Enzymatic and oxidative metabolites of lycopene

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD+, KCI, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of 2H10 lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C18H24O2 or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z =272 and (2) 3,4-dehydro-5,6-dihydro-15-apo-lycopenal (C20H28O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-l-al) with lambdamax= 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-ene-5,8-lycopenal-furanoxide (C37H50O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C40H56O2) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z =568; (5) lycopene-5,8-furanoxide isomer (I) (C40H56O2) with lambdamax = 410 nm, 440 nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C40H56O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C40H54O2) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.     

Enzymatic and oxidative metabolites of lycopene

Using the post-mitochondrial fraction of rat intestinal mucosa, we have investigated lycopene metabolism. The incubation media was composed of NAD(+), KCl, and DTT with or without added lipoxygenase. The addition of lipoxygenase into the incubation significantly increased the production of lycopene metabolites. The enzymatic incubation products of (2)H(10) lycopene were separated using high-performance liquid chromatography and analyzed by UV/Vis spectrophotometer and atmospheric pressure chemical ionization-mass spectroscopy. We have identified two types of products: cleavage products and oxidation products. The cleavage products are likely: (1) 3-keto-apo-13-lycopenone (C(18)H(24)O(2) or 6,10,14-trimethyl-12-one-3,5,7,9,13-pentadecapentaen-2-one) with lambdamax = 365 nm and m/z = 272 and (2) 3,4-dehydro-5,6-dihydro-15,15'-apo-lycopenal (C(20)H(28)O or 3,7,11,15-tetramethyl-2,4,6,8,12,14-hexadecahexaen-1-al) with lambdamax = 380 nm and m/z = 284. The oxidative metabolites are likely: (3) 2-apo-5,8-lycopenal-furanoxide (C(37)H(50)O) with lambdamax = 415 nm, 435 nm, and 470 nm, and m/z = 510; (4) lycopene-5, 6, 5', 6'-diepoxide (C(40)H(56)O(2)) with lambdamax = 415 nm, 440 nm, and 470 nm, and m/z = 568; (5) lycopene-5,8-furanoxide isomer (I) (C(40)H(56)O) with lambdamax = 410 nm, 440nm, and 470 nm, and m/z = 552; (6) lycopene-5,8-epoxide isomer (II) (C(40)H(56)O) with lambdamax = 410, 440, 470 nm, and m/z = 552; and (7) 3-keto-lycopene-5',8'-furanoxide (C(40)H(54)O(2)) with lambdamax = 400 nm, 420 nm, and 450 nm, and m/z = 566. These results demonstrate that both central and excentric cleavage of lycopene occurs in the rat intestinal mucosa in the presence of soy lipoxygenase.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.     

H-1, C-13, and F-19 nuclear magnetic resonance assignments of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate.

The molecular structure of 3,3-difluoro-5 alpha-androstane-17 beta-ol acetate was analyzed by 1H, 13C, and 19F nuclear magnetic resonance (NMR) techniques; two-dimensional NMR was used to assigned 1H and 13C resonances. The 1H NMR spectrum in deuterated chloroform shows three sharp singlets (delta = 0.74, 0.79, and 2.00 ppm) integrating for three protons each, an isolated triplet at 4.55 ppm integrating for one proton, and overlapping multiplets between 0.72 and 2.12 ppm integrating for 31 protons. The 13C spectrum shows 18 resonances between 10 and 55 ppm, and three additional resonances at 82.9, 124.0, and 171.5 ppm. The 19F[1H] spectrum shows two sets of doublets (observed 2J = 150 Hz) at 5.00 and -4.80 ppm. Multiplets arising from 19F-13C J-coupling provide the starting assignment for all resonances by means of 1H homonuclear correlation (COSY) and 1H-13C heteronuclear correlation spectroscopy.     

Absorption of oleic and taurocholic acids from the intestine of the chick. Interactions and interference by proteins.

4-Nitro-1-cyclohexyl-3-ethoxy-2-oxo-3-pyrroline reacts with both amino and sulfhydryl groups. The instability of the product with sulfhydryl groups makes the reagent a useful amino-group specific protein reagent. The advantages of this compound include (1) rapid reaction with protein (less than 15 min at pH 9), (2) EASE OF REVERSAL UNDER MILDLY ALKALINE CONDITIONS (PH larger than or equal to 8) with formation of a water-soluble by-product (lambdamax = 363 nm), and (3) ease of quantitation utilizing the high extinction coefficients of the amino derivative (lambdamax = 383 and 397 nm, epsilon397 = 20 200 M-1 . cm-1) and the reversal by-product (lambdamax = 363 nm, epsilon = 16 300 M-1 . cm-1). With these characteristics and the stability of the amino derivative under physiological conditions (t1/2 for reversal = 167 h at pH 7.0 and room temperature), nitrocyclohexylethoxyoxopyrroline can be a useful reagent in a wide variety of protein sequencing and structure studies.     

