Biomedical subjects

J K Sanders

Publications and source records attributed to J K Sanders.

36 records · Page 2Linked to original sources

PubMed1989

In vivo enzymology: a deuterium NMR study of formaldehyde dismutase in Pseudomonas putida F61a and Staphylococcus aureus.

High-resolution deuterium NMR spectroscopy has been used to follow the detoxifying metabolism of [D2]formaldehyde in vivo in several bacterial species. Production of [D2]methanol in Escherichia coli confirms that the oxidation and reduction pathways of metabolism are independent in this organism. Efficient production of equimolar quantities of [D]formate and [D3]methanol in Pseudomonas putida F61a and Staphylococcus aureus implicates a formaldehyde dismutase, or "cannizzarase", activity. These observations imply that the unusual formaldehyde resistance in P. putida F61a is a direct result of efficient dismutation acting as a route for detoxification. Cross-dismutation experiments yield an enzymic kinetic isotope effect of ca. 4 for H vs D transfer and a similar spectrum of substrate specificity to the isolated enzyme. [D]benzyl alcohol produced by cross-dismutation of [D2]formaldehyde and benzaldehyde in P. putida is demonstrated to have the R configuration by a novel deuterium NMR assay. Additionally, S. aureus produces methyl formate as a product of formaldehyde detoxification, apparently by oxidizing the methanol hemiacetal of formaldehyde.

Alcohol Oxidoreductases↗

PubMed1989

The poly-beta-hydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells.

High resolution 13C NMR spectroscopy of live cells has been used to show that poly-beta-hydroxybutyrate (PHB) is predominantly in a mobile state within the storage granules of Alcaligenes eutrophus, Methylobacterium extorquens, and Methylobacterium AM1. Comparison of chemical and NMR analysis of PHB indicates that about 70% of the polymer in A. eutrophus gives sharp observable resonances. Temperature-dependent line widths and relaxation rates together with nuclear Overhauser effect measurements demonstrate that the observed material is effectively a mobile amorphous elastomer that is well above its glass transition temperature. The hydroxyvalerate-hydroxybutyrate copolymer produced by propionate-fed A. eutrophus has virtually the same mobility as the homopolymer. Evidence is presented indicating that water is an integral component of the PHB granule and that this component acts as a plasticizer for the polymer. These observations strongly suggest that the enzyme(s) responsible for PHB biosynthesis and consumption operate only on mobile hydrated material and that the solid granules characteristic of dried cells are partially artifactual. This model is supported by a reinterpretation of previously inexplicable biochemical results.

Alcaligenes↗

PubMed1988

Formaldehyde adducts of glutathione. Structure elucidation by two-dimensional n.m.r. spectroscopy and fast-atom-bombardment tandem mass spectrometry.

Aqueous mixtures of formaldehyde and glutathione react to form a variety of cyclized adducts in addition to S-hydroxymethylglutathione. The adducts are in labile equilibrium with each other and are not readily separated. The structures of two of the other major adducts were determined by concerted application of 13C-1H two-dimensional chemical-shift correlation, fast-atom-bombardment mass spectrometry and tandem mass spectrometry to the adduct mixtures in aqueous solution.

Chemical Phenomena↗

PubMed1986

Formaldehyde metabolism by Escherichia coli. Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethyl)glutathione as a transient intracellular intermediate.

In vivo 13C NMR has been used to detect the transient formation of S-(hydroxymethyl)glutathione (GSCH2OH) from glutathione and [13C]formaldehyde in Escherichia coli. Two-dimensional 1H-13C shift correlation was used to locate the chemical shift of the formaldehyde-derived protons of the adduct. The adduct GSCH2OH is formed by chemical reaction in the first few minutes after cells are challenged with formaldehyde and remains within the cell until consumed by metabolism.

Carbon Isotopes↗

PubMed1985

Formaldehyde metabolism by Escherichia coli. Carbon and solvent deuterium incorporation into glycerol, 1,2-propanediol, and 1,3-propanediol.

Escherichia coli were grown on 14.3% uniformly 13C-labeled glucose as the sole carbon source and challenged anaerobically with 90% 13C-labeled formaldehyde. The major multiply labeled metabolites were identified by 13C NMR spectroscopy to be glycerol and 1,2-propanediol, and a minor metabolite was shown to be 1,3-propanediol. In each case, formaldehyde is incorporated only into the C1 position. A novel form of 13C NMR isotope dilution analysis of the major products reveals that all the 1,2-diol C1 is formaldehyde derived but that about 40% of the glycerol C1 is derived from bacterial sources. Glycerokinase converted the metabolite [1-13C]glycerol to equal amounts of [3-13C]glycerol 3-phosphate and [1-13C]glycerol 3-phosphate, demonstrating that the metabolite is racemic. When [13C]formaldehyde incubation was carried out in H2O/D2O mixtures, deuterium incorporation was detected by beta- and gamma-isotope shifts. The 1,3-diol is deuterium labeled only at C2 and only once, while the 1,2-diol and glycerol are each labeled independently at both C2 and C3; C3 is multiply labeled. Deuterium incorporation levels are different for each metabolite, indicating that the biosynthetic pathways probably diverge early.

