A phenomenological difference between membrane skeletal protein complexes isolated from normal and hereditary spherocytosis erythrocytes.
Membrane skeletons may be obtained from human erythrocytes by extraction with non-ionic detergent. When treated under defined conditions with a cAMP-independent kinase preparation from normal membranes, a suspension of these membrane skeletons sets to a gelatinous mass. Membrane skeletons from the cells of hereditary spherocytosis patients fail to show this response. Those from subjects with some other haemolytic anaemias do not share the abnormality. The gelation process could be shown also to occur with normal membrane skeletons, extracted at high ionic strength, and containing essentially only the structural protein constituents, spectrin, actin, 4.1 and 4.9. It also occurred rapidly when a column-purified kinase preparation was used, so that no significant amounts of contaminating proteins were introduced. Added spectrin, 4.1 or actin in moderate amounts did not induce gelation in the presence of ATP. Cytochalasin E did not perturb the gelation process. Gelation required ATP as well as kinase, and did not occur when the non-hydrolysable analogue, AMP X PNP, was used instead. Gelation was accompanied by phosphorylation of the spectrin alone, and is thus evidently a consequence of the modification of its properties by this means. Inhibition of phosphorylation by added adenosine retarded gelation. It may be inferred that phosphorylation of spectrin generates new, probably weak, non-covalent interactions between cytoskeletal constituents that cause association of the isolated cytoskeletons. A semi-quantitative method of observing the gelation process, based on the time of incubation before the membrane skeleton suspension ceases to flow under gravity at a low shear, is described.
D000199:Actins; D000255:Adenosine Triphosphate; D001798:Blood Proteins / Q000302:isolation & purification* / Q000502:physiology; D004910:Erythrocyte Membrane / Q000032:analysis*; D005782:Gels; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000502:physiology; D010766:Phosphorylation; D011489:Protein Denaturation; D011494:Protein Kinases; D013049:Spectrin; D013103:Spherocytosis, Hereditary / Q000097:blood*
D000199:Actins; D001798:Blood Proteins; D005782:Gels; D008565:Membrane Proteins; D013049:Spectrin; D000255:Adenosine Triphosphate; D011494:Protein Kinases