Development of Highly Sensitive Methods for the Detection of Fungi Utilizing the Horseshoe Crab Hemolymph Coagulation Cascade.

马蹄蟹血液细胞裂解物与(1*3)-β-D-葡聚糖反应，启动一系列酶促反应，最终形成血栓。我们已经纯化了丝氨酸蛋白酶酶原，因子G，它直接被(1*3)-β-D-葡聚糖激活，启动血淋巴凝血级联反应。因子G是一种异源二聚体蛋白，由两个非共价结合的亚基α(72 KDa)和β(37 KDa)组成。在(1*3)-β-D-葡聚糖的存在下，G因子被自动催化活化为具有活性的丝氨酸蛋白酶，称为G^-因子。这种激活伴随着两个亚基的有限蛋白降解：72 kDa的亚基α被切割成55 kDa和17 kDa的片段，37 kDa的亚基被缩短为34 kDa。与(1*3)-β-D-葡聚糖孵育较长时间会导致55 kDa的片段裂解为46 kDa，34 kDa的片段裂解为32 kDa，伴随着酰胺酶活性的丧失。利用参与血淋巴凝血级联反应的纯化蛋白进行重组实验表明，G^^-因子能够直接激活凝血酶，使凝血原转化为凝血凝胶。因此，纯化的因子G被证明是马蹄蟹血细胞裂解物中(1*3)-β-D-葡聚糖敏感的凝血途径的主要启动者。

Horseshoe crab hemocyte lysate responds to (1*3)-beta-D-glucans, initiating an enzymatic cascade which culminates in clot formation. We have purified to homogeneity the serine protease zymogen, factor G,which is directly activated by (1*3)-beta-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently-associated subunits alpha (72 kDa) and beta (37 kDa). In the presence of (1*3)-beta-D-glucans such as curdlan and paramylon, factor G is autocatalytically activated to an active serine protease, named factor G^^-. This activation is accompanied by limited proteolyses of both subunits : the 72-kDa subunit alpha is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is shortened to 34-kDa. Longer incubations with (1*3)-beta-D-glucans result in cleavege of the 55-kDa fragment to 46-kDa and the 34-kDa fragment to 32-kDa, with concomitant loss of amidase activity. Reconstitution experiments using purified proteins participating in the hemolymph clotting cascade demonstrate that factor G^^- is capable of activating proclotting enzyme directly, resulting in the conversion of coagulogen to coagulin gel. Thus, purified factor G is shown to be the primary initiator of the (1*3)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.