Mechanisms of formation and decomposition of hypervalent chromium metabolites in the glutathione-chromium (VI) reaction.

A long-lived chromium(IV) intermediate is generated during the reaction between Cr(VI) and glutathione in glycine below pH 3. The intermediate reacts with the tripeptide to produce Cr(III) and oxidized glutathione. A dynamic magnetic susceptibility measurement based on a nuclear magnetic resonance method yielded a 2.8 microB magnetic movement for the chromium(IV) species. The intermediate is formed by parallel third-order and second-order processes. The third-order process (k = 5.9 x 10(2) M-2 s-1) involves first-order participation by each of the oxidant, reductant, and hydrogen ions. A hydrogen ion independent pathway leads to a sluggish second-order process (k = 0.11 M-1 s-1) that is first order with respect to reduced glutathione [GSH] and [Cr(VI)]. Chromium(IV) species is reduced to Cr(III) by a second-order process (k = 0.13 M-1 s-1) that is first order in each of [Cr(IV)] and [GSH] and does not depend on [H+]. At pH 3.4, a chromium(V) species was detected as a minor intermediate as well. In the pH range 6.5-7.5, three dominant chromium(V) intermediates were detected. The existence of Cr(IV) in low pH offers an opportunity to examine the mechanism of DNA damage by this rare oxidation state.

Monovalent ion-phosphatidylcholine interactions: an electron paramagnetic resonance study.

The apparent Mn2+ binding constant for L-alpha-dipalmitoylphosphatidylcholine (DPPC) bilayers dispersed in monovalent salt and MnCl2 dispersions was determined as a function temperature using electron paramagnetic resonance (ERP). Reproducibility in the data sets requires the use of a standard salt solution and dual cavity techniques. Changes in the binding constant at different phase states and temperatures were observed and correlated to the influence of monovalent salts on the thermal properties of DPPC. The turning points (i.e. changes in slope) in the curves of the apparent Mn2+ binding constant versus temperature can be understood in terms of differences in ion binding to headgroups with different bilayer surface areas. The influence of Li+ and SCN- on Mn2+ binding is viewed as a function of their presence in the ionic media in contact with the bilayer rather than as a competitive event. Other monovalent ions studied appear to have little effect on the measured apparent Mn2+ binding constants for DPPC headgroups.

Alterations of oxygen uptake and the redox state of ubiquinone in rabbit sperm exposed to a variety of physiologic treatments.

The rate of TEMPONE reduction by electrons originating from ubiquinone in intact rabbit spermatozoa was observed for control, high ionic strength (HIS) medium-treated, and HIS-seminal plasma-treated (HIS-SP) samples. The presence of TEMPONE in the incubation medium had no effect on oxygen consumption, demonstrating the utility of TEMPONE as a nonperturbing probe of the ubiquinol redox state. The rate of TEMPONE reduction was significantly increased over control levels for sperm incubated in hypertonic medium and was correlated to a decrease in oxygen consumption and a relative increase in ATP in the total adenine nucleotide pool. This increase in TEMPONE reduction in HIS sperm was reversed by treatment of sperm with seminal plasma, but seminal plasma had no effect on oxygen consumption or relative amounts of ATP in the adenine nucleotide pool. These observations are consistent with state 3 respiration in control sperm and state 4 respiration in HIS- and HIS-SP-treated sperm. Arrhenius data were obtained for ejaculated and epididymal sperm subjected to a variety of treatments. Lines fitted to plots of Arrhenius data revealed that each treatment affected the activation energy and intercept relative to controls. Evidence is presented for a phase transition occurring at 13 degrees C based on changes in the rate of TEMPONE reduction by ubiquinol. It was noted that, above the phase transition, rate constants for the reaction were dependent upon both treatment and temperature, but below the transition the differential effects of treatment were no longer apparent. The present study has demonstrated that events taking place in the respiratory chain can be closely monitored by measuring oxygen uptake and TEMPONE reduction, and that these events are affected by alterations in the sperm environment.

Reduction of the spin-label TEMPONE by ubiquinol in the electron transport chain of intact rabbit spermatozoa.

When the hydrophilic spin label TEMPONE (deuterated 2,2,6,6-tetramethylpiperidine-N-oxyl) was incubated with intact rabbit spermatozoa at concentrations greater than 0.3 X 10(9) cells/ml, the electron spin resonance signal height decreased with time. This loss of signal amplitude was reversed by the oxidizing reagent potassium dichromate, indicating that the signal loss was due to a reduction of the paramagnetic nitroxide species to the nonparamagnetic hydroxylamine. Using inhibitors that act on the respiratory chain, we observed that, relative to controls, the rate of TEMPONE reduction was decreased in the presence of rotenone, but increased in the presence of antimycin A and potassium cyanide (KCN). Parallel studies measuring oxygen consumption showed decreases with all three inhibitors. We interpret these observations to mean that TEMPONE is reduced by ubiquinol in the respiratory chain. Supporting this conclusion is the observation that the midpoint potential of TEMPONE was determined by be +48mV, which is close to the midpoint potential of +40 mV for the ubiquinone/ubiquinol couple. Furthermore, in a cell-free test system, ubiquinol reduced TEMPONE, but ubiquinone, NADH, and succinate did not.

Cholestane spin label study of filipin action on lipid planar multibilayers.

EPR spectra of a cholestane probe dissolved in egg yolk lecithin and lecithin-cholesterol planar multibilayers were observed as a function of the filipin dose. The probe is structurally similar to cholesterol; its normal position when dissolved is with the long axis approximately along the bilayer normal. Both cholesterol-containing and cholesterol-free samples showed spectral components characteristic of bilayer fragmentation (tilted domains) which increased with dose. Furthermore, the cholesterol-free spectra indicated that some of the probe was frozen with the long molecular axis perpendicular to the slide normal. The frozen spectral component increased with dose. Spectra from a fatty acid probe did not have this feature. We interpret this as due to probe complexed with filipin (in place of cholesterol) in accordance with the filipin-cholesterol aggregate model of deKruijff and Demel. An ultraviolet study of filipin-probe interaction indicates that the probe is capable of complexing in just such a manner but has less affinity for the drug than cholesterol. Spectra from the cholesttane probe in liposomes were also observed.