Increased adsorption of cytoplasmic proteins to the erythrocyte membrane in ATP-depleted normal and pyruvate kinase-deficient mature cells and reticulocytes

Abstract

How the metabolic defect of pyruvate kinase deficiency (PK(-)) accelerates red blood cell (RBC) destruction is not established, but may be related to RBC membrane abnormalities associated with altered cellular metabolism. Furthermore, it has been shown that PK(-) reticulocytes are especially sensitive to metabolic depletion. Therefore, we compared the membranes of reticulocyte-rich PK(-) RBC, both fresh and ATP depleted, with membranes of fresh and ATP depleted normal mature RBC and reticulocytes. There was no difference between the specific gravity (SG) of the membranes of normal mature RBC (SG 1.152 +/- 0.004) and membranes of reticulocyte-rich RBC from several anemias (SG 1.150 +/- 0.002). However, membranes from fresh, reticulocyte-rich PK(-) RBC were dense with SG of 1.165 +/- 0.004 which correlated with a corresponding increase of protein to lipid phosphorus ratio of 66 +/- 8 micrograms protein/micrograms lipid phosphorus (normal 52 +/- 6 micrograms/micrograms). The membrane density of PK(-) RBC was further increased when the PK(-) RBC ATP was depleted by anaerobic incubation (SG 1.188 +/- 0.004) or cyanide inhibition (SG 1.182 +/- 0.001). When ATP was depleted in normal RBC and in non-PK(-) reticulocytes, corresponding increases in membrane SG occurred. A distinctive 50,000 MW peptide is adsorbed from the cytoplasm to the membranes of reticulocytes (both normal and PK(-) when these cells were depleted of ATP. The increased membrane adsorption of cytoplasmic proteins by PK(-) RBC was not associated with increased RBC calcium uptake, sulfhydryl oxidation, or altered membrane protein phosphorylation. All the observed abnormalities of PK(-) RBC membranes could by reproduced by ATP depletion of reticulocyte-enriched non-PK(-) RBC.