the vitamin mixture used in the corn and sorghum diets. The activities of the following enzymes were studied: betaHOB-D, G6P-D, NADH2-TR, NADPH2-TR, and 3 HOA-0. Urinary excretion of N'MN was statistically different among the three groups, the corn group having the lowest level. The corn-fed animals appeared to have more obvious alterations in liver enzyme activity. The changes found in corn and sorghum-fed animals are different and cannot be explained as due to niacin deficiency. The different amino acid compositions of the two grains and their relationship with the discrepancies in the result are also discussed.

Amino Acids, Essential↗
PubMed1990

Monoclonal antibodies to chick renal calcium regulating hemeproteins. Biochemical and immunohistochemical interactions with mitochondrial 25-hydroxyvitamin D3 hydroxylases.

The kidney mitochondrial monooxygenases known as 25-hydroxyvitamin D3 1 alpha- and 24R-hydroxylases are two analogous enzymes which utilize the vitamin as a common substrate for the catalytic production of 1 alpha,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3. These two enzymes are complexes of NADPH-ferredoxin reductase, and an (Fe-S)-cluster containing ferredoxin with a redox potential that allows the ultimate transfer of reducing equivalents to the terminal oxidases distinctly known as cytochromes P-450(1) alpha and P-450(24). We have used in vitro immunizations of splenocytes obtained from mice sensitized with the purified cytochrome P-450(1) alpha to generate three hybridoma clones from fusion with p3 x 63.Ag8.653 myeloma ATCC cells which selectively secrete monoclonal antibodies (MAbs) of the IgM class. The MAbs have been partially purified by ammonium sulfate fractionation followed by size separation chromatography on Sephacryl S-200-HR. We have compared the structural similarities and differences between the two kidney enzymes in Western Blot analyses using horseradish peroxidase conjugated goat anti-mouse Igs specific for the heavy and light chains of mouse IgA, IgG and IgM. The MAbs from all three clones recognized and interacted with apparent common epitopes of the two hydroxylases but selectively discriminated against liver microsomal P-450LM2 type and adrenal mitochondrial P-450SCC cytochromes. The cytochromes P-450(1) alpha and P-450(24) were detected as two separate bands with approximate molecular weights of 57 and 55 KDa, respectively. In reconstitution of hydroxylase activities in vitro, the MAbs were equally effective in inhibiting the 1 alpha-hydroxylation and 24R-hydroxylation reactions. The ratio of micrograms of Igs to pmol cytochrome P-450 for a 50% inhibition of either activity was approximately 25. These results, collectively, seem to suggest the existence of a precursor-product relationship between the kidney mitochondrial 1 alpha- and the 24R-hydroxylases, or perhaps, a common ancestral origin. Immunochemical peroxidase anti-peroxidase staining of kidney tissue first exposed to the MAbs revealed that only the proximal tubular segment of the nephron was specifically enriched with the cytochromes.

Adrenal Glands↗
PubMed1991

Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc.

Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.

Amino Acid Sequence↗
PubMedDate not supplied

[A ferredoxin-linked sulfite reductase from Spinacia oleracea (author's transl)].

A sulfite reductase from spinach has been purified 125 fold. Throughout all stages of purification the reduction of sulfite has been found dependent on ferredoxin. Reduced ferredoxin has been provided either by photosynthetic reduction in isolated, broken chloroplasts or by NADPH via the ferredoxin-NADP-oxidoreductase. During the purification procedure ferredoxin as electrondonor has been replaced by reduced methylviologen.

Chloroplasts↗
PubMed1979

Differences between the reactivities of two pyridine nucleotides in the rapid reduction process and the reoxidation process of adrenodoxin reductase.

The reaction process of adrenodoxin reductase with NADPH and NADH were investigated. The appearance of new intermediate with a broad absorption band at around 520 nm has been detected by rapid-scan stopped-flow spectrophotometry. Although the formation of this intermediate is more rapid with NADPH than with NADH, the rates of the subsequent decay to the fully reduced state are almost identical (Kobs values were 20.5 and 16.0s-1). These results indicate that the new intermediate is the complex formed between the oxidized enzyme and reduced pyridine nucleotide (enzyme-substrate complex), and that subsequent decay of the intermidiate is caused by a two-electron transfer process from the reduced pyridine nucleotide to the enzyme flavin. On the other hand, spectral and kinetic properties in the steady state of the reoxidation reaction of the enzyme reduced with NADPH and NADH were somewhat different. The rate of reoxidation of the enzyme under aerobic conditions from the reduced state to the oxidized state was 6.5 times faster when a 10-fold molar excess of NADH was used than when NADPH of the same concentration was used. This result is consistent with the fact that the NADH-dependent oxidase activity was 6.4 times greater than that dependent on NADPH. During reoxidation of the reduced enzyme under aerobic conditions in the presence of an excess of NADPH or NADH, the EPR spectra indicated the formation of the flavin semiquinone radical species. Similarly, the formation of semiquinone was observed in the absorption spectrum with either NADPH or NADH under the same conditions as in the EPR measurement. The intensity of the semiquinone signal on EPR was considerably smaller with NADH than with NADPH. These results suggest that NADP+ complex with the enzyme semiquinone protects the radical from oxidation by oxygen to a greater extent than NAD+, and consequently the semiquinone is easier to detect with NADPH than with NADH.

Adrenal Cortex↗
PubMed1979

Isolation, structural organization and mechanism of action of mitochondrial steroid hydroxylating systems.

A scheme for the isolation of individual proteins of the 20S,22R-cholesterol hydroxylating system is presented. Physico-chemical and structural parameters of cytochrome P-450 are furnished. Self-reconstitution of indivudal proteins of the system into an enzyme unit is shown. Furthermore a regulation process of electron transport in the 20S,22R-cholesterol system is considered.

Adrenodoxin↗