Bioluminescence Spectra of Native and Mutant Firefly Luciferases as a Function of pH

Bioluminescence spectra of the wild-type recombinant Luciola mingrelica firefly luciferase and its mutant form with the His433Tyr point mutation were obtained within the pH 5.6-10.2 interval. The spectra are shown to be a superposition of the spectra of the three forms of the electronically excited reaction product oxyluciferin: ketone (lambdamax = 618 nm), enol (lambdamax = 587 nm), and enolate-ion (lambdamax = 556 nm). The shift in lambdamax by 40 nm to the red region in the mutant luciferase bioluminescence at the pH optimum of enzyme activity (pH 7.8) is explained by the change in the relative content of different oxyluciferin forms due to the shift in the ketone <--> enol <--> enolate equilibria. A computer model of the luciferase-oxyluciferin-AMP complex was constructed and the structure of amino acid residues participating in the equilibrium is proposed. Computer models of the protein region near the His433 residue for the wild type and mutant luciferases are also proposed. Comparison of the models shows that the His433Tyr mutation increases flexibility of the polypeptide loop that binds the N and C domains of luciferase. As a result, the flexibility of the C domain amino acid residues in the emitter microenvironment increases, and this increase may be the reason for the observed differences in the bioluminescence spectra of the native and mutant luciferases.     

A soluble biotinyl-alpha-amanitin affinity probe for the isolation of RNA polymerase B

Utilizing the 6'-hydroxyindole moiety of alpha-amanitin for substitution, biotinyl-alpha-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-alpha-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 X 10(-8) M as compared with a Ki of 5 X 10(-9) M for natural alpha-amanitin. RNA polymerase B complexed with biotinyl-alpha-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the anatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-alpha-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.     

类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑的酶促合成及表征

肌肽(β-Ala-L-His)是一种高效抗氧化剂,广泛应用于生物、化工、医药等领域。应用微水相酶促合成类肌肽,效率高价格低,且具有相似性质,开发前景广阔。本研究以L-丙氨酸和4,5-二羧酸咪唑制备类肌肽4(5)-丙氨酰胺-5(4)-羧酸咪唑,正交实验下的最佳合成条件为:四氢呋喃:pH8磷酸缓冲溶液=10:1.6(V/V),L-丙氨酸:4,5-二羧酸咪唑=1:3(m/m),α-胰凝乳蛋白酶:底物=1:200(m/m),35oC下磁力搅拌1.5h。硅胶G60薄层色谱(TLC)分离反应产物,Rf=0.81处出现新斑点;刮下该点纯化后进行紫外扫描,高效液相色谱(HPLC)和核磁共振,紫外光谱253nm处吸收明显增强,310nm处出现新吸收峰;253nm、310nm、330nm高效液相色谱保留时间均为4.0min;13C核磁共振显示8组碳原子。结合胰凝乳蛋白酶的催化机理,得出产物结构为4(5)-丙氨酰胺-5(4)-羧酸咪唑。     

Spectral tuning of obelin bioluminescence by mutations of Trp92

The Ca(2+)-regulated photoprotein obelin was substituted at Trp92 by His, Lys, Glu, and Arg. All mutants fold into stable conformations and produce bimodal bioluminescence spectra with enhanced contribution from a violet emission. The W92R mutant has an almost monomodal bioluminescence (lambdamax=390 nm) and monomodal fluorescence (lambdamax=425 nm) of the product. Results are interpreted by an excited state proton transfer mechanism involving the substituent side group and His22 in the binding cavity.     

The molecular basis of adaptive evolution of squirrelfish rhodopsins

The wavelengths of maximal absorption (lambdamax) of the rhodopsins of nine squirrelfishes (N. sammara, N. argenteus, S. punctatissimum, S. microstoma, S. diadema, S. xantherythrum, S. spiniferum, N. aurolineatus, and S. tiere) and two soldierfishes (M. violacea and M. berndti) vary between 481 and 502 nm. Phylogenetic and mutagenesis analyses suggest that the common ancestor of these pigments had a lambdamax value of approximately 493 nm, and the contemporary lambdamax values were generated mostly by amino acid replacements E122M, F261Y, and A292S. The probability of observing all these amino acid replacements at specific branches of the phylogenetic tree is only 2.5 x 10(-9); it is highly unlikely that these changes have occurred by neutral evolution. Because of a close association between the lambdamax values of these pigments and the wavelengths of light available to the corresponding species, the excess number of amino acid changes at specific branches in the phylogenetic tree strongly suggests that the rhodopsins have undergone adaptive changes at various stages of the holocentrid evolution.     

