The changes in erythrocyte Ca2+-ATPase activity induced by PEG-1500 and low temperatures

Аннотация

Various organic compounds are applied upon cryopreservation and their adding into cell suspension causes modification of subcellular systems, providing cell survival during freeze–thawing. The aim of the study was to assess the modifying effect of cryoprotectant PEG-1500 and low temperatures on Ca²⁺-ATPase activity in saponin-permeabilized erythrocytes. PEG-1500 was revealed to inhibit erythrocyte Ca²⁺-ATPase activity despite the presence of endogenous effectors able to stimulate the enzyme function. Presumably, the Ca²⁺-ATPase modification was determined by the physicochemical properties of the polymer solution, since the removal of PEG-1500 out of the medium recovered the enzyme activity. Reversibility of Ca²⁺-ATPase inhibition was characteristic of erythrocytes both exposed to cryoprotectant without freezing and frozen–thawed in the PEG-1500 presence. The cell freeze–thawing without cryoprotectant had no effect on Ca²⁺-ATPase, suggesting that membrane form of enzyme is cryoresistent. Although the efficiency of erythrocyte cryopreservation with PEG-1500 depends on the incubation temperature before freezing stage, the functional indices of Ca²⁺-ATPase in erythrocytes exposed to PEG-1500 at 37 and 5–7°C had no significant distinctions if the subsequent ATP hydrolysis was conducted at 37°C. However, the enzyme activity was additionally slowed down when the temperature of enzymatic reaction was decreased to 5–7°C after erythrocyte preincubation with PEG-1500 under the same conditions. The identified changes in Ca²⁺-ATPase activity in erythrocytes in the PEG-1500 presence were most likely determined by a modifying effect of the cryoprotectant on the membrane structure; as a result, the Ca²⁺-ATPase endogenous effectors present in the medium could not overcome the restrictions imposed on the enzyme function by a modified membrane macroenvironment.