gamma-Carboxyglutamic acid (Gla)-domainless blood coagulation factor IXa species: preparation and properties.
To investigate the function of the gamma-carboxyglutamic acid (Gla) residues of factor IXa in the activation of factor X, a new species of bovine factor IXa, designated "factor IXa beta'," and its corresponding Gla-domainless form, designated "Gla-domainless factor IXa beta'," were prepared under controlled conditions and characterized. First, bovine factor IXa alpha was converted by alpha-chymotrypsin in the presence of calcium ions to factor IXa beta' (Mr 47,000). Compared with factor IXa beta, factor IXa beta' had essentially identical activities towards a synthetic substrate, benzoyl-L-arginine ethylester (BAEE), towards an active site titrant, p-nitrophenyl-p'-guanidinobenzoate, and towards protein substrate, namely, factor X. Next, the Gla-rich region (residues 1-41) of the light chain was removed from factor IXa beta' by additional selective cleavage by alpha-chymotrypsin in the absence of calcium ions. Gla-domainless factor IXa beta' was purified to homogeneity on a column of DEAE-Sepharose CL-6B. The heavy chain was not altered by either chymotryptic digestion. Functional comparisons of the three activated forms, namely, factor IXa alpha, factor IXa beta', and Gla-domainless factor IXa beta', with factor IXa beta revealed that all four activated forms of factor IX had one active-site residue per molecule and essentially identical specific esterase activity towards BAEE. However, the clotting activity of Gla-domainless factor IXa beta' was less than 0.5% of that of factor IXa beta'.(ABSTRACT TRUNCATED AT 250 WORDS)
Morita
T
T
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Fukudome
K
K
Miyata
T
T
Iwanaga
S
S
D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015949:Factor IXa / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry
D010455:Peptides; D015055:1-Carboxyglutamic Acid; D015949:Factor IXa