Biomedical subjects

M J Barber

Publications and source records attributed to M J Barber.

At least 37 records · Page 2Linked to original sources

PubMed1999

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol.

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.

Adenylate Kinase↗

PubMed1999

Activity-driven synapse elimination leads paradoxically to domination by inactive neurons.

In early postnatal life, multiple motor axons converge at individual neuromuscular junctions. However, during the first few weeks after birth, a competitive mechanism eliminates all the inputs but one. This phenomenon, known as synapse elimination, is thought to result from competition based on interaxonal differences in patterns or levels of activity (for review, see Lichtman,1995). Surprisingly, experimental data support two opposite views of the role of activity: that active axons have a competitive advantage (Ribchester and Taxt, 1983; Ridge and Betz, 1984; Balice-Gordon and Lichtman, 1994) and that inactive axons have a competitive advantage (Callaway et al., 1987, 1989). To understand this paradox, we have formulated a mathematical model of activity-mediated synapse elimination. We assume that the total amount of transmitter released, rather than the frequency of release, mediates synaptic competition. We further assume that the total synaptic area that a neuron can support is metabolically constrained by its activity level and size. This model resolves the paradox by showing that a competitive advantage of higher frequency axons early in development is overcome at later stages by greater synaptic efficacy of axons firing at a lower rate. This model both provides results consistent with experiments in which activity has been manipulated and an explanation for the origin of the size principle (Henneman, 1985).

Aging↗

PubMed1998

Crystallographic study of azurin from Pseudomonas putida.

Azurin from Pseudomonas putida is a blue copper protein which functions as an electron carrier. Two crystal forms of azurin were grown, one in the presence and the other in the absence of zinc acetate; each belongs to space group P21 and contains two molecules per asymmetric unit. The zinc-free crystals have cell dimensions a = 43.25, b = 50.65, c = 54.60 A, beta = 107.79 degrees, while the crystals grown from zinc-containing solution have cell dimensions a = 40.76, b = 51.22, c = 54.96 A, beta = 103.12 degrees. The latter crystals were found to have four zinc ions incorporated into the crystal lattice. Both crystal structures were solved by the molecular-replacement method using the program MERLOT. The search model was the structure of azurin from Alcaligenes denitrificans. The crystallographic R factor for native azurin is 0.169 (Rfree = 0. 257) from 8 to 1.92 A resolution, while that for zinc azurin is 0. 181 (Rfree = 0.248) from 10 to 1.6 A resolution; for each structure the root-mean-square deviation in bond lengths from ideal values is 0.007 A. In both crystal structures the Cu atom forms three strong bonds in the equatorial plane, two with Ndelta1 from His46 and His117, and one with the thiolate S atom of Cys112. Two longer axial approaches are made by the Sgamma from Met121 and the carbonyl O atom from Gly45. This results in a distorted trigonal bipyramidal co-ordination around the Cu atom. It further confirms the presence of a weak fifth bond to the copper in P. putida azurin, as with other azurin structures described at high resolution. The Ndelta1 atom of His35 is protonated, as it is in the low-pH form of azurin from Pseudomonas aeruginosa but unlike the low-pH form of the azurins from Alcaligenes denitrificans or Alcaligenes xylosoxidans. In each crystal form the two molecules of azurin in the asymmetric unit are related by a local twofold axis and form a dimer stabilized by the interaction of a pair of hydrophobic patches surrounding the partially exposed His117 side chain. In the other known azurin crystal structures, analogous dimer formation is observed, but with different relative orientations of the molecules. The four zinc ions introduced during crystallization of zinc azurin are bound to the protein and participate in five- and sixfold ligand coordination with no affect on the copper binding site. The zinc ligands are Ndelta from His, carboxylate O atoms from Asp and Glu, Ogamma from Ser and water molecules. One of the zinc ions, located on a non-crystallographic twofold axis, links the dimers of the asymmetric unit into continuous chains parallel to the crystallographic (-101) direction and is primarily responsible for the altered unit-cell parameters. Two of the other zinc ions bind to His83, one in each molecule.

Amino Acid Sequence↗

PubMed1998

The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase.

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes. It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor. Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases. Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme. To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis. The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold. The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively. The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner. Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively). Further, Y121F and Y121H proved less thermostable than the wild type. Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.

