Association of Glyceraldehyde-3-phosphate Dehydrogenase with the Plasma Membrane of the Intact Human Red Blood Cell

Abstract

Glycolytic enzymes have been observed to associate in vitro with membranes and cytoplasmic filaments in a variety of systems, but their distribution in vivo is contested.We have therefore examined the distribution of glyceraldehyde-3-phosphate dehydrogenase (G3PD) in the intact human erythrocyte using indirect immunofluorescence and affinity-purified rabbit antibodies to G3PD.Antibody specificity was demonstrated by immunoblotting as well as immunofluorescence experiments with ghosts specifically depleted of and reconstituted with G3PD.Anti-G3PD immunolabeling experiments utilized both fixed whole cells and fixed cell suspensions infused with 2.3 M sucrose, frozen and thick-sectioned.In all experiments a two-step fixation protocol was employed which ensured that cytoplasmic hemoglobin was retained when cells were subjected to Triton X-100 permeabilization, the antigenicity of G3PD was preserved, and antibody penetration was complete.We used mixtures of biotinylated affinity-purified antibodies to GSPD and dichlorotriazinylaminofluorescein-labeled, affinity-purified antibodies to hemoglobin, followed by rhodamine-streptavidin, in double-label experiments.In both whole and sectioned human erythrocytes, G3PD staining was predominantly membrane associated while hemoglobin staining was diffusely distributed throughout the cytoplasm.In isolated ghosts, some G3PD was tightly bound to the membrane and was resistant to elution with phosphate-buffered saline and NAD+/arsenate.However, in immunolabeled rat reticulocytes and erythrocytes G3PD was cytoplasmic.Nucleated human blood cells and platelets also exhibited cytoplasmic G3PD.In approximately 10% of the human erythrocyte population G3PD was also cytoplasmic.These cells were flatter in shape and exhibited strong cytoplasmic immunolabeling for hemoglobin which was sometimes concentrated along the cell membrane; possibly, these cells were late reticulocytes or early erythrocytes.We conclude that G3PD is preferentially associated with the plasma membrane of human erythrocytes in a specific fashion.