Cr(III) binding to transferrin (Tf; the main Fe(III) transport protein) has been postulated to mediate cellular uptake of Cr(III) to facilitate a purported essential role for this element. Experiments using HepG2 (human hepatoma) cells, which were chosen because of high levels of the transferrin receptor, showed that Cr(III) binding to vacant Fe(III) -binding sites of human Tf effectively blocks cellular Cr(III) uptake. Through bio-layer interferometry studies of the Tf cycle, it was found that both exclusion and efflux of Cr2 (III) Tf from cells was caused by 1) relatively low Cr2 Tf affinity to cell-surface Tf receptors compared to Fe2 Tf, and 2) disruption of metal release under endosomal conditions and post-endosomal Tf dissociation from the receptor. These data support mounting evidence that Cr(III) is not essential and that Tf binding is likely to be a natural protective mechanism against the toxicity and potential genotoxicity of dietary Cr through blocking Cr(III) cellular accumulation.

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Edited by Phyllis I. Hanson Cholesterol's effects on Na ؉ ,K ؉-ATPase reconstituted in phospholipid vesicles have been extensively studied. However, previous studies have reported both cholesterol-mediated stimulation and inhibition of Na ؉ ,K ؉-ATPase activity. Here, using partial reaction kinetics determined via stopped-flow experiments, we studied cholesterol's effect on Na ؉ ,K ؉-ATPase in a near-native environment in which purified membrane fragments were depleted of cholesterol with methyl-␤-cyclodextrin (m␤CD). The m␤CD-treated Na ؉ ,K ؉-ATPase had significantly reduced overall activity and exhibited decreased observed rate constants for ATP phosphorylation (ENa 3 ؉ 3 E2P, i.e. phosphorylation by ATP and Na ؉ occlusion from the cytoplasm) and K ؉ deocclusion with subsequent intracellular Na ؉ binding (E2K 2 ؉ 3 E1Na 3 ؉). However, cholesterol depletion did not affect the observed rate constant for K ؉ occlusion by phosphorylated Na ؉ ,K ؉-ATPase on the extracellular face and subsequent dephosphorylation (E2P 3 E2K 2 ؉). Thus, partial reactions involving cation binding and release at the protein's intracellular side were most dependent on cholesterol. Fluorescence measurements with the probe eosin indicated that cholesterol depletion stabilizes the unphosphorylated E2 state relative to E1, and the cholesterol depletion-induced slowing of ATP phosphorylation kinetics was consistent with partial conversion of Na ؉ ,K ؉-ATPase into the E2 state, requiring a slow E2 3 E1 transition before the phosphorylation. Molecular dynamics simulations of Na ؉ ,K ؉-ATPase in membranes with 40 mol % cholesterol revealed cholesterol interaction sites that differ markedly among protein conformations. They further indicated state-dependent effects on membrane shape, with the E2 state being likely disfavored in cholesterol-rich bilayers relative to the E1P state because of a greater hydrophobic mismatch. In summary, cholesterol extraction from membranes significantly decreases Na ؉ ,K ؉-ATPase steady-state activity.

RH421 is a widely used voltage-sensitive fluorescent membrane probe. It is also photochemically reactive and its photochemistry is dependent on its solvent surroundings. The aim of this study is to use the solvent dependence of the dye's photochemistry as an indicator of its environment within a lipid membrane. It was found that the dye actually undergoes two competing photochemical reactions. One photochemical pathway is characterized by an increase in fluorescence, the other by a fluorescence decrease. The pathway followed depends on the light intensity, the excitation wavelength and the solvent. The pathway leading to a fluorescence decrease is very dependent on the polarity of the solvent. It is promoted by solvents of high polarity (e.g., methanol, ethanol and propanol), by high intensity illumination and long wavelength excitation (i.e., 546 or 577 nm). In less polar solvents (butanol, pentanol, hexanol and heptanol) the pathway leading to a fluorescence increase dominates. The photochemical behavior observed when the dye is bound to a lipid membrane was not comparable to its behavior in any of the isotropic solvents studied. This is most likely due to the anisotropic environment of the membrane, which results in a steep gradient in polarity along the length of the dye molecule.

While Cr(III) dietary supplements are widely consumed, some commercial supplements have yet to be structurally characterized. X-ray absorption spectroscopy and other spectroscopic methods were used to characterize Cr(III) nicotinato nutritional supplements that have long been used in complementary medicine. Different ratios of nicotinic acid and CrCl3·6H2O (trans-[CrCl2(OH2)4]Cl·2H2O) at different pH values gave a range of products. The local structures of Cr(III) nicotinato complexes obtained at pH 7 and of the patented complex were characterized by performing multiple-scattering analysis of their EXAFS spectra as well as EPR, UV-vis, and IR spectroscopies. For the first time, these complexes have been definitively characterized as nicotinato-bridged polymers of dihydroxido-bridged dinuclear Cr(III) cores. In the patented complex used in commercial preparations, each Cr is octahedral with an additional terminal O-bound nicotinato ligand, two bridging nicotinato (one O and one N bound), and an aqua ligand. The other species also have two or three bridging nicotinato ligands and an aqua and, in some cases, a terminal hydroxido ligand, which is dependent upon the stoichiometry of the reactants and the pH value of the solution in which they are prepared.