No abstract

We investigated the erythrocyte mem-

The aim of the present work was to understand the pathophysiology of the severe human thalassemias as represented by beta-thalassemia intermedia and hemoglobin (Hb) H (alpha-thalassemia) disease. We have previously shown that the material properties of the red blood cell (RBC) and its membrane differ in severe alpha- and beta-thalassemia, and we now show that this difference is probably caused by accumulation of alpha-globin chains at the cytoskeleton in beta-thalassemia, whereas beta-globin chains are associated with the cytoskeleton in alpha- thalassemia. In both alpha- and beta-thalassemia, some of these globin chains have become oxidized as evidenced by loss of the free thiols. Furthermore, there is similar evidence of oxidation of protein 4.1 in beta-thalassemia, whereas beta-spectrin appears to be subject to oxidation in alpha-thalassemia. These observations support the idea that the association of partly oxidized globin chains with the cytoskeleton results in oxidation of adjacent skeletal proteins. The abnormality of protein 4.1 in beta-thalassemia is consistent with a prior observation, and is also in accord with the known importance of protein 4.1 in maintenance of membrane stability, a property that is abnormal in beta-thalassemic membranes.

We studied the RBC membrane proteins of four patients, including a mother and daughter, with hereditary stomatocytosis. One- and two-dimensional gel electrophoresis revealed that a 28 kDa integral protein, present in normal RBC membranes, was absent in all four patients. This abnormality, reported once previously (Lande et al, 1982), appears to be a characteristic feature of hereditary stomatocytosis, and may be related to the underlying permeability defect in this disorder.

The marked increase in cation (Na+, K+) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). 45Ca influx in intact RBC, 45Ca efflux in red ghosts, and 45Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT32P--only 20--40% of normal control values in both shortterm (5 min) and more extended (60 min) incubations--is not improved by DMA. After reaction of 14C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes in corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3--9 A, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.