Jacobs M, Freedman S J, Furie B C, Furie B
Center for Hemostasis and Thrombosis Research, New England Medical Center, Boston, Massachusetts.
J Biol Chem. 1994 Oct 14;269(41):25494-501.
The fully gamma-carboxylated peptides based upon the complete and truncated Gla/aromatic amino acid stack domains of human Factor IX were prepared by solid phase peptide synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. A 47-residue peptide Factor IX-(1-47) and a 42-residue peptide Factor IX-(1-42), both containing 12 residues of L-gamma-carboxyglutamic acid, were purified by high performance liquid chromatography and oxidized to form the disulfide bond. Quantitative gamma-carboxyglutamic acid analysis of Factor IX-(1-47) and Factor IX-(1-42) indicated the presence of 12.1 and 11.2 gamma-carboxyglutamic acid residues/mol of peptide, respectively; no glutamic acid was detected. As monitored by fluorescence quenching, calcium ions induced the prototypical conformational transition in Factor IX-(1-47), but not in Factor IX-(1-42), that is observed with Factor IX. Half-maximal quenching of the intrinsic fluorescence of Factor IX-(1-47) was observed at Ca(II) concentrations of about 50 microM. Factor IX-(1-47) bound to the conformation-specific antibodies, anti-Factor IX:Mg(II) and anti-Factor IX:Ca(II)-specific in the presence of metal ions. Factor IX-(1-47) bound to phospholipid membranes, as monitored by energy transfer from intrinsic fluorophores to dansyl (5-dimethylaminonaphthalene-1-sulfonyl)-phosphatidylethanolamine incorporated into a lipid bilayer composed of phosphatidylserine:phosphatidylcholine. In contrast, Factor IX-(1-42) bound poorly to these same membranes. Factor IX-(1-47) did not inhibit Factor XIa activation of Factor IX but did inhibit the activation of Factor X by Factor IXa bound to Factor VIII in the presence of calcium ions and phospholipid. These results show that phospholipid membrane binding is a property of the Gla/aromatic amino acid stack domain and that the Factor IX-(1-47) peptide, prepared by chemical synthesis, preserves the membrane binding properties and the metal-induced conformational transitions observed in native Factor IX. These results indicate that Factor IX-(1-47) but not Factor IX-(1-42) is a suitable model for structural studies of Factor IX-membrane interaction.
基于人凝血因子IX完整和截短的Gla/芳香族氨基酸堆叠结构域的完全γ-羧化肽，采用Fmoc（N-（9-芴基）甲氧基羰基）化学方法通过固相肽合成制备。一种含有12个L-γ-羧基谷氨酸残基的47个残基的肽凝血因子IX-(1-47)和一种含有12个L-γ-羧基谷氨酸残基的42个残基的肽凝血因子IX-(1-42)，通过高效液相色谱法纯化并氧化形成二硫键。对凝血因子IX-(1-47)和凝血因子IX-(1-42)的定量γ-羧基谷氨酸分析表明，每摩尔肽分别存在12.1和11.2个γ-羧基谷氨酸残基；未检测到谷氨酸。通过荧光猝灭监测，钙离子在凝血因子IX-(1-47)中诱导了典型的构象转变，但在凝血因子IX-(1-42)中未诱导，这与凝血因子IX中观察到的情况相同。在Ca(II)浓度约为50 microM时观察到凝血因子IX-(1-47)内在荧光的半数最大猝灭。凝血因子IX-(1-47)在金属离子存在下与构象特异性抗体抗凝血因子IX:Mg(II)和抗凝血因子IX:Ca(II)特异性结合。通过从内在荧光团到掺入由磷脂酰丝氨酸:磷脂酰胆碱组成的脂质双层中的丹磺酰（5-二甲基氨基萘-1-磺酰基）-磷脂酰乙醇胺的能量转移监测，凝血因子IX-(1-47)与磷脂膜结合。相比之下，凝血因子IX-(1-42)与这些相同的膜结合较差。凝血因子IX-(1-47)不抑制凝血因子XIa对凝血因子IX的激活，但在钙离子和磷脂存在下确实抑制与凝血因子VIII结合的凝血因子IXa对凝血因子X的激活。这些结果表明磷脂膜结合是Gla/芳香族氨基酸堆叠结构域的一种特性，并且通过化学合成制备的凝血因子IX-(1-47)肽保留了在天然凝血因子IX中观察到的膜结合特性和金属诱导的构象转变。这些结果表明凝血因子IX-(1-47)而非凝血因子IX-(1-42)是凝血因子IX-膜相互作用结构研究的合适模型。