Isolation and partial characterization of the multiple forms of deoxyribonucleic acid-dependent ribonucleic acid polymerase in the fungus Podospora anserina

Three DNA-dependent RNA polymerases have been isolated and partially purified from the mycelium of the fungus Podospora anserina. Separated by DEAE-Sephadex chromatography, they have been designated RNA polymerases I, II, and III according to their order of elution. Their catalytic properties and alpha-amanitin sensitivity are in agreement with those of the homologous enzymes found in other eukaryotic organisms. The three enzymes exhibit rather sharp monophasic ammonium sulfate dependence with optima which are, respectively, 0.035 M, 0.050 M, and 0.075 M. Enzyme I has the largest Mn2+/Mg2+ activity ratio, shows a marked preference for native DNA, and is insensitive to alpha-amanitin. Enzyme III uses poly(dA-dT) in preference to native DNA as template and is only partially sensitive to alpha-amanitin. Enzyme II is sensitive to alpha-amanitin, but high concentrations of the toxin are required for inhibition compared to other eukaryotic class II enzymes. Three similar RNA polymerases with comparable levels of activity were found in the temperature-dependent VR strain when cellular incompatibility, leading to a rapid cessation of RNA synthesis, was induced.     

1H-NMR assignments of GM1-oligosaccharide in deuterated water at 500 MHz by two-dimensional spin-echo J-correlated spectroscopy

The ¹H-NMR spectra of the oligosaccharide derived from monosialoganglioside G_M1 (G_M1 = β-d-galactosyl-(1–3)-β-d-N-acetylgalactosaminyl-(1–4)-[α-N-acetylneuraminyl-(2–3)]-β-d-galactosyl-( 1–4)-β-d-glucosylceramide) (G_M1OS) and its reduced form (G_M1OS-R) have been obtained at 500 MHz in D₂O. Through the combined use of one-dimensional and homonuclear two-dimensional spin-echo J-correlated (2D SECSY) spectra of G_M1OS-R, the assignments for the ring protons of G_M1OS are made. Data on chemical shifts and coupling constants of G_M1OS including the α-linked neuraminic acid protons, in aqueous solution, are tabulated. Due to the very small coupling constants (<2 Hz) and the closeness in chemical shifts (<0.04 ppm) for the pair of correlated peaks in the two-dimensional spectrum, the information on the connectivities of the H5 ring protons of the neutral sugar residues is missing. Second-order coupling also blurs this information. Data are compared with those obtained for ganglioside G_M1 in dimethyl sulfoxide (DMSO;the actual composition therein was 97% DMSO-d₆ and 3% D₂O) by T.A.W. Koerner, J. H. Prestegard, P. C. Demou, and R. K. Yu (1983, Biochemistry22, 2676). While the heterogeneity of chemical shifts for the H5, H6a, and H6b protons diminishes in D₂O, that for A-9a and A-9b remains. The latter suggests an intraneuraminic acid conformation involving the glycerol side chain unaffected by the solvent. Moreover, the chemical shifts of the III-1, III-2, and A-4 protons (and perhaps the II-4, IV-2, and A-8 protons) in D₂O exhibit unusual upfield shifts compared with those in DMSO. This indicates that the intramolecular interactions between GalNAc residue III and neuraminic acid present in DMSO are weakened in D₂O. The effect of temperature on the conformation is also examined and appears to be minimal (<0.02 ppm) in the range 22–50 °C.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

Stereochemistry of the reactions of glutamate-1-semialdehyde aminomutase with 4,5-diaminovalerate

Conversion of glutamate 1-semialdehyde to the tetrapyrrole precursor, 5-aminolevulinate, takes place in an aminomutase-catalyzed reaction involving transformations at both the non-chiral C5 and the chiral C4 of the intermediate 4,5-diaminovalerate. Presented with racemic diaminovalerate and an excess of succinic semialdehyde, the enzyme catalyzes a transamination in which only the l-enantiomer is consumed. Simultaneously, equimolar 4-aminobutyrate and aminolevulinate are formed. The enzyme is also shown to transaminate aminolevulinate and 4-aminohexenoate to l-diaminovalerate as the exclusive amino product. The interaction of the enzyme with pure d- and l-enantiomers of diaminovalerate prepared by these reactions is described. Transamination of l-diaminovalerate yielded aminolevulinate quantitatively showing that reaction at the C5 amine does not occur significantly. A much slower transamination reaction was catalyzed with d-diaminovalerate as substrate. One product of this reaction, 4-aminobutyrate, was formed in the amount equal to that of the diaminovalerate consumed. Glutamate semialdehyde was deduced to be the other primary product and was also measured in significant amounts when a high concentration of the enzyme in its pyridoxal form was reacted with d-diaminovalerate in a single turnover. Single turnover reactions showed that both enantiomers of diaminovalerate converted the enzyme from its 420-nm absorbing pyridoxaldimine form to the 330-nm absorbing pyridoxamine via rapidly formed intermediates with different absorption spectra. The intermediate formed with l-DAVA (lambdamax = 420 nm) was deduced to be the protonated external aldimine with the 4-amino group. The intermediate formed with d-DAVA (lambdamax = 390 nm) was deduced to be the unprotonated external aldimine with the 5-amino group.     

