Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen

• Published in: The Journal of biological chemistry Vol. 262; no. 36; pp. 17497 - 17503
• Main Authors: Agre, P, Saboori, A M, Asimos, A, Smith, B L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.12.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Biological and medical sciences; Cell coat. Cell surface; Cell structures and functions; Electrophoresis, Polyacrylamide Gel; Erythrocytes - analysis; Fundamental and applied biological sciences. Psychology; Humans; Membrane Proteins - analysis; Membrane Proteins - isolation & purification; Molecular and cellular biology; Molecular Weight; Rh-Hr Blood-Group System; Spectrin; Membrane protein; Purification; Gel electrophoresis; Actins; Red blood cell; Molecular interaction; Oligomer; Cell surface; Rh nul blood group; Antigen; Gel filtration; Dimer
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)45408-7

Erythrocytes bearing the Rh(D) antigen have an Mr 30,000 integral membrane protein which can be surface-labeled with 125I and can be quantitatively immunoprecipitated from Triton X-100-solubilized spectrin-depleted membrane vesicles. The 125I-labeled Rh(D)-associated protein was purified to radiochemical homogeneity from membrane skeletons solubilized in sodium dodecyl sulfate and urea by hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. The Rh(D)-associated protein was purified nearly 200-fold from 2 units of erythrocytes from DD individuals by employing similar methods on a large scale using the purified 125I-labeled Rh(D)-associated protein as a tracer. The product appeared to be greater than 95% pure and migrated as a diffuse band of Mr approximately 30,000-32,000 on silver-stained sodium dodecyl sulfate electrophoresis gels poured from 12% acrylamide. It is estimated that the Rh(D)-associated protein makes up approximately 0.5% of the original membrane protein. When concentrated, partially purified Rh(D)-associated protein forms dimers and larger oligomers which are stable in sodium dodecyl sulfate and urea. The Rh(D)-associated protein was protected from degradation when intact erythrocytes or inside out membrane vesicles were enzymatically digested. These studies indicate that the Mr 30,000 protein associated with the Rh(D) antigen is linked to the membrane skeleton, resides within the lipid bilayer with minimal extra- or intracellular protrusions, exists normally as an oligomer, and can be purified in denatured form.