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PubMed1996

Derivation of input function from FDG-PET studies in small hearts.

UNLABELLED: The extraction of pure arterial time-activity curves (TACs) from dynamic PET images of a small animal heart using factor analysis of dynamic structures (FADS) was found to be unsuccessful due to the small size of the cardiac chamber that causes extensive mixture of TACs of different structures. METHODS: In this study, we used digital phantoms of the left ventricle (LV cavity size: 1-2 cm) and small monkey (LV cavity size: approximately 2 cm) dynamic FDG PET studies to evaluate FADS for extracting the pure blood-pool TACs by adding a single blood sample (taken at a late scan time) constraint. RESULTS: In the digital phantom studies, spillover fractions in the extracted blood-pool TACs using FADS without a blood sample constraint (FADS(-)) and with a blood sample constraint (FADS(+)) were 3%-91% and < 3%, respectively. In the monkey studies (n = 4), FADS(+) extracted blood-pool TACs matched well with the arterialized well counter measurements (% differences of curve integration; FADS(-) < 82%; FADS(+) < 9%). The microparameters (K1*, k2*, k3*, k4*) and macroparameters (Knlr), obtained from the FADS(+) blood-pool TACs, were similar to those obtained from plasma samples in a three-compartment model fitting (% differences of Knlr:phantom studies < 5%; monkey studies < 9%). CONCLUSION: The FADS technique with a single-blood sample has the potential to extract the pure blood-pool TACs directly from dynamic PET images of a small animal without multiple blood sampling, region of interest definition or spillover correction.

Animals↗
PubMed1997

[Estrogen receptors and breast fibroadenoma with suspicious traits of malignancy].

Fibroadenomas (FAD) are the most common breast tumors. Their clinical and morphological characteristics often vary, and endocrine factors has been implicated in their etiology. The development of a breast carcinoma overlying a FAD is very rare. An immunohistochemical study is made of the presence of estrogen receptor (ER) in a series of 21 FAD with clinical, mammographic or cytological suspect of malignancy. An intraoperative biopsy evaluation was made in all cases. In three cases FAD was associated to breast cancer (three lobular carcinomas-two infiltrating and one in situ; one case was also adjacent to an infiltrating ductal carcinoma). Two of these three fibroadenomas were ER-positive. Globally, only 35% of the FAD evaluated proved ER-positive. Of all the parameters evaluated (age, tumor size, single or multiple presentation, history of breast carcinoma or oral contraceptives, and histological characteristics) no association was observed with respect to ER positivity, with the exception of patient age. Thus, ER-positive FAD were found in patients 5.4 years younger than the ER-negative women (ages 28.4 and 33.8 respectively). On the other hand, the mean age (46.6 years) of the patients with FAD and cancer was 10.2 years above the mean age (34.4 years) of the patients with FAD but no cancer; however, and despite this greater age, two of the three patients with both FAD and cancer exhibited ER positivity at FAD level.

Adult↗
PubMed1977

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Flavin-Adenine Dinucleotide↗
PubMed1993

Screening of the mis-sense mutation producing the 717Val-->Ile substitution in the amyloid precursor protein in Japanese familial and sporadic Alzheimer's disease.

We investigated a C to T transition at base pair 2149 in the amyloid precursor protein gene in 41 Japanese cases of early-onset familial Alzheimer's disease (FAD), late-onset FAD and sporadic Alzheimer's disease (AD) by polymerase chain reaction and restriction enzyme polymorphism with BclI. Among 9 early-onset FAD patients derived from independent families, only one patient had the mis-sense mutation. Neither 5 patients with late-onset FAD nor 27 patients with sporadic AD had the mutation. Our result and the previous reports from Japan indicate that this type of mis-sense mutation is present in several cases of Japanese early-onset FAD. On the other hand, our data suggest that this mutation is not a common cause of Japanese early-onset FAD. Moreover, this mutation could be absent in late-onset FAD and sporadic AD in Japan. Because the mutation has been reported to be rare in Caucasian early-onset FAD and to be absent in Caucasian late-onset FAD and sporadic AD, the situation of this mutation in Alzheimer's disease may be common beyond the ethnic background.

Adult↗
PubMed1993

The c-fos gene and early-onset familial Alzheimer's disease.

A gene for early-onset familial Alzheimer's disease (FAD) is located on chromosome 14q24.3. The c-fos gene (FOS) is also located in the same band of this chromosome and is thus a candidate for the FAD locus. A yeast artificial chromosome (YAC) clone was identified which contains FOS. This YAC also contains the short-tandem repeat polymorphic (STRP) locus D14S76, placing FOS in the FAD region between D14S53 and D14S43. No recombinants were observed between D14S76 and FAD, and a maximum positive LOD score of 7.20 at a recombination fraction of 0.001 was observed for linkage of this marker to FAD. DNA sequence analysis was used to examine FOS in two affected subjects from an FAD family in which the chromosome 14 FAD locus is clearly responsible for the disease. The coding regions and parts of the 5' and 3' untranslated sequences of FOS were sequenced; no FAD-related mutations were observed. This work suggests that the FOS gene is not the chromosome 14 FAD locus although we cannot exclude the possibility that a mutation in an as yet unknown regulatory region is responsible for the disease. A new polymorphism was detected in the third intron of the gene.

Alleles↗
PubMed1985

Electrochemical and glucose oxidase coenzyme activity of flavin adenine dinucleotide covalently attached to glassy carbon at the adenine amino group.

Flavin adenine dinucleotide (FAD) was covalently attached to an electron-conducting support, i.e., glassy carbon. The support was activated by oxidation to create surface carboxylic acid groups, followed by reaction with a water-soluble carbodiimide. FAD was then attached to the activated support by three different methods: (1) directly; (2) through 6-aminocaproic acid as a spacer; and (3) through ethylenediamine glutaraldehyde as a spacer. Coupling occurred at the FAD adenine amino group, or possibly at a ribityl OH group. Cyclic voltammetry was used to determine Eo' values and FAD loadings. The immobilized FAD also acted as a catalyst for the oxidation of reduced nicotinamide adenine dinucleotide (NADH) in that it reduced overpotential by about 195 mV. When the apoenzyme of glucose oxidase was added to the glassy carbon-FAD or glassy carbon-spacer-FAD preparations, no reconstitution of enzyme activity could be observed. This suggests strongly that the adenine amino group of FAD cannot be modified by attachment of something as large as easily visible solid particles. However, it leaves unanswered the question of larger molecular weight material can be accommodated in the FAD-apoenzyme cleft and retain glucose oxidase activity.

Adsorption↗
PubMed2003

Electron transfer is activated by calmodulin in the flavin domain of human neuronal nitric oxide synthase.

The objective of this study was to clarify the mechanism of electron transfer in the human neuronal nitric oxide synthase (nNOS) flavin domain using the recombinant human nNOS flavin domains, the FAD/NADPH domain (contains FAD- and NADPH-binding sites), and the FAD/FMN domain (the flavin domain including a calmodulin-binding site). The reduction by NADPH of the two domains was studied by rapid-mixing, stopped-flow spectroscopy. For the FAD/NADPH domain, the results indicate that FAD is reduced by NADPH to generate the two-electron-reduced form (FADH(2)) and the reoxidation of the reduced FAD proceeds via a neutral (blue) semiquinone with molecular oxygen or ferricyanide, indicating that the reduced FAD is oxidized in two successive one-electron steps. The neutral (blue) semiquinone form, as an intermediate in the air-oxidation, was unstable in the presence of O(2). The purified FAD/NADPH domain prepared under our experimental conditions was activated by NADP(+) but not NAD(+). These results indicate that this domain exists in two states; an active state and a resting state, and the enzyme in the resting state can be activated by NADP(+). For the FAD/FMN domain, the reduction of the FAD-FMN pair of the oxidized enzyme with NADPH proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The formation of semiquinones from the FAD-FMN pair was greatly increased in the presence of Ca(2+)/CaM. The air-stable semiquinone form, FAD-FMNH(.), was further rapidly reduced by NADPH with an increase at 520 nm, which is a characteristic peak of the FAD semiquinone. Results presented here indicate that intramolecular one-electron transfer from FAD to FMN is activated by the binding of Ca(2+)/CaM.

Binding Sites↗
PubMed2001

A rate-limiting conformational change of the flavin in p-hydroxybenzoate hydroxylase is necessary for ligand exchange and catalysis: studies with 8-mercapto- and 8-hydroxy-flavins.

The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2003

Electron transfer in flavocytochrome P450 BM3: kinetics of flavin reduction and oxidation, the role of cysteine 999, and relationships with mammalian cytochrome P450 reductase.

Cys-999 is one component of a triad (Cys-999, Ser-830, and Asp-1044) located in the FAD domain of flavocytochrome P450 BM3 that is almost entirely conserved throughout the diflavin reductase family of enzymes. The role of Cys-999 has been studied by steady-state kinetics, stopped-flow spectroscopy, and potentiometry. The C999A mutants of BM3 reductase (containing both FAD and FMN cofactors) and the isolated FAD domain are substantially compromised in their capacity to reduce artificial electron acceptors in steady-state turnover with either NADPH or NADH as electron donors. Stopped-flow studies indicate that this is due primarily to a substantially slower rate of hydride transfer from nicotinamide coenzyme to FAD cofactor in the C999A enzymes. The compromised rates of hydride transfer are not attributable to altered thermodynamic properties of the flavins. A reduced enzyme-NADP(+) charge-transfer species is populated following hydride transfer in the wild-type FAD domain, consistent with the slow release of NADP(+) from the 2-electron-reduced enzyme. This intermediate does not accumulate in the C999A FAD domain or wild-type and C999A BM3 reductases, suggesting more rapid release of NADP(+) from these enzyme forms. Rapid internal electron transfer from FAD to FMN in wild-type BM3 reductase releases NADP(+) from the nicotinamide-binding site, thus preventing the inhibition of enzyme activity through the accumulation of a stable FADH(2)-NADP(+) charge-transfer complex. Hydride transfer is reversible, and the observed rate of oxidation of the 2-electron-reduced C999A BM3 reductase and FAD domain is hyperbolically dependent on NADP(+) concentration. With the wild-type BM3 reductase and FAD domain, the rate of flavin oxidation displays an unusual dependence on NADP(+) concentration, consistent with a two-site binding model in which two coenzyme molecules bind to catalytic and regulatory regions (or sites) within a bipartite coenzyme binding site. A kinetic model is proposed in which binding of coenzyme to the regulatory site hinders sterically the release of NADPH from the catalytic site. The results are discussed in the light of kinetic and structural studies on mammalian cytochrome P450 reductase.

Amino Acid Sequence↗
PubMed2005

Characterization of a bifunctional PutA homologue from Bradyrhizobium japonicum and identification of an active site residue that modulates proline reduction of the flavin adenine dinucleotide cofactor.

PutA is a bifunctional flavoenzyme in bacteria that catalyzes the four-electron oxidation of proline to glutamate. In certain prokaryotes such as Escherichia coli, PutA is also a transcriptional repressor of the proline utilization (put) genes and thus is trifunctional. In this work, we have begun to assess differences between bifunctional and trifunctional PutA enzymes by examining the PutA protein from Bradyrhizobium japonicum (BjPutA). Primary structure analysis of BjPutA shows it lacks the DNA-binding domain of E. coli PutA (EcPutA). Consistent with this prediction, purified BjPutA does not exhibit DNA-binding activity in native gel mobility shift assays with promoter regions of the putA gene from B. japonicum. The catalytic and redox properties of BjPutA were characterized and a reduction potential (E(m)) value of -0.132 V (pH 7.5) was determined for the bound FAD/FADH(2) couple in BjPutA that is significantly more negative ( approximately 55 mV) than the E(m) for EcPutA-bound FAD. The more negative E(m) value thermodynamically limits proline reduction of the FAD cofactor in BjPutA. In the presence of phospholipids, reduction of BjPutA is stimulated, suggesting lipids influence the FAD redox environment. Accordingly, an E(m) value of -0.114 V (pH 7.5) was determined for BjPutA-bound FAD in the presence of polar lipids. The molecular basis for the lower reduction potential of FAD in BjPutA relative to EcPutA was explored by site-directed mutagenesis. Amino acid sequence alignment between BjPutA and EcPutA indicates only one difference in active site residues near the isoalloxazine ring of FAD: Val402 in EcPutA is substituted at the analogous position in BjPutA with Ala310. Replacement of A310 by Val in the BjPutA mutant A310V raised the reduction potential of bound FAD relative to wild-type BjPutA to an E(m) value of -0.09 V (pH 7.5). The >40-mV positive shift in the potential of the BjPutA mutant A310V suggests that the corresponding Val residue in EcPutA helps poise the FAD redox potential for thermodynamically favored proline reduction thereby allowing EcPutA to be efficiently regulated by proline availability. Limited proteolysis of BjPutA under reducing conditions shows FAD reduction does not influence BjPutA conformation indicating further that the redox dependent regulation observed with EcPutA may be limited to trifunctional PutA homologues.

Alanine↗
PubMed2006

Reaction mechanism of the iron-sulfur flavoenzyme adenosine-5'-phosphosulfate reductase based on the structural characterization of different enzymatic states.

The iron-sulfur flavoenzyme adenosine-5'-phosphosulfate (APS) reductase catalyzes a key reaction of the global sulfur cycle by reversibly transforming APS to sulfite and AMP. The structures of the dissimilatory enzyme from Archaeoglobus fulgidus in the reduced state (FAD(red)) and in the sulfite adduct state (FAD-sulfite-AMP) have been recently elucidated at 1.6 and 2.5 A resolution, respectively. Here we present new structural features of the enzyme trapped in four different catalytically relevant states that provide us with a detailed picture of its reaction cycle. In the oxidized state (FAD(ox)), the isoalloxazine moiety of the FAD cofactor exhibits a similarly bent conformation as observed in the structure of the reduced enzyme. In the APS-bound state (FAD(ox)-APS), the substrate APS is embedded into a 17 A long substrate channel in such a way that the isoalloxazine ring is pushed toward the channel bottom, thereby producing a compressed enzyme-substrate complex. A clamp formed by residues ArgA317 and LeuA278 to fix the adenine ring and the curved APS conformation appear to be key factors to hold APS in a strained conformation. This energy-rich state is relaxed during the attack of APS on the reduced FAD. A relaxed FAD-sulfite adduct is observed in the structure of the FAD-sulfite state. Finally, a FAD-sulfite-AMP1 state with AMP within van der Waals distance of the sulfite adduct could be characterized. This structure documents how adjacent negative charges are stabilized by the protein matrix which is crucial for forming APS from AMP and sulfite in the reverse reaction.

Adenosine Monophosphate↗
PubMed1999

Properties of p-cresol methylhydroxylase flavoprotein overproduced by Escherichia coli.

The alpha(2)beta(2) flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida is composed of a flavoprotein homodimer (alpha(2) or PchF(2); M(r) = 119 kDa) with a cytochrome monomer (beta, PchC; M(r) = 9.3 kDa) bound to each PchF subunit. Escherichia coli BL21(DE3) has been transformed with a vector for expression of the pchF gene, and PchF is overproduced by this strain as the homodimer. During purification, it was recognized that some PchF had FAD bound, while the remainder was FAD-free. However, unlike PchF obtained from PCMH purified from P. putida, FAD was bound noncovalently. The FAD was conveniently removed from purified E. coli-expressed PchF by hydroxyapatite chromatography. Fluorescence quenching titration indicated that the affinity of apo-PchF for FAD was sufficiently high to prevent the determination of the dissociation constant. It was found that p-cresol was virtually incapable of reducing PchF with noncovalently bound FAD (PchF(NC)), whereas 4-hydroxybenzyl alcohol, the intermediate product of p-cresol oxidation by PCMH, reduced PchF(NC) fairly quickly. In contrast, p-cresol rapidly reduced PchF with covalently bound FAD (PchF(C)), but, unlike intact PCMH, which consumed 4 electron equiv/mol when titrated with p-cresol (2 electrons from p-cresol and 2 from 4-hydroxybenzyl alcohol), PchF(C) accepted only 2 electron equiv/mol. This is explained by extremely slow release of 4-hydroxybenzyl alcohol from reduced PchF(C). 4-Hydroxybenzyl alcohol rapidly reduced PchF(C), producing 4-hydroxybenzaldehyde. It was demonstrated that p-cresol has a charge-transfer interaction with FAD when bound to oxidized PchF(NC), whereas 4-bromophenol (a substrate analogue) and 4-hydroxybenzaldehyde have charge-transfer interactions with FAD when bound to either PchF(C) or PchF(NC). This is the first example of a "wild-type" flavoprotein, which normally has covalently bound flavin, to bind flavin noncovalently in a stable, redox-active manner.

Bacterial Proteins↗
PubMed1998

31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states.

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by 31P-NMR in the oxidised and reduced states. The 31P chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP+ reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-13C2]FAD, [4,10a-13C2]FAD, or [U-15N4]FAD. The reconstituted proteins were studied by 13C- and 15N-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)-OH group of FAD. The sp2 hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp3 hybridised in comparison to those of other flavoproteins.