5-Aminolevulinate Synthetase↗

PubMed1997

Direct electrochemistry of the flavin domain of assimilatory nitrate reductase: effects of NAD+ and NAD+ analogs.

Direct electrochemical studies, utilizing two voltammetric methods-square-wave voltammetry (SWV) and cyclic voltammetry (CV)-have been performed on recombinant forms of the flavin domain of spinach assimilatory nitrate reductase in the presence of NAD+ analogs. The reduction potentials (E degrees ') of the flavin domains have been determined at an edge pyrolytic graphite electrode utilizing MgCl2 as a redox-inactive promoter. Under identical experimental conditions (pH 7.0, 25 degrees C), the two-electron reduction potential for the FAD/FADH2 couple has been determined to be -274 and -257 mV by SWV and CV, respectively. In contrast, the reduction potentials of free FAD have been determined to be -234 and -227 mV by SWV and CV, respectively. The reduction potentials of the complex formed between the FAD prosthetic group in the recombinant flavin domain and various NAD+ analogs have been determined to be as follows: NAD+ (E degrees ' = -192 mV), 5'-ADP ribose (E degrees ' = -199 mV), ADP (E degrees ' = -154 mV), AMP (E degrees ' = -196 mV), adenosine (E degrees ' = -192 mV), adenine (E degrees ' = -220 mV), and NMN (E degrees ' = -208 mV). In contrast to these positive shifts in reduction potential, nicotinamide (E degrees ' = -268 mV) had very little effect on the reduction potential of this flavin complex. Moreover, addition of NAD+ to the FAD prosthetic group in a variety of mutant forms of the recombinant flavin domain resulted in positive shifts in the reduction potential of the complex, although the magnitude of the shifts varied from a minimum of 6 mV obtained for the C240A mutant to a maximum of 79 mV obtained for the C62S mutant. These results represent the first extensive application of direct electrochemistry to examine the redox properties of assimilatory nitrate reductase and indicate that complex formation with NAD+, or various NAD+ analogs, results in a positive shift in the flavin reduction potential, with the magnitude of the shift correlating well with the efficiency of the inhibitor.

Adenosine Diphosphate↗

PubMed1997

Biotin sulfoxide reductase. Heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 micromol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 microM for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.

Chromatography, High Pressure Liquid↗

PubMed1996

Mutations at a glycine loop in aminolevulinate synthase affect pyridoxal phosphate cofactor binding and catalysis.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in animals, fungi, and some bacteria. The enzyme belongs to a large family of enzymes that use pyridoxal 5'-phosphate as an essential cofactor. We previously analyzed the informational content contained in each residue of a conserved glycine loop, which we proposed to form part of the cofactor binding site [Gong, J., & Ferreira, G. C. (1995) Biochemistry 34, 1678-1685]. We found that Gly-142 and -144 contain high informational content, and we identified G144A, G144S, G144T, and G142C as functional mutants. Here, the catalytic parameters, cofactor affinities, and spectral and thermostability properties of these four glycine mutants are determined to examine the function of the glycine loop. In addition, computer models of the glycine loops from the wild-type and mutant enzymes were generated, using glycogen phosphorylase b as the structural template. G144A, G144S, G144T, and G142C displayed lower affinity than the wild-type enzyme for the cofactor, reflected in the 8.5-, 8-, 24.5-, and 15-fold increases, respectively, in the dissociation constant value for binding of the cofactor. While the turnover numbers for G144A, G144S, G144T, and G142C were 43%, 39%, 21%, and 6% of the wild-type value, respectively, the K(m) values for both substrates remained unchanged, with the exception of the G142C K(m)Gly, which showed a 4-fold increase. The UV-visible and CD spectra of Gly-144 mutants were similar to those of the wild type; however, the spectral properties of G142C suggest that this mutant binds the cofactor in a different mode at the active site. G144A, G144S, G144T, and G142C were also found to be less stable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 3.5, 3, 3.5, and 5 degrees C, respectively, lower than that of the wild-type enzyme. Collectively, computer modeling of the wild-type and mutant forms of the ALAS glycine loop and biochemical and spectroscopic characterization of G144A, G144S, G144T, and G142C strongly suggest that the conserved glycine loop in 5-aminolevulinate synthase is a pyridoxal 5'-phosphate cofactor binding motif.