Deuterium↗

PubMed1984

Formaldehyde metabolism by Escherichia coli. In vivo carbon, deuterium, and two-dimensional NMR observations of multiple detoxifying pathways.

13C NMR has been used to demonstrate the metabolism of dilute solutions of labeled formaldehyde by Escherichia coli to methanol, formate, carbon dioxide, and several other unidentified metabolites which contain labeled CH2 groups. Aeration of bacterial suspensions within the spectrometer dramatically increased the rate of oxidation to formate and carbon dioxide. Deoxygenation with nitrogen gas virtually abolished all metabolism, as did the exposure of bacteria to very high formaldehyde concentrations. Deuterium NMR of whole cells in deuterium-depleted water further demonstrated the conversion of formaldehyde-d2 to methanol-d2, ruling out a formaldehyde dismutase as an important species. Two-dimensional proton-carbon chemical shift correlation was used to reveal the chemical shifts of the protons attached to 13C labels in metabolites. The results indicate that formaldehyde is efficiently detoxified by the bacterial cell through a route or routes which do not appear to involve tetrahydrofolate. This detoxification may be in competition with the lethal antibacterial processes associated with formaldehyde.

Carbon Isotopes↗

PubMed1983

Mechanisms of antibacterial formaldehyde delivery from noxythiolin and other 'masked-formaldehyde' compounds.

Formaldehyde release in aqueous solutions of noxythiolin (N-methyl-N'-hydroxymethyl thiourea) has been monitored by nuclear magnetic resonance (n.m.r.) spectroscopy. The results suggest that antibacterial activity in such solutions resides mainly in the free formaldehyde. N.m.r. spectroscopy also demonstrated slow C-N bond rotation in noxythiolin and N-methylthiourea, with delta G of ca 15 kcal mol-1 (63 kJ mol-1). N-Hydroxymethyl imidazole is marginally more effective than corresponding hydrated formaldehyde solutions, an effect which is attributed to more rapid turnover of unhydrated formaldehyde as detected by saturation transfer n.m.r. spectroscopy. These observations are combined with the known delivery of lethal iminium ions, R2N+ = CH2, by compounds of the form R2NCH2X (X = OH, NR2; R is alkyl) to suggest a single consistent explanation of the antibacterial properties of a wide range of masked formaldehyde compounds.

Biological Assay↗

PubMed1983

Recent applications of N.M.R. to the investigation of steroid structures.

A detailed and clear understanding of the solution state structure and conformation of steroids is now possible. This is achieved using high field super conducting magnets and sophisticated computer control systems in nuclear magnetic resonance (n.m.r.) spectroscopy. The combination of greater signal dispersion and sensitivity obtained at high magnetic fields (cf. greater than 9.4 T--1H resonant frequency 400 MHz) has increased the utility of traditional assignment techniques such as nuclear Overhauser enhancement (nOe) and spin decoupling measurements. It has also permitted the application of various two dimensional (2D) n.m.r. experiments and resulted in complete unambiguous analysis of the proton spectra of various steroids [1-3]. The power of the various methods is illustrated by their application to the solution conformation of 17 alpha-acetoxy-6 alpha-methylpregn-4-en-3,20-dione (Medroxyprogesterone acetate, MPA).

Contraceptive Agents, Female↗

PubMed1982

Reductive methylation of proteins with sodium cyanoborohydride. Identification, suppression and possible uses of N-cyanomethyl by-products.

Reductive methylation of protein amino groups with formaldehyde and sodium cyanoborohydride is shown to give up to 25% yield of N-cyanomethyl (-CH2CN) product; on work up of the reaction this is hydrolysed back to starting amine, lowering the methylation yield. Addition of metal ions such as Ni2+, which complex with free cyanide ion, improve reductive methylation yields by suppressing by-product formation. The N-cyanomethyl group itself, produced in good yield when cyanide ion replaces cyanoborohydride, may have some value as a reversible modifier of amino groups in proteins.

Borohydrides↗

PubMed1981

Acetaldehyde--enkephalins: structure proof and some conformational deductions from one- and two-dimensional proton nuclear magnetic resonance spectra.

The structure of the adduct formed by reaction of acetaldehyde and Met5-enkephalin has been determined by analysis of 400-MHz proton spectra: two-dimensional J spectroscopy was used to resolve and measure virtually all the overlapping resonances, and decoupling difference spectroscopy was used to assign the resonances. Suitable manipulation of the two-dimensional data allowed analysis of alpha-CH resonances which were completely buried under a water signal and of amide NH resonances which overlapped in both dimensions. The adduct was shown to be a mixture of two diastereoisomers, each containing a 2-methylimidazolidin-4-one ring formed by condensation of an acetaldehyde molecule with the N-terminal amino group and Gly2 amide nitrogen. Analysis of the NMR data suggests that the folded conformation characteristic of native enkephalins in dimethyl-d6 sulfoxide is not important in these derivatives.

Acetaldehyde↗

PubMed1981