The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.
Multidimensional solid-state nuclear magnetic resonance (NMR) under magic-angle spinning (MAS) conditions has been developed to determine the dihedral angle for a Hα1–Cα13–Cβ13–Hβ1 moiety in powdered states. The pulse sequence for this experiment includes C113H dipolar evolution periods for Cα and Cβ, which are correlated through a coherent Cα1313Cβ dipolar mixing period. Theoretical analysis based on the symmetry of the spin system indicates that the dipolar correlation spectrum only due to the CαHα and CβHβ dipolar couplings is strongly dependent on the dihedral angle χ about the CαCβ bond axis, but two χ angles give the same spectrum in the χ range from 0° to about 140°, where χ=0° corresponds to the cis conformation. Inclusion of the CαCβ dipolar coupling together with the weak CαHβ and CβHα dipolar couplings, however, breaks the symmetry of the system with respect to χ in the range from 0° to 180°. These properties are confirmed by the spectra calculated for the pulse sequence as a function of χ and the root-mean-square deviation between them. The bond lengths, bond angles, and dihedral angle also alter the dipolar correlation spectrum differently. This enables us the experimental determination of all the structural parameters, which improves the accuracy of the dihedral angle determination. The high resolution due to C13 isotropic chemical shifts under MAS conditions in this multidimensional NMR permits its application to molecules having a number of C13-labeled sites. Experimental results are presented for powdered L-valine uniformly labeled with C13 and N15 nuclei. Effects of the structural parameters and noise on the dihedral angle determination are evaluated numerically. The accuracies of the determined structural parameters are discussed.
Competitive spin-trapping behavior of 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) and 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) indicated that the affinity of DPhPMPO for hydroxyl radicals was 1.7 times higher than that of DMPO. Human erythrocyte ghosts (HEGs) with encapsulated FeII catalysts (HEG–Fe) were prepared for ESR measurement. By trapping the hydroxyl radical (OH•) in the presence of polyethylene glycol (PEG-4000) outside the HEG–Fe, the generation of OH• inside the catalyst was confirmed. The trapping of hydroxyl radicals generated in HEG was accomplished using DPhPMPO, and the permeability of the erythrocyte membrane to DPhPMPO was 2.4 times greater than that of DMPO.
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