5-Aminolevulinate Synthetase↗

PubMed1996

Thiol modification and site directed mutagenesis of the flavin domain of spinach NADH:nitrate reductase.

Incubation of either Chlorella nitrate reductase or the recombinant flavin domain of spinach nitrate reductase with reagents specific for modification of cysteine residues, such as N-ethylmaleimide, resulted in a time-dependent inactivation of NADH:ferricyanide reductase activity which could be prevented by incubation in the presence of NADH. At 25 degrees C and employing a fixed enzyme:modifier ratio, the rate of inactivation for both the Chlorella and spinach enzymes followed the order p-chloromercuribenzoate > methyl methanethiosulfonate > 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid > N-ethylmaleimide. For the spinach flavin domain, inactivation by methyl methanethiosulfonate or p-chloromercuribenzoate was found to be concentration independent suggesting the absence of nonspecific modifications. Initial rate studies of the methyl methanethiosulfonate-modified flavin domain indicated a reduction in NADH:ferricyanide activity (Vmax) from 85 to 44 micromol NADH consumed/min/nmol FAD and an increase in the Km for NADH from 12 to 35 microM when compared to the native enzyme, confirming a role for cysteine residue(s) in maintaining diaphorase activity. Site-directed mutagenesis of the four individual cysteines (residues 17, 54, 62, and 240) in the recombinant spinach flavin domain resulted in mutant proteins with visible and CD spectra very similar to those of the wild-type domain. Initial rate studies indicated that only substitutions of serine for cysteine 240 decreased diaphorase activity with maximal NADH:ferricyanide activity for the C240S mutant corresponding to 51 micromol NADH consumed/min/nmol FAD with a Km for NADH of 14 microM. Mutation of C240 to Ala or Gly resulted in greater loss of activity. The thermal stability of the four serine mutants was slightly decreased compared to the wild-type domain with the C62S mutant exhibiting the greatest instability. In contrast to the effects on diaphorase activity, square wave voltammetric studies indicated changes in the oxidation-reduction midpoint potential for the FAD/FADH2 couple in the C54S (E0'= -197 mV), C62S (E0' = -226 mV), and C240S (E0' = -219 mV) mutants compared to the wild-type domain (E0' = -268 mV). These results indicate that of the four cysteine residues in the spinach nitrate reductase flavin domain, only C240 plays a role in maintaining diaphorase activity, while C54 has the greatest influence on flavin redox potential and that no correlation between changes in catalytic activity and flavin redox potential was observed.

Amino Acid Sequence↗

PubMed1996

Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.

Amino Acid Sequence↗

PubMed1996

Superoxide production during reduction of molecular oxygen by assimilatory nitrate reductase.

Assimilatory NADH:nitrate reductase catalyzes the transfer of reducing equivalents from NADH to molecular oxygen. Initial rate studies performed under conditions of optimal pH (8.0) and constant ionic strength (mu = 0.2) revealed that the maximal rate of activity with molecular oxygen was 0.5% (0.44 mumol NADH consumed/min/nmol heme) with a Km for O2 of 586 microM. NADH:molecular oxygen reductase activity exhibited a pH optimum of 9.2, was inhibited by cyanide, and was unaffected by changes in ionic strength or the presence of phosphate ions. Spectroscopic studies indicated NADH:molecular oxygen reductase activity resulted in the production of the superoxide radical, detected as the formation of adrenochrome from epinephrine and by the formation of adrenochrome from epinephrine and by the reduction of nitroblue tetrazolium, both of which could be inhibited by the addition of superoxide dismutase and were unaffected by the addition of catalase. Direct observation of superoxide production using spin-trapping in combination with EPR spectroscopy resulted in the detection of the spin adduct 5.5-dimethyl-5-hydroxy-1-pyrrolidinyloxy (DMPO-OH). The formation of this spin adduct was abolished either in the absence of nitrate reductase, NADH, or DMPO or the the addition of superoxide dismutase or nitrate and was greatly reduced by the presence of cyanide. Inclusion of catalase or ethanol had no effect on the formation of the spin adduct. These results indicate that nitrate reductase can utilize molecular oxygen as an electron acceptor and that the product, O2.(-), is primarily generated via the Mopterin center.

