James A. Magnuson

Nuclear magnetic resonance studies of sodium and potassium in etiolated pea stem.

Based on nuclear magnetic resonance (NMR) spectroscopy and other evidence, it has been argued that tissues accumulate, and retain, ions in a binding process by a highly structured water-protoplasm system; thus active membrane transport need not be involved. Recent evidence has accounted for the loss of resonance intensity usually found when investigating quadrupolar ions in animal tissue. Using continuous wave NMR spectroscopy, we have examined two quadrupolar ions, Na(+) and K(+), in pea stem cells where about 90% of the ion content is in the largely aqueous vacuoles having a membrane barrier. The NMR resonances from these ions correspond to almost 100% of that expected from independent measurements of total ion content. This indicates that the ions are retained as free ions after accumulation. The small fraction which is NMR invisible may represent ions in an ordered, anisotropic environment, such as that in the wall or cytoplasm.

The Chemical Structure of UF Resins

Abstract The structure of urea-formaldehyde (UF) resin was studied in light of the influence of various synthesis parameters on the chemical structure of the resulting resin solutions. The structural variability was analyzed by 13C nuclear magnetic resonance spectroscopy (NMR). Using appropriate pulse techniques, quantitative determination of the proportion of various functional groups was obtained. The parameters of formaldehyde/urea mole ratio, pH control, extent of reaction, and concentration of reactants were shown to affect the final resin structure. Variation of these parameters influenced the extent of branching, the rate of condensation, and the proportion of di-methylene ethers and cyclic uron structures.

Calorimetric study of carbohydrate binding to concanavalin A

Flow microcalorimetry has been used to examine the delta H of binding of two types of saccharides, a series of simple monosaccharides and a series of alpha-(1----4)-linked glucosides, to the lectin Concanavalin A. It has been found that the delta H decreases with any change in the stereochemistry of a hydroxyl group relative to methyl alpha-D-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The delta Hs of binding for the alpha-(1----4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for alpha-(1----4)-linked glucopyranosides, in contrast to that proposed for alpha-(1----2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.

A nuclear magnetic resonance study of sodium ion interaction with erythrocyte membranes

Abstract Sodium-23 nuclear magnetic resonance line widths have been used to study sodium ion interaction with erythrocyte membranes and erythrocyte membrane proteins. Detergent solubilization has been effective in freeing sodium and potassium binding sites which are not available for rapid chemical exchange with the whole membrane. Evidence for sodium ion binding has been obtained by showing that the resonance line width increases in the presence of solubilized membrane. Competition by potassium for binding sites is demonstrated by a decrease of the sodium line width in the presence of potassium ion.

Membrane phase transitions and the transport of chlortetracycline

Abstract When chlortetracycline is added to a suspension of respiring Staphylococcus aureus cells, the active transport of the antibiotic may be monitored by its fluorescence enhancement as it moves from a polar aqueous environment into the apolar regions of the membrane. The initial rates of transport are temperature dependent with a maximal rate between 35 and 45 °C. Arrhenius plots of the initial rates are biphasic with a transition temperature of 27 °C for control cells. This transition temperature is sensitive to the fatty acid composition of the S. aureus cells. By culturing the cells in the presence of oleic acid or at 10 °C, the S. aureus cells incorporate a larger percentage of unsaturated and branched chain fatty acids into their membranes, resulting in transition temperatures 8–9 °C lower than the control cells. Studies of depolarization of fluorescence also indicate that the mobility of the bound chlortetracycline is temperature-dependent. Temperature transitions occur at the same temperatures as those measured by Arrhenius plots. The transition temperatures indicated by the Arrhenius plots and the polarization studies are believed to reflect order-disorder phase transitions associated with the melting of the phospholipids in the cell envelope.

Kinetics of chlorotetracycline uptake in Staphylococcus aureus by a fluorescence technique

Abstract The antibiotic chlorotetracycline (CTC) is used as a fluorescent chelate probe to investigate the kinetics of its uptake into Staphylococcus aureus . CTC binds to divalent cations in an aqueous solution with enhanced fluorescence. This fluorescence is polarity dependent, being higher in apolar solutions. Upon addition of CTC to dispersions of S. aureus , a time dependent fluorescence enhancement is detected demonstrating that the CTC-divalent cation complex migrates into the apolar regions of the membrane. This uptake, which follows saturation kinetics, is energy dependent. A Km of 162 μ M was obtained for CTC concentration ranges of 0.2–100 μgm/ml.

Location of Mn2+ in concanavalin A containing only a Mn2+ ion.

Abstract The metal-sugar distances in two metallized forms of concanavalin A have been compared by 19 F magnetic resonance techniques. Using relaxation times measured at two different frequencies we have shown that the distance between the Mn 2+ ion and the bound sugar in concanavalin A containing only Mn 2+ is essentially identical to that found in concanavalin A containing both Mn 2+ and Ca 2+ . Our results rule out the possibility that Mn 2+ activates concanavalin A by binding at the Ca 2+ site (S2) and would suggest that Mn 2+ alone can induce an active saccharide binding conformation by binding at the transition metal site (S1).

Conformational changes following Mn(II) binding to demetalized concanavalin A

Abstract A temperature-dependent conformational change occurs following the binding of only one Mn(II) to a concanavalin A monomer. This change is independent of Ca(II) near pH 7 and is characterized by an activation energy of 22.3 kcal mol −1 , a value similar to that attributed to a cis-trans peptide isomerization. Two conformations have been detected in magnetic resonance experiments on solvent water protons where spin lattice relaxation times are influenced by bound Mn(II). Both conformations possess saccharide binding activity and Ca(II) stoichiometrically enhances the rate of conversion to the final, more stable conformation.

A new nmr technique to study disulfide reduction: Comparison of lysozyme and α-lactalbumin

Abstract A nuclear magnetic resonance technique employing 35 Cl resonances has been used to study the reduction of disulfides in lysozyme and α-lactalbumin. The nmr technique allows rapid analysis of approximately 2 × 10 −5 moles of disulfides. Reductions were carried out with dithiothreitol (DTT) in 4 M urea. From a time study of the amount of reduction for the two proteins, it appears that the disulfides of α-lactalbumin are more accessible for reduction than those of lysozyme.

Reductive degradation of dieldrim and endrin in the field using acidified zinc

The reductive dechlorination of dieldrin and endrin was investigated as a possible procedure for field disposal of small quantitites of these pesticides. The objective was to convert the parent compounds to environmentally less objectionable materials. Emulsifiable concentrate formulations of the pesticides in a soil slurry were mixed with powdered zinc, dilute acetic acid, and acetone to facilitate reaction. Analysis of the mixtures by GC-MS indicated essentially complete conversion of endrin and partial conversion of dieldrin to products probably formed by replacement of the bridge anti chlorines with hydrogen. Partial confirmation of the desired anti dechlorodieldrin product was made by nmr. Disappearance of the parent compounds and production of conversion products was monitored for 2 1/2 years by GC in an outside soil plot, and accelerated destruction of parent compounds was achieved by the treatment. By analogy to the bridge anti dechloro products of aldrin and isodrin, the anti dechloro products probably are much less toxic than the parent compounds and are therefore environmentally less objectionable.