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

发现猪卵巢中含有水解肽4-甲基香豆酰-7-酰胺（MCA）底物的酶活性，并优先选择Arg-MCA键。这些活性几乎完全存在于卵泡液中，并在卵泡成熟过程中增加数倍。负责活性的酶被纯化至表观均一性。纯化的wnzyme由两条不同的多肽链组成，每条多肽链的Mr分别为45，000和32，000，共价结合。该酶活性可被二异丙基氟磷酸、苄脒、亮抑酶肽和抗痛剂强烈抑制，表明它是一种丝氨酸蛋白酶。使用合成的肽底物含有MCA，酶被证实优先水解Arg-X键，但不是LyS-X键。45 kDa和32 kDa多肽的氨基端氨基酸序列以及分别与人lpasma激肽释放酶和人因子XIa的重链和轻链的氨基酸序列高度同源。免疫学分析和底物特异性研究以及其它已有的证据表明，该酶与激肽释放酶和因子XIa不同，是一种新的丝氨酸蛋白酶，我们将其命名为卵泡素。卵泡蛋白酶在猪卵巢的卵泡液和基质细胞中均存在。提示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.