Biomedical subjects

F A Walker

Publications and source records attributed to F A Walker.

At least 37 records · Page 2Linked to original sources

PubMed1994

High affinity histamine-binding and antihistaminic activity of the salivary nitric oxide-carrying heme protein (nitrophorin) of Rhodnius prolixus.

The salivary glands of Rhodnius prolixus contain a nitrosyl-heme protein, named nitrophorin, that releases the vasodilatory and antiplatelet compound nitric oxide (NO). Because imidazole compounds such as histamine can interact with Fe(III) heme proteins, we investigated whether such substances could interact with Rhodnius nitrophorins. Both imidazole and histamine, but not histidine can produce full of the difference spectra of the Soret band in the 1-3 microM concentration range (at a heme protein concentration of 0.4 microM). The apparent K0.5 for the binding of histamine with the heme protein is below 1 microM. Furthermore, the complex histamine-heme protein does not dissociate after molecular sieving chromatography. To investigate whether histamine could displace NO from the native nitrosyl nitrophorins, histamine was added to the native heme proteins, leading to displacement of the bound NO as observed by changes in the absorption spectra as well as by the production of nitrite. Finally, the antihistamine effect of the heme protein was demonstrated by its inhibition of the histamine-provoked contractures of the guinea pig ileum. It is concluded that histamine, a common autacoid found at the site of injury and exposure to antigenic substances such as the site of feeding by hematophagous arthropods, can be scavenged by the nitrosyl nitrophorin of R. prolixus, which, in return, will release the vasodilatory and platelet inhibiting NO to counteract the host hemostatic response.

Animals↗

PubMed1994

Hydration and localization of diacylglycerol in the insect lipoprotein lipophorin. A 13C-NMR study.

In order to probe the organization of diacylglycerol (DG) in lipophorin, 13C-enriched lipophorin was prepared for NMR investigations. We obtained 13C-enriched lipophorin labeled exclusively in DG by feeding insects tobacco leaves coated with [1-13C]palmitic acid or [1-13C]oleic acid. Lipophorins enriched up to 5% with a [13C]fatty acid were obtained by this procedure. NMR studies of the isolated lipophorin DG showed that palmitic acid accumulates almost entirely (> 90%) in the sn-1 position. Oleic acid was found equally distributed between the sn-1 and sn-2 positions, yielding a DG enriched equally at both positions. The 13C-NMR spectra of both [13C]palmitate- and [13C]oleate-enriched lipophorins showed that DG had one narrow carbonyl resonance indicative of rapid motion. A comparative analysis of the 13C carbonyl chemical shift data for DG in organic solvents, aqueous solutions, and dispersions with the DG carbonyl chemical shift of native lipophorin enriched in [13C]palmitate or [13C]oleate shows a high degree of water exclusion from the DG carbonyls in lipophorin. This result is consistent with the existence of a lipophorin lipid core containing most of the lipophorin DG. This study represents the first attempt to elucidate the organization of DG in lipophorin. The possibility of obtaining [13C]DG-enriched lipophorins, selectively enriched in one or both acyl chains of DG, should provide a powerful tool for further analysis of the organization and the dynamic properties of DG in native lipoproteins.

Animals↗

PubMed1994

Cation-promoted cyclic voltammetry of recombinant rat outer mitochondrial membrane cytochrome b5 at a gold electrode modified with beta-mercaptopropionic acid.

Reversible cyclic voltammetry of recombinant rat outer membrane (OM) cytochrome b5 was observed at a gold electrode modified with beta-mercaptopropionic acid. Electron transfer between the negative electrode surface and the negatively charged OM cytochrome b5 was promoted by the addition of divalent metal ions such as Mg2+ or Ca2+ and by the positively charged species poly-L-lysine. The titration of OM cytochrome b5 (0.1 mM) with poly-L-lysine resulted in a gradual positive shift of the E1/2 value which leveled off at +8 mV vs NHE when the poly-L-lysine:cytochrome b5 ratio reached a value of 2:1. Since the further addition of poly-L-lysine had no effect on the E1/2 value of the protein, it was concluded that a complex is formed in which two molecules of poly-L-lysine bind to each molecule of OM cytochrome b5. When the OM cytochrome b5-poly-L-lysine complex (0.1 mM) was titrated with Mg2+ or Ca2+ ions, the E1/2 value shifted gradually in the negative direction and leveled off at -40 mV vs NHE when the concentration of divalent ions reached 85 mM. When the voltammetric response of 0.1 mM cytochrome b5 was promoted by Mg2+ or Ca2+ ions, the minimum concentration of divalent cation necessary to produce a reversible voltammogram was 40 mM and the observed E1/2 was -46 mV vs NHE. On the other hand, only 0.2 mM [Cr(NH3)6]3+ was necessary to promote the reversible electrochemistry of 0.1 mM cytochrome b5. The half-wave potential observed under these conditions was -78 mV vs NHE. This indicates that there is a large dependence of the reduction potential of cytochromes b5 on the kind and concentration of multivalent ions in solution. A reduction potential of -102 mV vs NHE was obtained for OM cytochrome b5 (0.60 mM) by spectroelectrochemical titration in the presence of 0.4 mM [Ru(NH3)6]3+ and 1 mM methyl viologen, pH 7.0, mu = 0.1 M. This value is approximately 100 mV more negative than the reduction potentials reported for microsomal cytochromes b5 obtained from other sources under the same conditions. The binding interactions between OM cytochrome b5 and poly-L-lysine or Mg2+ ions were probed by investigating the isotropically shifted 1H NMR resonances arising from the heme in the OM cytochrome b5.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Mercaptopropionic Acid↗

PubMed1993

Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect.

