Radek J T, Jeong J M, Wilson J, Lorand L
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208.
Biochemistry. 1993 Apr 13;32(14):3527-34. doi: 10.1021/bi00065a002.
Interactions of a recombinant human placental protein (rA2) expressed in yeast and considered to be identical to the catalytic A2 subunits of factor XIII, the fibrin stabilizing factor zymogen, were examined with the native carrier subunits (B2) of the factor isolated from human plasma. Nondenaturing electrophoresis and HPLC gel-filtration experiments showed a tight binding of rA2 to B2 for forming an ensemble similar to that of plasma factor XIII (A2B2). In the presence of excess B2, however, some higher ordered oligomers (rA2Bn, where n > 2) were also seen in electrophoresis. The same technique revealed a microheterogeneity in the rA2 preparation; nevertheless, all isoforms could bind to B2. By employing an ELISA procedure for measuring free B2 in mixtures with rA2, an apparent binding constant of 4 x 10(7) M-1 was derived for the association of rA2 with B2. Fluorescence depolarization was used to monitor the heterologous association of rA2 with fluorescein-labeled B2F as well as the dissociation of the rA2B2F structure. The former was characterized by an increase, and the latter by a decrease, in the fluorescence anisotropy of the system. Binding of rA2 to B2F (pH 7.5, mu = 0.315, 37 degrees C) was not influenced by low concentrations of Ca2+ (< or = 30 mM), and rA2B2F proved to be quite stable under these conditions. Much higher concentrations of Ca2+, as well as higher ionic strengths, were required to dissociate this assembly. By contrast, release of B2F from the thrombin-modified rA2'B2F occurred rapidly in the presence of low concentrations of Ca2+ at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)
对在酵母中表达的一种重组人胎盘蛋白（rA2）进行了研究，该蛋白被认为与纤维蛋白稳定因子（凝血因子XIII）的催化性A2亚基相同，研究其与从人血浆中分离出的该因子的天然载体亚基（B2）的相互作用。非变性电泳和高效液相色谱凝胶过滤实验表明，rA2与B2紧密结合，形成类似于血浆凝血因子XIII（A2B2）的聚集体。然而，在过量B2存在的情况下，电泳中也出现了一些高阶寡聚体（rA2Bn，其中n>2）。同样的技术揭示了rA2制剂存在微不均一性；不过，所有同工型都能与B2结合。通过采用酶联免疫吸附测定法（ELISA）测量与rA2混合后的游离B2，得出rA2与B2结合的表观结合常数为4×10⁷ M⁻¹。利用荧光偏振监测rA2与荧光素标记的B2F的异源结合以及rA2B2F结构的解离。前者表现为体系荧光各向异性增加，后者表现为降低。rA2与B2F的结合（pH 7.5，μ = 0.315，37℃）不受低浓度Ca²⁺（≤30 mM）影响，并且rA2B2F在这些条件下相当稳定。需要更高浓度的Ca²⁺以及更高的离子强度才能使该聚集体解离。相比之下，在低离子强度下，低浓度Ca²⁺存在时，凝血酶修饰的rA2'B2F中的B2F会迅速释放。（摘要截短于250词）
