title: Binding of intracellular protein to the erythrocyte membrane during incubation: the production of Heinz bodies.
abstract: During sterile incubation of normal human erythrocytes at 37 degrees C., intracellular nonhemoglobin protein is bound to the membrane prior to hemolysis. These studies have characterized this phenomenon further. Protein binding to the membrane began after 12 hours incubation when cellular ATP was depleted and increased to 36 hours incubation. The binding was prevented by adding adenosine or glucose at the start of incubation and was arrested by adding adenosine to regenerate ATP during the course of incubation. However, protein, once bound, was not released by regeneration of ATP. The amount of protein bound was not altered by: (1) addition of Ca++ or EDTA to the medium, (2) blockade of sulfhydryl groups with N-ethylmaleimide, or (3) stabilization of heme-globin bonds by conversion of hemoglobin to cyanmethemoglobin. Conversion of hemoglobin to carboxyhemoglobin by incubations under carbon monoxide inhibited protein binding, but this appeared to be an effect of exclusion of oxygen rather than stabilization of heme-globin bonds since incubation under nitrogen had a similar effect. The morphological counterpart of this chemically-measured membrane-bound protein was visible in red cell ghosts stained with crystal violet as small membrane-associated particles resembling Heinz bodies. Sodium dodecyl sulfate acrylamide gel electrophoresis of membranes of incubated cells revealed a new protein band that was identical to globin monomers. This membrane binding of globin during incubation provides a model for the study of Heinz body formation in clinical disorders.
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002248:Carbon Monoxide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006366:Heinz Bodies / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D002248:Carbon Monoxide; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D005947:Glucose; D000241:Adenosine; D005033:Ethylmaleimide; D002118:Calcium