[The effect of cryoprotective agents on proteins of the erythrocyte membrane-cytoskeleton complex]. / Vliianie krioprotektornykh agentov na belki membranno-tsitoskeletnogo kompleksa éritrotsitov.

The aim of the study was to evaluate of the effects of glycerol and DMSO, belonging to the endocellular type of cryoprotective agents (CPAs), as well as polyethylene glycol, dextran, sucrose, and mannitol, related to exocellular CPAs, on proteins of the membrane-cytoskeleton complex (MCC) of human erythrocytes at the stage preceding freezing. The assessment of protein modifications was performed by SDS-PAGE using different approaches when preparing samples for analysis. The use of ß-mercaptoethanol in the solubilizing buffer showed no changes in the MCC polypeptide profile of erythrocytes preincubated with CPAs thus suggesting good biocompatibility of the studied substances. The use of the cross-linking reagent diamide for assessment of protein modifications did not reveal structural abnormalities that would result in significant changes in the localization of -SH groups and an increase in the production of high-molecular-weight polypeptide complexes identified by SDS-PAGE without ß-mercaptoethanol. However, the recognized changes in the electrophoretic mobility of proteins in the area of band 5 in erythrocytes incubated with CPA in the presence of diamide suggest a reorganization of the structural state of actin protofilaments, which can be caused by alterations of actin monomers themselves or initiated by modifications of actin-binding proteins in the presence of CPAs. In addition, an increase in the amount of the protein fraction located between bands 5 and 6 in the MCC profiles of erythrocytes incubated with CPA and diamide was revealed. Despite the similarity of the reaction of erythrocyte proteins to different CPAs, the properties of cells depending on MCC, may differ due to modifications in the macromolecule structures, which are not associated with changes in the localization of the -SH-groups of proteins. The results obtained indicate that CPAs may have a significant impact on the erythrocyte MCC, and this requires further research.

CHANGES IN ERYTHROCYTE CA2+-ATPASE ACTIVITY UNDER PEG-1500 AND LOW TEMPERATURE INFLUENCE.

During cryopreservation different organic compounds are applied. Their introduction into cell suspension causes modification of subcellular systems and provides cell survival during freeze­thawing. The aim of the study was to determine the modifying effect of cryoprotectant PEG-1500 and low temperatures on the Ca2+-ATPase activity using the model of the saponin-permeabilized erythrocytes. PEG-1500 was found to cause the inhibition of erythrocytes Ca2+-ATPase activity despite the presence in the medium of endogenous effectors capable of stimulating the enzyme function. Modification of Ca2+-ATPase is apparently due to the physical and chemical parameters of the cryoprotectant solution since the removal of the polymer out of the medium leads to recovery of its functional properties. The reversibility of the inhibition of Ca2+-ATPase is characteristic for erythrocytes exposed to the cryoprotectant without freezing as well as for those that have been frozen and thawed in the presence of PEG-1500. The processes of freezing­thawing of cells in the cryoprotectant-free medium did not affect the Ca2+-ATPase activity indicating cryoresistance of the membrane-bond enzyme. Despite the fact that the efficiency of erythrocyte cryopreservation with PEG-1500 depends on incubation temperature prior to freezing, functional induces of Ca2+-ATPase activity in the erythrocytes exposed to PEG-1500 at 37 of 5­7 °C show no significant differences when subsequent ATP hydrolyses reaction is carried out at 37 °C. However, reduction in the temperature upon enzymatic reaction progression down to 5­7 °C after treatment of erythrocytes with PEG-1500 under similar conditions, leads to additional inhibition of enzyme activity. The identified changes in Ca2+-ATPase activity in the presence of PEG-1500 are presumably due to the modifying effect of cryoprotectant on the membrane structure and even presence of endogenous effectors in the medium cannot overcome the restrictions imposed by the modified membrane environment on this enzyme functioning.

CHANGES IN ERYTHROCYTE SURFACE MARKER CD44 DURING HYPOTHERMIC AND LOW TEMPERATURE STORAGE.

We studied the changes in surface marker CD44 in erythrocytes, cryopreserved under the protection of glycerol and PEG­1500, or stored in hypothermic conditions. It was shown that during hypothermic storage the CD44 characteristics in erythrocyte suspension were unchanged within 10 days. In cryopreserved erythrocytes a reduction in CD44­positive cells and in the level of expression of the surface marker were marked. Using PEG­1500 resulted in more pronounced change in erythrocyte CD44 characteristics after freeze­thawing in comparison with glycerol. Removal of cryoprotectants and the loss of a part of cells during the washing process led to the restoration of the CD44 characteristics in freeze­thawed erythrocytes suspension which successfully survived after the stresses. The results indicate that revealed changes in cryopreserved erythrocytes cover only a part of the cells, and they are associated with the instability of the population of erythrocytes with altered CD44 characteristics wherethrough after the removal of cryoprotectants with concomitant hemolysis of unstable cells the CD44 parameters in erythrocyte suspensions recovered. The mechanisms underlying the changes in the parameters of the surface marker CD44 in freeze­thawed erythrocyte may be related to the disruption of intermolecular interactions in the membrane under the influence of physical and chemical environmental factors, followed by the membrane vesiculation with the inclusion of the CD44 into the vesicles.

Effect of substances with cryoprotective properties on surface marker CD44 in human erythrocytes.

In this study there were investigated the changes in surface marker CD44 in human erythrocytes exposed to cryoprotective media, as well as the impact of oxidative modification of membrane-cytoskeleton proteins on the CD44 characteristics under the changed physico-chemical parameters of the cellular environment. Glycerol, DMSO, sucrose, and PEG-1500 were shown to cause a decrease in CD44 expression level and amount of CD44-positive cells during prolonged exposure erythrocytes with them. That may reflect subtle adjustments in the system of protein-protein interactions in erythrocyte membrane-cytoskeleton complex, which may affect the stability of cells during cryopreservation. Extracellular substances (sucrose and PEG-1500) exhibited a more pronounced effect on the CD44 in erythrocytes in comparison with the examined substances of endocellular type. Modification of membrane-cytoskeleton proteins with oxidizing bifunctional reagent diamide enhanced the identified tendencies.