BIOCHEMICAL AND INNUNOHISTOCHEMICAL STUDIES OF A NOVEL PROTEINASE PRESENT IN THE FOLLICULAR FLUID OF MAMMALIAN OVARY

猪卵巢含有水解肽4-甲基香豆基-7-酰胺(MCA)底物的酶活性，并优先选择Arg-MCA键。这种活性几乎只存在于卵泡液中，并在卵泡成熟过程中数倍增加。负责该活性的一种酶被提纯到明显的均一状态。纯化的wnzyme由两条不同的多肽链组成，每条多肽链的MR-45,000和32,000是共价结合的。该酶对二异丙基氟磷酸盐、苯甲酰胺、亮氨酸酶和止痛剂均有较强的抑制作用，表明该酶是一种分泌型蛋白酶。使用含有MCA的合成肽底物，证实该酶优先水解Arg-X键，而不是Lys-X键。45 kDa和32 kDa多肽的氨基末端氨基酸序列分别与人Lpasma激肽释放酶和人Xia因子的重链和轻链高度同源。免疫学分析和底物专一性研究以及其他现有证据表明，该酶不同于激肽释放酶和因子XIa，因此该酶是一种新型的丝氨酸蛋白酶，我们将其命名为Follipsin。卵泡素免疫组织化学定位于猪卵巢的卵泡液和基质细胞中。结果表明，卵泡素来源于卵巢间质细胞。

Porcine ovary was found to contain enzyme activities hydrolyzing peptide 4-methylcoumaryl-7-amide (MCA) substrates with a preference for Arg-MCA bond. The activities were shown to be present almost exclusively in the follicular fluid and to increase several times during follicular maturation. An enzyme responsible for the activity was purified to apparent homogeneity. The purified wnzyme consists of two different polypeptide chains having Mr-45,000 and 32,000 each, associated covalently. The enzyme activity was strongly inhibited by diisopropylfluorophosphate, benzamidine, leupeptin and antipain, indicating that it is a scrine proteinase. Using synthetic peptide substrates containing MCA, the enzyme was confirmed to hydrolyze preferentially Arg-X bonds but not LyS-X bonds. The amino-terminal amino acid sequences of the 45kDa and 32kDa polypeptides as well as were highly homologous with those of the heavy and light chains, respectively, of human lpasma kallikrein and human factor XIa. Immunological analyzes and substrate specificity studies, together with other existing evidence, indicated that the enzyme is distict from the kallikrein and factor XIa.Thus this enzyme is a movel type of serine proteinase, and we named it follipsin. Follipsin is immunohistochemically localized in follicular fluid as well as in stroma cells of porcine ovary. The results strongly suggest that follipsin originates from interstitial cells of ovarian stroma.