Rongchang Li

The events relating to lanthanide ions enhanced permeability of human erythrocyte membrane: binding, conformational change, phase transition, perforation and ion transport.

The binding and uptake of Gd3+ ions by human erythrocytes in vitro were studied by determining the Gd contents in membrane and in cytosol by means of particle-induced X-ray emission (PIXE) spectrometry. Results obtained from varied incubation time revealed that the Gd3+ ions bind to the membrane proteins and lipids at first. Gd3+ binding to the membrane lipids and proteins lasts 0 approximately 20 and 20 approximately 100 ms respectively, as shown by the stopped-flow studies. Then a fraction of Gd3+ ions diffuses through the membrane. The kinetics of Gd3+ binding indicates that the binding to phospholipids is prior to that to the membrane proteins, but a portion of the lipid-bound Gd3+ redistributed later to the proteins. PIXE studies showed that the entry of Gd3+ increased the influx of Ca2+ and Cl-. By monitoring the changes in fluorescence of proteins and that of the Ln3+, the uptake of La3+, Eu3+, Gd3+ and Tb3+ was shown to be a process comprising a series of events. Binding to the membrane molecules induces the phase transition of lipid bilayer and conformational changes and aggregation of membrane proteins. Conformational changes of the proteins were characterized by Fourier transform IR spectroscopy (FT-IR) deconvolved spectra, i.e. alpha-helix content decreases while beta-sheet increases. ESR spectra of MSL-labeled proteins reflect the aggregation state related with the conformational change. [31P]NMR spectra of membrane lipid bilayer revealed the Ln3+ ions induced hexagonal (H(II)) phase formation. Phase transition and aggregation of membrane proteins cause the formation of domain structure and perforation in the membrane. These alterations in membrane structure are responsible for the Ln3+ enhanced membrane permeability. Thus the previous Ln3+ binding will facilitate the across-membrane transport of other Ln3+ ions through the membrane.

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin’s oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln(3+) binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln(3+) binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln(3+) binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N(I)), non-cooperative strong sites (N(S)) and non-cooperative weak sites (N(W)) with binding constants in decreasing order: K(I)>K(S)>K(W). The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln(3+) concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln(3+) binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln(3+) concentration. At the range of [Ln(3+)]/[Hb]<2, the marked increase of oxygen affinity (P(50) decrease) with the Ln(3+) concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln(3+) concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln(3+) binding. This was indicated by the changes in secondary structure characterized by the decrease of alpha-helix content.

Orally administrated cerium chloride induces the conformational changes of rat hemoglobin, the hydrolysis of 2,3-DPG and the oxidation of heme-Fe(II), leading to changes of oxygen affinity

The structure and oxygen affinity of hemoglobin from erythrocytes of CeCl(3) fed Wistar rats in the dose range of 0.2-20.0 mg/kg body weight/day were investigated by means of various spectroscopic methods. The changes in oxygen saturation curves of hemoglobin are dependent upon both feeding dose and feeding time. After 40 days feeding with 20 mg CeCl(3)/kg body weight/day, the curve changed to a double sigmoid shape and the oxygen affinity in low oxygen pressure increases. It regained the sigmoid form after 80 days feeding, but the degree of oxygen saturation in higher oxygen pressure became higher than that in the control. These results indicate that CeCl(3) can increase the oxygen affinity of hemoglobin of rat erythrocytes. This effect is further demonstrated by the analysis of Mössbauer spectra of erythrocytes. Increase of hemoglobin content in erythrocytes was found in rats fed with CeCl(3). It might be the offset response to the poor oxygen-releasing capability of the hemoglobin. CD and FT-IR deconvoluted spectra indicate that secondary structures of hemoglobin have remarkable changes, characterized by a gradual decrease of alpha-helix content, in a dose- and feeding time-dependent fashion. Meanwhile, the 31P NMR spectra demonstrate that the level of 2,3-diphosphoglyceric acid (2,3-DPG) in erythrocytes, an allosteric regulator of oxygen release from hemoglobin, decreases due to its hydrolysis. In addition, the Mössbauer and ESR spectra show clearly that a fraction of the heme-iron changes from Fe (II) to Fe (III) in CeCl(3) fed rats. The results indicate that the oral administration of CeCl(3) leads to a microenvironment changes of heme in intracellular hemoglobin. Oxygen affinity changes might be attributed to a series of events triggered by the binding of Ce (III) to hemoglobin and 2,3-DPG, including conformational changes of hemoglobin and 2,3-DPG hydrolysis, respectively and also the partial transformation from heme-Fe (II) to heme-Fe (III).

Effects of lanthanide ions on hydrolysis of phosphatidylinositol in human erythrocyte membranes

The effects of lanthanides on the hydrolysis of phosphatidylinositol in human erythrocyte membranes were studied.3H-inositol labeling chromatography and HPLC were used to determine inositol 1, 4, 5-triphosphate and diacylglycerol separately, the hydrolytic products of phosphatidylinositol due to the reaction of lanthanide ions with human erythrocyte membranes. The unhydrolyzed phosphatidylinositol in membranes was also determined. The results indicate that the hydrolysis of phosphatidylinositol can be promoted by lanthanides (La3+, Ce3+, Y3+, Tb3+) and the effects of La3+ and Ce3+ are stronger than those of Y3+ and Tb3+.

Lanthanides enhance pulmonary absorption of insulin

In an effort to investigate the enhancement effect of lanthanide ions (Ln3+) on the absorption of larger molecules from the pulmonary pathway, insulin (mol. wt.=5730) was chosen as a model peptide. The absorption of insulin preadministered or coadministered with Ln3+ from the lung was investigated by means of an in situ pulmonary absorption experiment. The enhancement absorption of insulin by Ln3+ ions was evaluated by calculating the various bioavailabilities (Fr) of insulin from pulmonary absorption. Moreover, the temporal change of Gd content in serum was also investigated. Results showed that the promoting effect of Ln3+ on the bioavailability of insulin is closely related to its species, concentration, and delivery order. The effect of the median Ln3+ series was remarkably greater than that of light and heavy Ln3+. The anionic form of Gadolinium (Fr=68.4%) seemed to be more effective compared with its cationic form (Fr=59.5%). Coadministration of Gd3+ with insulin (Fr=80.1%) was the most effective in increasing insulin absorption from the lung. Gd3+ was rapidly absorbed and metabolized to a normal level after 4 h. It was suggested that lanthanides in a very low concentration might become potent absorption enhancers to improve absorption of larger molecules via the pulmonary pathway.

Tb3+ binding to human erythrocyte spectrin resulting in conformation change and aggregation.

The Tb3+ binding to spectrin tetramer (SPT) was studied by Tb3+ fluorescence titration and CD spectra. The results indicated that the total high-affinity Tb3+ binding sites are n1 = 330, with average Kd = 3.6 x 10(-6) M. Among them, ca. 90 sites are of higher affinity and are probably more specific to Tb3+ than the remaining sites. There are 520 low affinity Tb3+ binding sites with average Kd = 1.5 x 10(-5) M. Fluorescence and CD spectra revealed that the alpha-helix content of SPT decreased with Tb3+ binding to specific sites and further binding did not result in conformation change. Tb3+ binding to SPT and the subsequent reactions were studied by employing stopped-flow fluorescence and light scattering methods. The studies demonstrate that this is a multistep reaction assembly: high-affinity terbium binding-conformation change-aggregation-low-affinity terbium binding--the second conformation change. The critical Tb3+ concentration-induced spectrin dimer (SPD) aggregation was determined with a light scattering method.

Kinetic studies of mobilization of copper(II) from human serum albumin with chelating agents

The kinetics of the mobilizing reactions of five chelating agents for human serum albumin (HSA)-bound copper(II) [Cu(II)] have been studied spectrophotometrically. The decreasing sequence of reaction rate has been determined to be EDTA greater than DTPA greater than EGTA greater than NTA greater than IDA. A group of mathematical models were established to define the mechanisms of the competitive reactions between low-molecular-weight ligand and macromolecular ligand. All reactions of the five chelating agents follow a process involving the intermediate ternary complexes: (formula; see text) The reactions of DTPA and EDTA were found to be different from those of EGTA, NTA, and IDA. In the former cases, the reactions are likely following an overlapping mechanism in which the rate constant k1 was closed to k2. The reactions involving the other three chelators are different in k1 much greater than k2.

Lanthanides' enhancing absorption of insulin and reduction of blood glucose of rat by pulmonary administration

To explore the possibility of lanthanides as the enhancer of insulin absorption, the promoting effects of lanthanide ions on the absorption of intrapulmonary delivered insulin and reduction of blood glucose were investigated by means of two approaches, preadministration and coadministration of lanthanide ions with insulin. The results indicate that, compared with the results of those given insulin only, lanthanide ions can effectively enhance the level of insulin and the reduction of glucose level in blood. These effects are dependent on lanthanide species and their concentrations. The mechanism was discussed.

Formation of domain structure of erythrocyte membrane in Wistar rat fed with CeCl3 per os

To explore the possibility of absorption of lanthanides via digestive duct and their effects on the membrane structure and permeability of erythrocytes, the fine structure of erythrocyte membrane from Wistar rats, fed for 70 days of daily administrationper os with 20 mg CeCl3/kg weight, was imaged by means of atomic force microscopy and FT-IR deconvolution spectra. The results show that, although the erythrocytes maintain the intact shape, the change of secondary structure, aggregation and crosslinking of the protein particles of membrane surface and the enlarged lipid regions lead to the domain structure formation. This structure might be responsible for the increasing permeability of erythrocyte membrane.

SPECTROSCOPIC STUDIES ON Cd2+ BINDING TO HUMAN ERYTHROCYTE SPECTRIN

The binding of Cdto the human erythrocyte spectrin tetramer (3PT) and the consequent conformation change were studied as a function of the Cd/SPD mole ratio (R) by fluorescence and circular dichroism (CD) spectra. The conformation dependence on R has a biphasic feature: in the range less than 60, the Cd ions are likely bound to a small fraction of the thiol groups and the non-thiol groups on the surface of SPT and the conformational changes are not significant as characterized by the labeled N-(l-pyrenyl)maleimide (NPM)fluorescence and CD spectra. When R reaches 60, as a result of a conformational change, the SH-related strong binding sites are exposed and bind with Cd. The biphasic conformational change at this turning point might be interpreted on the basis of thiol groups as critically important for maintaining the secondary structure of SPT.