Solubilization and Structural Integrity of the Human Red Cell Membrane (original) (raw)

Abstract

Solubilization of structural protein components of red-cell ghosts and alterations of their biological activities by treatment with various solvents was attempted. Ghosts were suspended in the solvents and then separated into supernates and residues by high-speed ultracentrifugation. Treatment with 8 M urea yielded the highest degree of solubilization (60 per cent) and approximately 80 per cent of the protein-bound sialic acid was found in the supernate. Non-urea solvents yielded supernates rich in proteins but low in phosphorus and carbohydrates. Urea starch gel electrophoresis demonstrated the heterogeneity of the solubilized material. Serological studies showed high sensitivity of the Rho (D)-antigen to treatment with urea, mercaptoethanol and iodoacetamide. The M-antigen was present in all residues and some M activity was found in the urea supernate. No acetylcholinesterase (AChE) activity was detected in the residue and supernate of stroma treated with 8 M urea. To ascertain the critical molarity, red blood cells and ghosts were incubated at increasing urea concentrations (0.5-6.0 M) and complete loss of AChE activity was observed with 4 M urea. Considerable reduction of AChE (70 per cent) was found after mild reduction and alkylation of stroma. When mercaptans and iodoacetamide were tested separately, no significant effect on AChE activity was observed except with high concentrations of cysteine. In our previous study (Poulik and Lauf, 1965) to elucidate the nature of protein structural components of human red-cell membranes, water-soluble high molecular weight proteins (molecular weight about 200,000) were extracted by a biphasic butanol-water system of Maddy (r964). The water-soluble material showed, however, a considerable heterogeneity when tested by urea starch gel electrophoresis. The high carbohydrate, low nitrogen and phosphorus contents, as well as the presence of A, M and N substances, further confirmed the heterogeneous nature of this material. The yield of this material was increased when the stroma was cleaved reductively prior to butanol extraction. Although A, M and N substances were found serologically active after treatment with butanol, 8 M urea, or reduction and alkylation in the presence and absence of urea, the Rh,(D)-antigen could not be detected. On the basis of these findings, studies were undertaken to explore the effects of urea, mercaptans, and other solvents on the serological activity (e.g. blood group antigens) and acetylcholinesterase activity (AChE) of intact red cells and their ghosts. These studies have shown that urea and reductive cleavage followed by alkylation irreversibly inactivated AChE and the Rh-antigen. High concentrations of cysteine also reduced the activity of AChE in intact cells. The results partially confirm those observed by Sirchia, Ferrone, Milani and Mercuriali (1966).

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