The bloodsucking bug Rhodnius prolixus has a salivary vasodilator, previously characterized as a nitrovasodilator, with salivary smooth muscle-relaxing and antiplatelet activity. Rhodnius salivary glands are bright red owing to the abundance of heme proteins. Electron paramagnetic resonance and optical spectroscopic experiments indicated that the salivary vasodilator is a nitrosylheme protein with an Fe(III) heme that binds nitric oxide (NO) reversibly. Dilution of the protein in neutral pH promoted NO release. This protein thus appears to be the NO carrier that helps R. prolixus to feed on blood.

Animals↗

PubMed1993

Laser flash photolysis studies of electron transfer to the cytochrome b5-cytochrome c complex.

Rate constants for electron transfer in the complex between recombinant rat mitochondrial outer membrane cytochrome b5 or the tryptic fragment of bovine liver cytochrome b5 and horse mitochondrial cytochrome c were measured by laser flash photolysis of 5-deazariboflavin-EDTA solutions. When an excess of cytochrome b5 was titrated with increasing amounts of cytochrome c at low ionic strength and electron transfer was initiated by a laser flash, both proteins were rapidly reduced by deazariboflavin semiquinone. The initial photoreduction was followed by a slower second-order reduction of b5 complexed oxidized cytochrome c by free reduced cytochrome b5. At an 8:1 ratio of cytochromes b5 to c, the pseudo-first-order rate constant for reduction of complexed cytochrome c increased 3-5-fold between ionic strengths of 5 and 40 mM, and then dropped precipitously at higher ionic strengths. The ionic strength dependent increase in rate constant is likely to be due to relief of steric hindrance via rearrangement of cytochrome c in the complex. The reaction rate showed no sign of saturation at any ionic strength, indicating a first-order rate constant greater than 10(4) s-1 within a transient ternary protein complex; i.e., interprotein electron transfer approaches the largest values previously reported for the stable binary protein complex (approximately 4 x 10(5) s-1). Our results emphasize the flexibility of electron-transfer protein complexes, which had previously been modeled in a single conformation with specific salt bridges. It appears that a variety of orientations can exist within such protein-protein complexes and that the population of conformations changes with ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5.

The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.

Amino Acid Sequence↗

PubMed1991

1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: evidence for S = 1/2 in equilibrium S = 5/2 spin equilibrium for intact His/Met ligation.

The solution 500-MHz 1H NMR spectral parameters for ferricytochrome b562, a soluble 12-kDa electron carrier from Escherichia coli with axial His/Met coordination, are shown to be strongly influenced by protein concentration and ionic strength at low pH and 25 degrees C in a manner consistent with significant aggregation at low ionic strength. At high ionic strength a well-resolved 1H NMR spectrum reveals over 40 hyperfine-shifted resonances which arise from two isomeric species in the ratio 2:1. 2D COSY and NOESY maps at 25 degrees C for the hyperfine-shifted resonances allow the assignment of a number of axial His resonances and all heme peripheral substituent peaks. The resulting asymmetric heme contact shift patterns, together with the halving of the number of lines when reconstituting with 2-fold symmetric hemin, demonstrate the molecular basis of the solution heterogeneity to be heme orientational disorder. The strongly upfield-shifted axial Met-7 resonances, characteristic of low-spin ferricytochromes c with His/Met ligation, appear upfield only at very low temperatures. At elevated temperatures, all resonances, in particular those of the axial Met, move strongly downfield. Detailed analysis of the deviation from Curie behavior for different functional groups demonstrates the presence of a low spin in equilibrium high spin equilibrium with an intact His-Fe-Met coordination. The weaker axial field in ferricytochrome b562, relative to the purely low-spin ferricytochromes c, is attributed to a perturbed iron-Met bond. The contact shifts for a coordinated Met in the high-spin state are estimated. A link between equatorial hemin and axial ligand interactions is indicated by a differential population of the high-spin form for the two hemin orientations.

Cytochrome b Group↗

PubMed1984

Considerations in determining cost effectiveness in psychiatric rehabilitation.

Over the past decade there has been an increasing tendency by mental health professionals and economists to evaluate programs in terms of costs and benefits. The current paper deals with some of the more important issues which should be accounted for when contemplating undertaking a cost benefit analysis or a cost effectiveness exercise in the area of psychiatric rehabilitation.

Community Mental Health Services↗

PubMed1979