Chlorella↗

PubMed1996

High-level expression in Escherichia coli of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase.

A T7 expression system has been produced for the high-level production of the soluble, catalytic domain of rat hepatic cytochrome b5 reductase in Escherichia coli. The recombinant protein was purified to homogeneity using affinity chromatography on 5'-ADP agarose and gel exclusion chromatography and exhibited a molecular mass of approximately 30 kDa by polyacrylamide gel electrophoresis and a molecular mass of 30,588 by mass spectrometry. Direct sequencing of the initial 12 residues of the amino-terminus of the purified domain yielded the sequence MITLENPDIKYP, identical to that predicted from the DNA sequence. The domain incorporated a full complement of FAD with a visible absorption spectrum typical of a flavoprotein exhibiting maxima at 389 and 461 nm and a distinct shoulder at 485 nm. Addition of NADH to the protein resulted in an extensive bleaching of the visible spectrum. The recombinant domain retained both NADH:ferricyanide and NADH:cytochrome b5 reductase activities with Vmax of 48 and 26 micromol NADH consumed/min/nmol FAD, respectively, and Km of 6, 7, and 11 microM for NADH, ferricyanide, and cytochrome b5. Comparison of the activities obtained using NADH and NADPH indicated a substantial preference for NADH as the reducing substrate. The results indicate that the recombinant protein retains the physical and catalytic properties of the native protein and represents an excellent system for probing the role of specific amino acid residues using site-directed mutagenesis.

Animals↗

PubMed1995

The amino acid sequence of Rhodobacter sphaeroides dimethyl sulfoxide reductase.

The complete amino acid sequence of the soluble, monomeric molybdenum-containing enzyme dimethyl sulfoxide reductase from Rhodobacter sphaeroides f sp. denitrificans has been determined using a combination of gas-phase Edman sequencing of isolated peptides and direct sequencing of PCR products generated from R. sphaeroides genomic DNA. The protein comprises 777 residues corresponding to an apoenzyme molecular weight of 84,748 Da. The amino acid sequence was rich in Ala and Gly residues which represented 21% of the protein's composition. The DNA sequence was 67% rich in G and C nucleotides. The amino acid sequence contained 10 cysteine residues which were relatively evenly distributed throughout the sequence and featured regions of sequence corresponding to the prokaryotic molybdopterin-binding signatures 2 and 3. While exhibiting limited sequence similarity to the corresponding membrane-bound molybdenum-containing subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase, the Rhodobacter sequence showed extensive sequence similarity to that of the E. coli molybdoprotein, trimethylamine N-oxide reductase (torA). Comparison with other related prokaryotic molybdenum-containing enzymes indicated the presence of two highly conserved cysteine residues (Cys-268 and Cys-616) which may function in molybdenum coordination.

Amino Acid Sequence↗

PubMed1995

Detection of hydroxyl radicals upon interaction of ozone with aqueous media or extracellular surfactant: the role of trace iron.

As part of a study on mechanisms modulating ozone-induced surfactant perturbations, we used the electron paramagnetic resonance (EPR) spin trapping technique to determine the type and origin of radicals generated following interaction of ozone with aqueous solutions and cell-free bronchoalveolar lavage fluid (BAL) fractions. All aqueous media were exposed to ozone at 25 degrees C with or without added chelator, 1 mM diethylenetriaminepentaacetic acid, and spintrap, 100 mM 5,5'-dimethyl-1-pyrroline-1-oxide (DMPO). Exposure of distilled water to 0.5, 1.0, 2.0, and 3.0 ppm ozone for 1 h yielded four-line spectra, 1:2:2:1, consistent with hydroxyl radical adduct formation (DMPO-OH), the amplitudes of which increased with the ozone concentration. No signals were obtained from air-exposed samples. Similar four-line spectra were also produced following interaction of 3 ppm ozone with Hank's balanced salt solution (HBSS) alone or containing BAL fractions. Addition of the hydroxyl radical scavenger dimethyl sulfoxide (DMSO) to the incubation medium strongly inhibited formation of DMPO-OH adduct during ozone exposure. As an alternate method of demonstrating the generation of hydroxyl radicals, aqueous solutions of 1 mM L-phenylalanine were exposed to high concentrations of ozone and shown, using ion-exchange chromatography, to contain small amounts of L-tyrosine. Production of hydroxyl radicals upon interaction of ozone and water was further substantiated using the spintrap PBN (phenyl-N-tert-butylnitrone) in the presence of DMSO which reacts with the hydroxyl radical resulting in the formation of methyl radical. The methyl radical subsequently reacts with spintrap PBN, yielding PBN-methyl adduct. In the absence of DMSO there was no detectable formation of methyl radical adduct. EPR double distilled water containing DMPO showed a small amount of DMPO-OH adduct upon exposure to ozone. Addition of 10 microM ferrous sulfate to this mixture produced a 10-fold increase of the signal, which was attenuated in the presence of 1500 U catalase, strongly attenuated with 50-500 microM deferoxamine or 8000 U catalase and abolished by higher concentration of deferoxamine (1 mM). The signal was not influenced by 1000 U superoxide dismutase. These results indicate that hydroxyl radicals are produced via iron-dependent reactions during the initial interaction of ozone with aqueous media, including bronchoalveolar fluid.

