The formaldehyde-morpholine method for the conversion of γ-carboxyglutamyl (Gla) residues to γ-methyleneglutamyl (γ-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 K(m)(app) and 25 K(m)(app) the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 γ-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 K(m)(app) provided a homogeneous protein containing 3 γ-MGlu and 7 Gla residues, bovine 3 γ-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperativity associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 γ-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology