Posttranslational changes in band 3 in adult and aging brain following treatment with ergoloid mesylates, comparison to changes observed in Alzheimer's disease.

Band 3, the most heavily used anion transport system in vertebrates, ages as cells and tissues age. Posttranslational changes in band 3 in adult and aging brain were investigated following treatment with ergoloid mesylates and compared to changes observed in Alzheimer's disease (AD). The study was conducted in a double blind fashion and was decoded only after the study was completed. The following posttranslational changes in brain band 3 occur with age: increased breakdown of band 3; decreased phosphorylation; and decreased anion transport. Autoantibodies to senescent cell antigen (SCA) synthetic peptides residue 538-554 and 812-827 increase with age, but antibodies to the former peptide are significantly reduced in ergoloid mesylate treated old mice. This is a critical transport region of band 3. Results showed the aged/altered band 3 increased in Alzheimer's disease (AD) as determined by quantitative antibody binding. Ergoloid mesylates altered the age-related posttranslational changes as follows: the observed age-related decrease in brain band 3 was partially reversed and anion transport was increased. This is consistent with the data indicating decreased autoantibodies to a critical anion transport segment of band 3. Aging appears to result in damage to a critical transport region of the anion transporter which is reflected by decreased anion transport, increased breakdown, alteration of the molecule itself, and an increase in autoantibodies to the region. Ergoloid mesylates seem to protect against this damage.

Differential sensitivity of stilbenedisulfonates in their reaction with band 3 HT (Pro-868-->Leu).

Band 3 HT (Pro-868-->Leu) is a mutant anion exchange protein which has several phenotypic characteristics, including a 2- to 3-fold larger Vmax, and reduced covalent binding of the anion transport inhibitor 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS). We have used fluorescence kinetic methods to study inhibitor binding to band 3 to determine if the point mutation in band 3 HT produces localized or wide-spread conformational changes within the membrane-bound domain of this transporter. Our results show that covalent binding of H2DIDS by band 3 HT is slower by a factor of 10 to 20 compared with the wild-type protein. In contrast, no such difference in the kinetics was observed for covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In addition, the kinetics of H2DIDS release from band 3 HT was abnormal, while the kinetics of 4,4'-dibenzamidostilbene-2,2'-disulfonate (DBDS) release showed no difference when compared with the wild-type protein. We conclude that substitution of leucine for proline at position 868 does not perturb the structure of "lysine A" in the membrane-bound domain of band 3 but rather produces an apparently localized conformational change in the C-terminal subdomain of the protein which alters H2DIDS affinity. When combined with the observation of an increased Vmax, these results suggest that protein structural changes at position 868 influence a turnover step in the transport cycle.

Natural killer cell defects resulting from thermal injury.

Peripheral blood mononuclear lymphocytes (PBL) from thermal injury patients were examined for their ability to mediate spontaneous (NK) cytotoxic activity against K562 tumor cells. NK cell numbers were quantitated in burn patients using the fluorescein isothiocyanate-conjugated monoclonal antibody Leu-7 and an Ortho cell sorter. NK activity of burn patients was only 30% of control levels. Thus, at a 50:1 effector:target cell ratio, burn patients' PBL gave a mean of 18% killing as compared to 60% killing mediated by control PBL (14 individuals). Pretreatment of PBL with interferon-gamma or interferon-alpha enhanced NK activity of normal PBL but had no effect on the NK activity of PBL from burn patients. The number of cells bearing antigen recognized by Leu-7 was variable in burn patients, with some patients having decreased (less than 5%), normal (8-10%), or elevated (20-40%) numbers of Leu-7 bearing PBL. No differences were observed in the total numbers of PBL in burn patients when compared to control individuals. These results indicate that NK activity does not correlate with the percentage of Leu-7 bearing cells present in PBL and that the inability of thermal injury patients to mediate normal NK function may contribute to their susceptibilities to viral infections.