Mosher D F
J Biol Chem. 1976 Mar 25;251(6):1639-45.
Experiments were performed to investigate whether proteins other than fibrin are substrates for activated fibrin-stabilizing factor (FSF, blood coagulation Factor XIII, plasma transglutaminase) in clotting whole plasma. Three fluorescently labeled polypeptides were identified in serum prepared by clotting normal, but not FSF-deficient, plasma in the presence of the fluorescent amine, N-(5-aminopentyl)-5-dimethyl-aminonaphthalene-1-sulfonamide (dansylcadaverine). The major labeled polypeptide had a Mr (estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate) of 1.6 times 10(5) and was found in the protein fraction precipitated by 33 to 50% saturated ammonium sulfate. The second had a Mr of 2.0 times 10(5), was found in the protein fraction insoluble in 33% saturated ammonium sulfate, and was precipitated by gamma-globulin directed against cold-insoluble globulin. The third had a Mr of 1.1 times 10(5) and was precipitated by 33 to 50% saturated ammonium sulfate. All three polypeptides were found in the first protein peak when labeled serum was chromatographed on Sephadex G-200. The immunoprecipitin arc containing alpha2-macroglobulin was fluorescent when labeled serum was analyzed by immunoelectrophoresis. These results indicate that alpha2-macroglubulin, cold-insoluble globulin, and an unidentified third protein with a subunit of Mr = 1.1 times 10(5) are transamidated by FSF in clotting plasma. The concentration of cold-insoluble globulin was decreased in serum formed at 37 degrees from normal, but not from FSF-deficient, plasma. The depletion of cold-insoluble globulin in normal serum was partially blocked by clotting in the presence of dansylcadaverine and completely blocked by clotting in the absence of calcium ions. Sera formed at 2 degrees from both normal and FSF-deficient plasma contained less cold-insoluble globulin than plasma. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of clots formed at 2 degrees demonstrated cross-linking of cold-insoluble globulin to fibrin in the normal, but not the FSF-deficient, sample. The serum concentration of alpha2-macroglobulin was the same as the plasma concentration irrespective of the conditions of clotting. Thus, the experiments suggest that FSF catalyzes the cross-linking of cold-insoluble globulin (but not alpha2-macroglobulin) to fibrin in clotting plasma.
进行实验以研究在凝血全血浆中，除纤维蛋白之外的蛋白质是否是活化纤维蛋白稳定因子（FSF，血液凝固因子XIII，血浆转谷氨酰胺酶）的底物。在荧光胺N-(5-氨基戊基)-5-二甲基氨基萘-1-磺酰胺（丹磺酰尸胺）存在的情况下，通过使正常血浆而非FSF缺陷血浆凝固制备的血清中，鉴定出三种荧光标记的多肽。主要的标记多肽的相对分子质量（通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳估算）为1.6×10⁵，存在于用33%至50%饱和硫酸铵沉淀出的蛋白质组分中。第二种多肽的相对分子质量为2.0×10⁵，存在于不溶于33%饱和硫酸铵的蛋白质组分中，并且可被抗冷不溶性球蛋白的γ球蛋白沉淀。第三种多肽的相对分子质量为1.1×10⁵，可被33%至50%饱和硫酸铵沉淀。当标记血清在葡聚糖凝胶G-200上进行层析时，所有这三种多肽都出现在第一个蛋白质峰中。当通过免疫电泳分析标记血清时，含有α2-巨球蛋白的免疫沉淀弧呈荧光。这些结果表明，α2-巨球蛋白、冷不溶性球蛋白以及一种相对分子质量为1.1×10⁵亚基的未鉴定的第三种蛋白质在凝血血浆中被FSF转酰胺化。在37℃由正常血浆而非FSF缺陷血浆形成的血清中，冷不溶性球蛋白的浓度降低。在丹磺酰尸胺存在的情况下凝血可部分阻断正常血清中冷不溶性球蛋白的消耗，而在无钙离子的情况下凝血可完全阻断这种消耗。在2℃由正常血浆和FSF缺陷血浆形成的血清中，冷不溶性球蛋白的含量均低于血浆。在2℃形成的凝块的十二烷基硫酸钠聚丙烯酰胺凝胶电泳显示，在正常样品而非FSF缺陷样品中，冷不溶性球蛋白与纤维蛋白发生交联。无论凝血条件如何，α2-巨球蛋白的血清浓度与血浆浓度相同。因此，这些实验表明，FSF催化凝血血浆中冷不溶性球蛋白（而非α2-巨球蛋白）与纤维蛋白的交联。