Apoproteins↗
PubMed2002

Partially folded structure of flavin adenine dinucleotide-depleted ferredoxin-NADP+ reductase with residual NADP+ binding domain.

Maize ferredoxin-NADP(+) reductase (FNR) consists of flavin adenine dinucleotide (FAD) and NADP(+) binding domains with a FAD molecule bound noncovalently in the cleft between these domains. The structural changes of FNR induced by dissociation of FAD have been characterized by a combination of optical and biochemical methods. The CD spectrum of the FAD-depleted FNR (apo-FNR) suggested that removal of FAD from holo-FNR produced an intermediate conformational state with partially disrupted secondary and tertiary structures. Small angle x-ray scattering indicated that apo-FNR assumes a conformation that is less globular in comparison with holo-FNR but is not completely chain-like. Interestingly, the replacement of tyrosine 95 responsible for FAD binding with alanine resulted in a molecular form similar to apo-protein of the wild-type enzyme. Both apo- and Y95A-FNR species bound to Cibacron Blue affinity resin, indicating the presence of a native-like conformation for the NADP(+) binding domain. On the other hand, no evidence was found for the existence of folded conformations in the FAD binding domains of these proteins. These results suggested that FAD-depleted FNR assumes a partially folded structure with a residual NADP(+) binding domain but a disordered FAD binding domain.

Alanine↗
PubMed2005

Switching pyridine nucleotide specificity in P450 BM3: mechanistic analysis of the W1046H and W1046A enzymes.

Flavocytochrome P450 BM3 is a member of the diflavin reductase enzyme family. Members include cytochrome P450 reductase, nitric-oxide synthase, methionine synthase reductase, and novel oxidoreductase 1. These enzymes show a strong preference for NADPH over NADH as reducing coenzyme. An aromatic residue stacks over the FAD isoalloxazine ring in each enzyme, and in some cases it is important in controlling coenzyme specificity. In P450 BM3, the aromatic residue inferred from sequence alignments to stack over the FAD is Trp-1046. Mutation to Ala-1046 and His-1046 effected a remarkable coenzyme specificity switch. P450 BM3 W1046A/W106H FAD and reductase domains are efficient NADH-dependent ferricyanide reductases with selectivity coefficients (k(cat)/K(m)(NADPH)/k(cat)/K(m)(NADH)) of 1.5, 67, and 8571 for the W1046A, W1046H, and wild-type reductase domains, respectively. Stopped-flow photodiode array absorption studies indicated a charge-transfer intermediate accumulated in the W1046A FAD domain (and to a lesser extent in the W1046H FAD domain) and was attributed to formation of a reduced FADH(2)-NAD(P)(+) charge-transfer species, suggesting a relatively slow rate of release of NAD(P)(+) from reduced enzymes. Unlike wild-type enzymes, there was no formation of the blue semiquinone species observed during reductive titration of the W0146A/W146H FAD and reductase domains with dithionite or NAD(P)H. This was a consequence of elevation of the semiquinone/hydroquinone couple of the FAD with respect to the oxidized/semiquinone couple, and a concomitant approximately 100-mV elevation in the 2-electron redox couple for the enzyme-bound FAD (-320, -220, and -224 mV in the wild-type, W1046A, and W1046H FAD domains, respectively).

Alanine↗
PubMed1993

Search for the genes responsible for familial Alzheimer's disease.

Inherited or Familial Alzheimer's Disease (FAD) has clearly been shown to be a genetically heterogeneous disorder. Mutations in the gene on chromosome 21 encoding the beta-amyloid protein precursor (APP) have been shown to be linked to 2-3% of FAD kindreds examined around the world. A late onset FAD locus has been mapped to a region of chromosome 19 in which a recently isolated APP-like gene, APLP1 has also been localized, making this gene a strong candidate to harbor a late-onset FAD defect. More recently, a major FAD locus has been mapped to the long arm of chromosome 14. The chromosome 14 locus appears to be mainly linked to the gene defect in early onset FAD pedigrees. Besides the FAD loci on chromosome 21, 19, and 14, at least two other loci must exist since the gene defect in some early- and late-onset FAD pedigrees do not appear to segregate with markers from any of these autosomes. As different gene defects responsible for various forms of FAD are discovered, perhaps, a common basis for the etiology of this devastating disorder can be discerned.

