Purification and partial characterization of the M(r) 30,000 integral membrane protein associated wih the erythrocyte Rh(D) antigen

P. Agre, A. M. Saboori, A. Asimos, B. L. Smith

Research output: Contribution to journalArticle

Abstract

Erythrocytes bearing the Rh(D) antigen have an M(r) 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 > 95% pure and migrated as a diffuse band of M(r) 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 M(r) 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 protrusion, exists normally as an oligomer, and can be purified in denatured form.

Original languageEnglish (US)
Pages (from-to)17497-17503
Number of pages7
JournalJournal of Biological Chemistry
Volume262
Issue number36
StatePublished - Dec 1 1987

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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