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Related Experiment Videos

Purification and partial characterization of rat factor D.

B C Baker1, C J Campbell, C J Grinham

  • 1Department of Biochemistry, Glaxo Group Research Ltd., Middx., U.K.

The Biochemical Journal
|November 1, 1991
PubMed
Summary

Rat factor D, a serum protein, shares functional similarities with human and mouse counterparts but is heavily glycosylated. Deglycosylation reveals structural resemblance to human factor D.

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Area of Science:

  • Biochemistry
  • Proteomics

Background:

  • Factor D is a key complement system enzyme.
  • Understanding species-specific Factor D characteristics is crucial for comparative immunology and drug development.

Purpose of the Study:

  • To purify and characterize rat factor D.
  • To compare its biochemical and structural properties with human and mouse factor D.

Main Methods:

  • Purification of rat factor D using multiple chromatography techniques (CM-Sepharose, phenyl-Sepharose, Mono S).
  • Enzymatic deglycosylation and SDS-PAGE analysis.
  • N-terminal amino acid sequencing.

Main Results:

  • Rat factor D purified to homogeneity exhibits similar substrate specificity and inhibitor susceptibility as human and mouse Factor D.

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  • The rat enzyme is heavily N-linked glycosylated, contributing approximately 15,000 Da to its molecular weight.
  • Deglycosylation results in a molecular weight comparable to human Factor D.
  • High sequence similarity observed with human Factor D (over 76%) and mouse adipsin (over 93%).

    • Conclusions:

    • Rat factor D is a heavily glycosylated protein with conserved functional domains but distinct post-translational modifications compared to its counterparts.
    • The deglycosylated form of rat factor D closely resembles human Factor D structurally.
    • N-terminal sequencing confirms significant evolutionary conservation between rat, human, and mouse Factor D.