Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase.

Abstract

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.

• Authors: K Suehiro, S Kawabata, T Miyata, H Takeya, J Takamatsu, K Ogata, T Kamiya, H Saito, Y Niho, S Iwanaga
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 264 Issue 35 Pg. 21257-65 (Dec 15 1989) ISSN: 0021-9258 [Print] United States
• PMID: 2592373 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• factor IX BM Nagoya
• Tryptophan
• Factor IX
• Arginine
• Serine Endopeptidases
• Chymotrypsin
• Chymases

Topics

• Amino Acid Sequence
• Animals
• Arginine
• Chymases
• Chymotrypsin (metabolism)
• Enzyme Activation
• Factor IX (genetics, metabolism)
• Humans
• Kinetics
• Mast Cells (enzymology)
• Molecular Sequence Data
• Mutation
• Peptide Mapping
• Rats
• Reference Values
• Serine Endopeptidases (metabolism)
• Tryptophan