Animals↗

PubMed1995

Molecular cloning and expression of biotin sulfoxide reductase from Rhodobacter sphaeroides forma sp. denitrificans.

Biotin sulfoxide reductase catalyzes the conversion of d-biotin d-sulfoxide (BSO) to d-biotin. Oligonucleotides directed against common sequences in Escherichia coli biotin sulfoxide reductase and in Rhodobacter sphaeroides f.sp. denitrificans dimethyl sulfoxide reductase have been utilized to amplify by PCR a 651-bp fragment from R. sphaeroides total genomic DNA that showed a high degree of sequence similarity with both E. coli biotin sulfoxide reductase and R. sphaeroides dimethyl sulfoxide reductase. Screening of a genomic cosmid library, prepared from R. sphaeroides genomic DNA, with this probe resulted in the isolation of a 7-kb EcoRI-EcoRI fragment that contained the complete coding region for R. sphaeroides BSO reductase which has been sequenced. The sequence data indicated a single open reading frame of 2231 nucleotides encoding a protein of 744 amino acid residues corresponding to a subunit molecular weight of 80,234 Da. The translated protein sequence contained the prokaryotic Mo-pterin signatures 2 and 3 (Mo-cofactor binding motifs) and a ATP/GTP-binding P-loop. The R. sphaeroides BSO reductase sequence showed 51% sequence similarity with the corresponding E. coli enzyme. In addition, there were only two conserved cysteines between the two BSO reductase sequences. The R. sphaeroides gene was demonstrated, by complementation, to rescue a mutant E. coli strain that was deficient in BSO reductase when grown on BSO as the sole source of biotin. When expressed from the FLAG*Shift 12c expression vector, in the presence of IPTG, the BSO reductase gene encoded a protein of approximately 80 kDa, which cross-reacted with the anti-FLAG monoclonal antibody and exhibited BSO reductase activity by the disk microbiological assay.

Amino Acid Sequence↗

PubMed1995

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

5-Aminolevulinate Synthetase↗

PubMed1995

0.0625% bupivacaine with 0.0002% fentanyl via patient-controlled epidural analgesia for pain of labor and delivery.

OBJECTIVE: To compare the utility of 0.0625% bupivacaine with fentanyl administered via patient-controlled epidural analgesia (PCEA) to a traditional continuous epidural infusion for pain of labor and delivery. DESIGN AND SUBJECTS: Forty-one women in established labor were randomized to receive either (a) 0.0625% bupivacaine with 2 micrograms/ml fentanyl via PCEA (demand dose = 3 ml, lockout interval = 6 min, background infusion = 6 ml/h, no 1 or 4 h limits) or (b) 0.125% bupivacaine with 2 micrograms/ml fentanyl via continuous epidural infusion (CEI) at 12 ml/h. Supplemental 0.25% bupivacaine (3 ml every 5 min, p.r.n., x 3) was administered for treatment of breakthrough pain upon patient request. The study protocol was double-blind and placebo-controlled. OUTCOME MEASURES: Visual analogue pain scores, motor strength, pinprick level of sensory analgesia and bupivacaine use were assessed by an anesthesiologist unaware of the individual patient's randomization to a particular study group. RESULTS: The cephalad extent of pinprick sensory analgesia was significantly lower during both the first (p < 0.03) and second (p < 0.03) stages of labor in patients receiving PCEA. However, visual analogue pain scores, intensity of motor blockade, and need for physician-administered supplemental bupivacaine were comparable in both groups. Patients receiving PCEA used 40% less bupivacaine per hour while achieving analgesia comparable to patients receiving CEI. CONCLUSIONS: The results of this study show that 0.0625% bupivacaine with 2 micrograms/ml of fentanyl is an effective analgesic combination when used via PCEA.

