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Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte
P Agre1, A M Saboori, A Asimos
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
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.
Insights
Researchers identified and purified the Rh(D)-associated protein, a key component of red blood cell membranes. This integral membrane protein, crucial for Rh(D) antigen function, is linked to the membrane skeleton and exists as an oligomer.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Erythrocytes (red blood cells) possess the Rh(D) antigen, a critical determinant in blood transfusion compatibility.
- The molecular composition and structure of the Rh(D) antigen complex within the erythrocyte membrane are not fully elucidated.
Purpose of the Study:
- To identify, purify, and characterize the integral membrane protein associated with the Rh(D) antigen.
- To investigate the structural properties and membrane localization of the Rh(D)-associated protein.
Main Methods:
- Surface labeling of intact erythrocytes with 125I followed by immunoprecipitation.
- Solubilization of membrane vesicles and purification using hydroxylapatite chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis.
- Large-scale purification using the radiolabeled protein as a tracer.
Main Results:
- A Mr 30,000-32,000 integral membrane protein associated with the Rh(D) antigen was identified and purified to >95% homogeneity.
- The Rh(D)-associated protein constitutes approximately 0.5% of the total erythrocyte membrane protein.
- Purified protein forms stable dimers and oligomers in denaturing conditions and is protected from enzymatic degradation in intact cells, indicating membrane skeleton linkage and limited extracellular/intracellular exposure.
Conclusions:
- The Mr 30,000 protein is intrinsically linked to the Rh(D) antigen complex and the erythrocyte membrane skeleton.
- This protein resides within the lipid bilayer with minimal protrusions, existing physiologically as an oligomer.
- The study provides a method for purifying this denatured protein, aiding further structural and functional analysis.