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Purification from guinea pig erythrocyte stroma of a decay-accelerating factor for the classical c3 convertase,
Insights
Researchers isolated a decay-accelerating factor of stroma (DAF-S) from guinea pig red blood cells. This protein regulates the complement system
Area of Science:
- Immunology
- Biochemistry
Background:
- The complement system is crucial for innate immunity.
- Regulation of complement activation is vital to prevent self-damage.
Purpose of the Study:
- To isolate and characterize a protein responsible for accelerating the decay of the classical C3 convertase (C4b2a).
- To investigate the properties and species-specific antigenicity of the isolated protein.
Main Methods:
- Protein isolation from guinea pig erythrocyte stroma using butanol extraction and multiple chromatography techniques.
- Molecular weight determination using SDS-PAGE and alkaline gradient gels.
- Generation of a monospecific antiserum to assess protein function and antigenicity.
Main Results:
- A protein, decay-accelerating factor of stroma (DAF-S), was purified.
- DAF-S exhibits varying molecular weights, suggesting subunit composition.
- The generated antiserum neutralized DAF-S activity and showed species-specific agglutination of guinea pig erythrocytes.
Conclusions:
- DAF-S is an integral membrane protein that accelerates the decay of the classical C3 convertase.
- DAF-S possesses species-specific antigens on the guinea pig erythrocyte surface.
Abstract:
A protein with decay-accelerating activity for the classical C3 convertase, C4b2a, has been isolated on the basis of this function from guinea pig erythrocyte stroma. The isolation procedure for decay-accelerating factor of stroma (DAF-S) utilizes butanol extraction and chromatography on DEAE-Sephacel, hydroxylapatite, and phenyl Sepharose. Purified DAF-S has a m.w. of 60,000 and 65,000 on reduced and unreduced SDS gels, respectively, and exhibits m.w. of 30,000 and 175,000 on alkaline gradient gels, suggesting multiples of a 60,000-65,000 subunit. Purified DAF-S elicited a monospecific antiserum whose IgG fraction neutralized the decay-accelerating activity for C4b,2a affixed to 10(7) sheep erythrocytes (EAC1,4,2) in a dose-response fashion. The monospecific antiserum diluted up to 1:5120 agglutinated 1 x 10(6) guinea pig erythrocytes, but not sheep or human erythrocytes, suggesting that DAF-S, an integral membrane protein, has species-specific antigens that are expressed on the surface of the guinea pig erythrocyte.