The inability of the Psammechinus miliaris H3 RNA to be processed in the Xenopus oocyte is associated with sequences distinct from those highly conserved amongst sea urchin histone RNAs.

3' processing of precursors of the H3 RNA of the sea urchin Psammechinus miliaris in Xenopus oocytes is dependent upon sea urchin U7 snRNA. Sequences necessary for this interaction are highly conserved in all sea urchin histone precursor RNAs (including the Psammechinus H3) which, in contrast, are efficiently processed in the Xenopus oocyte without the addition of the homologous U7 snRNA. We resolve this seeming paradox by demonstrating here that the inability of the sea urchin Psammechinus miliaris H3 histone RNA to be processed in the Xenopus oocyte is associated with nucleotides immediately 3' to the conserved downstream sea urchin histone sequence element. Thus, a sequence-specific element (or lack of it) is responsible for the poor recognition of the Psammechinus H3 precursor RNA by the Xenopus processing machinery.     

Photochemical and chemical studies on the chromophore of bacteriorhodopsin.

The chromophore (purple complex) of bacteriorhodopsin is reduced by sodium borohydride upon illumination to RPhv with a three-peaked absorption band at 360 nm. Treatment of this reduction product with ultraviolet light or acid yields a modified product from which retro-retinyllysine can be obtained by alkaline hydrolysis. No reduction of the 412 nm complex was found. Under specific conditions the purple complex equilibrates with a photochemically active 460 nm form that can be reduced by borohydride in the dark. This reduction product RP460 behaves idential to RPHV. Reconstitution of the purple complex from chromophore-free membrane (apomembrane) and retinal occurs via intermediates. The first (lambdamax 400nm) shows a three-peaked absorption band and is reduced to RP400 without a change of the three-peaked absorption (lambdamax 360 nm). The same product is obtained from apomembrane and retinol. Detergents shift the absorption band to 330 nm in all cases. From the experiments described no participation of retro-retinal structures during the photochemical cycle can be concluded but stereospecific interaction of the retinal moiety with the protein resulting in a specific retinal conformation os omdocated by the spectral changes observed.     

Intercalation of aflatoxin B1 in two oligodeoxynucleotide adducts: comparative 1H NMR analysis of d(ATCAFBGAT).d(ATCGAT) and d(ATAFBGCAT)2

8,9-Dihydro-8-(N7-guanyl-[d(ATCGAT)])-9-hydroxyaflatoxin B1.d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1.8,9-dihydro-8-(N7-guanyl-[d(ATGCAT)])-9-hydroxyaflatoxin B1 were prepared by direct addition of afltoxin B1 8,9-epoxide to d(ATCGAT)2 and d(ATGCAT)2, respectively. In contrast to reaction of aflatoxin B1 8,9-epoxide with d(ATCGAT)2 which exhibits a limiting stoichiometry of 1:1 aflatoxin B1:d(ATCGAT)2 [Gopalakrishnan, S., Stone, M. P., & Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239], reaction of aflatoxin B1 8,9-epoxide with d(ATGCAT)2 exhibits a limiting stoichiometry of 2:1 aflatoxin B1:d(ATGCAT)2. 1H NOE experiments, nonselective 1H T1 relaxation measurements, and 1H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5'-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide 1H NOEs are observed between aflatoxin and the 5'-neighbor base pair and include both the major groove and the minor groove. The aflatoxin methoxy and cyclopentenone ring protons face into the minor groove; the furofuran ring protons face into the major groove. No NOE is observed between the imino proton of the modified base pair and the imino proton of the 5'-neighbor base pair; sequential NOEs between nucleotide base and deoxyribose protons are interrupted in both oligodeoxynucleotide strands on the 5'-side of the modified guanine. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons; chemical shift perturbations of the oligodeoxynucleotide protons are confined to the immediate vicinity of the adducted base pair. The imidazole proton of the modified guanine exchanges with water and is observed at 9.75 ppm. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B1 8,9-epoxide and B-DNA. Insertion of aflatoxin B1-8,9 epoxide above the 5'-face of guanine in d(ATCGAT)2 would prevent the binding of a second molecule of aflatoxin B1 8,9-epoxide. In contrast, two intercalation sites would be available with d(ATGCAT)2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant expression and initial characterization of the putative human enteric ferric reductase Dcytb

Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.