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Cytotechnology

, Volume 14, Issue 2, pp 97–107 | Cite as

Identification and characterization of major bovine serum tyrosine-O-sulfate-binding protein as a complement factor H

  • Yoichi Sakakibara
  • Masahito Suiko
  • P. H. P. Fernando
  • Tomio Ohashi
  • Ming-Cheh Liu
Regular Research Papers

Abstract

A major tyrosine-O-sulfate (TyrS)-binding protein present in bovine serum was purified to electrophoretic homogeneity using a combination of TyrS-Affi-Gel 10 affinity chromatographyy, DEAE-Bio-Gel A ion-exchange chromatography, and hydroxylapatite chromatography. The purified TyrS-binding protein migrated as doublet protein bands with apparent molecular weights of ca. 160, 000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. N-termini of the two forms of purified TyrS-binding protein contain most likely identical sequence for the first fifteen amino acids residues, which displays a high degree of homology to those of human and mouse complement factor H. Furthermore, the purified TyrS-binding protein exhibited immunologic cross-reactivity with anti-human complement factor H. These results indicate the identity of the purified TyrS-binding protein being bovine complement factor H. The two forms of the purified bovine factor H were investigated with respect to the sensitivity to limited trypsin digestion. The high-molecular weight form was cleaved into two fragments with apparent molecular masses of, respectively, 45 kD and 125 kD. The low-molecular weight form was cleaved in a different manner to generate three major fragments with molecular masses of 25 kD, 45 kD and 100 kD, respectively. Limited V8 protease mapping of the two forms yielded similar, yet unidentical, peptide band patterns. Purified bovine factor H appeared to bind agarose-bonded heparin through its anion-binding domain and the binding was inhibited by the presence of free heparin or dextran sulfate.

Key Words

Tyrosine-O-sulfate protein-protein interaction complement factor H heparin 

Abbreviations

HEPES

N-2-hydroxylpiperazine-N′-2-ethanesulfonic acid

NP-40

Nonidet P-40

PBS

phosphate-buffered saline

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TyrS

tyrosine-O-sulfate

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Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • Yoichi Sakakibara
    • 1
    • 2
  • Masahito Suiko
    • 2
  • P. H. P. Fernando
    • 2
  • Tomio Ohashi
    • 2
  • Ming-Cheh Liu
    • 1
  1. 1.Department of BiochemistryThe University of Texas Health Center at TylerTylerUSA
  2. 2.Department of Biological Resource SciencesMiyazaki UniversityMiyazaki, MiyazakiJapan

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