Falanga A, Gordon S G
Biochemistry. 1985 Sep 24;24(20):5558-67. doi: 10.1021/bi00341a041.
Cancer procoagulant, a proteolytic procoagulant enzyme, has been purified from rabbit V2 carcinoma extracts by two procedures. In the first, the protein was purified by benzamidine--Sepharose affinity chromatography, gel filtration chromatography, and phenyl-Sepharose hydrophobic chromatography. Antiserum was raised against the purified protein and was used to prepare an immunoadsorbent column. In the second, tumor extracts were purified by immunoaffinity chromatography followed by p-(chloromercuri)benzoate affinity chromatography. The second procedure was substantially quicker and easier. The final product of both procedures was homogeneous on the basis of analytical sodium dodecyl sulfate--polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weight was 68 000 and the isoelectric point 4.8. The proteinase activity of cancer procoagulant directly activated factor X, in the absence of factor VII, and was inhibited by 1 mM iodoacetamide and 0.1 mM mercury which are classic cysteine proteinase inhibitors. A carbohydrate analysis showed less than 1 mol of hexose or sialic acid/mol of protein. The amino acid analysis showed that serine (19.1%), glycine (18.77%), and glutamic acid (12.5%) were the prevalent amino acids. The amino acid composition of cancer procoagulant was substantially different than other known factor X activating proteinases or other cysteine proteinases including cathepsin B.
癌促凝素是一种蛋白水解促凝酶，已通过两种方法从兔V2癌提取物中纯化出来。第一种方法是，通过苯甲脒-琼脂糖亲和色谱、凝胶过滤色谱和苯基-琼脂糖疏水色谱对该蛋白进行纯化。用纯化后的蛋白制备抗血清，并用于制备免疫吸附柱。第二种方法是，先通过免疫亲和色谱，再通过对氯汞苯甲酸亲和色谱对肿瘤提取物进行纯化。第二种方法要快得多且更简便。两种方法的最终产物在分析型十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和等电聚焦的基础上均呈均一状态。其分子量为68000，等电点为4.8。癌促凝素的蛋白酶活性在无因子VII的情况下可直接激活因子X，并被1 mM碘乙酰胺和0.1 mM汞抑制，这两种物质是典型的半胱氨酸蛋白酶抑制剂。碳水化合物分析显示，每摩尔蛋白中己糖或唾液酸含量不到1摩尔。氨基酸分析表明，丝氨酸（19.1%）、甘氨酸（18.77%）和谷氨酸（12.5%）是主要氨基酸。癌促凝素的氨基酸组成与其他已知的因子X激活蛋白酶或其他半胱氨酸蛋白酶（包括组织蛋白酶B）有很大不同。