Alzheimer Disease↗
PubMed1993

Multiple acyl-coenzyme A dehydrogenation disorder responsive to riboflavin: substrate oxidation, flavin metabolism, and flavoenzyme activities in fibroblasts.

Multiple acyl-CoA dehydrogenation disorders result from generalized defects in intramitochondrial acyl-CoA dehydrogenation. Fibroblasts from a riboflavin-responsive multiple acyl-CoA dehydrogenation disorder patient catabolized 14C-butyrate, -octanoate, and -leucine normally after culture in riboflavin-supplemented medium (2 mg/L). After culture in riboflavin-depleted medium (< or = 1.4 micrograms/L), his cells oxidized the same substrates poorly at 20 to 33% of control (p < 0.05). Patient cells incubated in a wide range of D-[2-14C]riboflavin concentrations (3, 31.4, and 100 micrograms/L) synthesized 14C-flavin mononucleotide and 14C-flavin adenine dinucleotide (FAD) normally and had normal cytosolic 14C-flavin mononucleotide and 14C-FAD contents, which argues against defects in cellular riboflavin uptake and conversion to flavin mononucleotide and FAD. After culture in 31.4 micrograms 14C-riboflavin/L for 2 wk, 14C-FAD specific radioactivities plateaued and were similar in patient and control cells. However, culturing these uniformly labeled cells in riboflavin-depleted medium for 2 wk lowered the patient's cellular 14C-FAD content to only 23% of control levels. Similarly, after incubation in low 14C-riboflavin concentrations (4.4 micrograms/L), the patient's mitochondrial 14C-FAD content was only 51% of control after 1 h and 29% of control at 4 h. After a 4-h incubation in a high physiologic concentration of 14C-riboflavin (31.4 micrograms/L), which raised the patient's cellular 14C-FAD levels 3- to 4-fold, his mitochondrial 14C-FAD content rose to normal; control values did not change. We also investigated possible defective FAD binding to flavoenzymes essential for acyl-CoA dehydrogenation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMedDate not supplied

Blue light mediated photoreduction of the flavoprotein NADPH-cytochrome P450 reductase. A Förster-type energy transfer.

The absorption spectra and the corresponding molar absorption coefficients of the fluorophores umbelliferone, FAD and FMN and of the FAD and FMN containing flavoprotein NADPH-cytochrome P450 reductase of different oxydation-reduction states are documented. Binding spectra of the ligand umbelliferone with the CYP2B1:NADPH-cytochrome P450 reductase-complex were determined by difference spectroscopy. The Scatchard plot of the equilibrium ligand binding shows a high affinity part and a low affinity part of 12 and 34 umbelliferone binding sites per CYP2B1:reductase-complex molecule, respectively. The fluorescence excitation and emission spectra of the donor molecule umbelliferone and the acceptor molecules FAD and FMN are given. The fluorescence spectra of the reaction components under test conditions of CYP2B1-dependent 7-ethoxycoumarin-O-deethylase are measured. The excitation energy transfer from the donor umbelliferone (lambda E = 380 nm; lambda F = 460 nm) to the acceptor molecule FMN (lambda E = 465 nm; lambda F = 525 nm) was examined under assay conditions. The results demonstrate that a radiationless Förster-type energy transfer takes place in the presence of the CYP2B1:reductase-complex. It turned out that this effect is a function of the protein complex-concentration. The data presented here combined with previously made observations by Müller-Enoch (Müller-Enoch D. (1994), Z. Naturforsch. 49c, 763-771) support the finding that the umbelliferone molecules, n = 12-34, bound per mole of CYP2B1:reductase-complex, transfer their absorbed light energy radiationless to the FAD binding domain. The complex formed containing 12 or 34 molecules of umbelliferone provides absorption coefficient values at lambda = 380 nm of 78 and 221 mM-1.cm-1, respectively. The Förster-type energy transfer from the donor umbelliferone to the acceptor FAD not only leads to a light activation of the singlet state of FAD but also to a conformational change of the amino acids close to the FAD binding side to favour the encaging of the FAD* triplet state which reacts with the NADPH to form the FADH2 reductase. Due to this process the overall reaction can start with the unquenched excited FAD* triplet state as an intermediate which is about 30 kJ/mol lower in energy than the dark reaction.

Animals↗