Adult↗

PubMed1994

Assimilatory nitrate reductase: reduction and inhibition by NADH/NAD+ analogs.

Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the rate-limiting step, the conversion of nitrate to nitrite, in nitrate assimilation. Initial rate studies of nitrate reductase activity, performed under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substrate inhibition at NADH concentrations below 300 microM and NO3- concentrations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (Vmax = 9.2 mumol NADH/min/nmol heme and Km = 2.3 microM) as the physiological electron donor but could also utilize alpha-NADH (Vmax = 5.6 mumol NADH/min/nmol heme and Km = 131 microM) and NADPH (Vmax = 0.6 mumol NADPH/min/nmol heme and Km = 910 microM) though with significantly decreased efficiency. Examination of various NADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleotide (NHDH) was used most efficiently (Vmax = 9.3 mumol NHDH/min/nmol heme and Km = 7.9 microM), while reduced nicotinamide mononucleotide (NMNH) was utilized least efficiently (Vmax = 0.07 mumol NMNH/min/nmol heme and Km = 676 microM). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result in the most significant decreases in Vmax, indicating poor reducing substrates. Product inhibition studies indicated both NAD+ (Ki = 2.2 mM) and NADP+ (Ki = 10.5 mM) to be competitive inhibitors of Chlorella NR. A variety of NAD+ analogs were also determined to act as competitive inhibitors with varying degrees of efficiency. 3-Pyridinealdehyde adenine dinucleotide was the most efficient inhibitor (Ki = 0.74 mM) while nicotinamide was the least efficient (Ki = 18.1 mM). Overall, changing substituents on the nicotinamide ring or its complete deletion produced the most effective inhibitors compared to NAD+. In contrast, changes in the adenine or ribose moieties produced less effective inhibitors when compared to NAD+. These results represent the most comprehensive analysis of the effect of modifications of the physiological reductant (NADH) and product (NAD+) on nitrate reductase activity.

Binding, Competitive↗

PubMed1994

Construction and expression of a flavocytochrome b5 chimera.

A gene has been constructed coding for a chimeric flavocytochrome b5 protein that comprises the soluble domain of rat hepatic cytochrome b5 as the NH2-terminal portion of the chimera and the flavin-containing domain of spinach assimilatory NADH:nitrate reductase as the C terminus. The chimeric protein has been expressed in Escherichia coli and purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose, anion-exchange chromatography, and fast protein liquid chromatography gel filtration with an estimated molecular mass of 43 kDa from polyacrylamide gel electrophoresis. Visible and fluorescence spectroscopy indicated the purified protein contained both a b-type cytochrome and FAD prosthetic groups. The chimeric hemoflavoprotein immunologically cross-reacted with both anti-rat cytochrome b5 and anti-spinach nitrate reductase polyclonal antibodies, indicating the conservation of antigenic determinants from both native domains. NH2-terminal and internal amino acid sequencing of the native and CNBr-digested protein confirmed the presence of peptides derived from both the heme- and flavin-binding portions of the sequence which were identical to the deduced amino acid sequence. The chimera exhibited both NADH: ferricyanide reductase and NADH:cytochrome c reductase activities with Vmax values of 88 and 37 mumol of NADH consumed per min/nmol of heme (mu = 0.05 and pH 7.0) and Km values of 2.1, 32, and 1.4 microM for NADH, ferricyanide, and cytochrome c, respectively. This work represents the first successful bacterial expression of a mammalian-plant chimeric metalloflavoprotein. The chimera exhibited properties extremely similar to those of the native cytochrome b5 heme and spinach nitrate reductase FAD components.

Amino Acid Sequence↗