Barns R J, Elmslie R G
Biochim Biophys Acta. 1977 Feb 9;480(2):450-60. doi: 10.1016/0005-2744(77)90037-7.
Porcine enteropeptidase (EC 3.4.21.9) purified from acetone powders of fresh duodenal fluid shows a molecular weight, as determined on Ultragel AcA-34, of 190000. Enteropeptidase has been solubilised from pig intestinal mucosa using 1% (v/v) Triton X-100. When Triton X-100 extracts of freeze-dried mucosa after partial fractionation on DEAE-cellulose were chromatographed on Sephadex G-200, the bulk of the activity eluted in the void volume rather than with an expected Ve/V0 ratio of about 1.24 corresponding to a molecular weight of around 200000. Gel filtration of aqueous mucosal extracts obtained in the absence of Triton X-100 showed two regions of enzymic activity in approximately equal proportions, one in the void volume, and the other with the expected Ve/V0 ratio of 1.24, whereas the Triton X-100 extracts of the residue from the above extract showed the presence of only the macromolecular species of enteropeptidase. This species was excluded from Sepharose 4B. It was confirmed that aminopeptidase was also extracted by Triton X-100 in a molecular form which was excluded from Sepharose 4B. The results suggest that Triton X-100 extracts enteropeptidase with a membrane component attached and in agreement with this it was found that proteolysis rapidly converted the macromolecular form to a stable smaller molecular species corresponding in size to that found in solution in the duodenal fluid. There was full recovery of the enzymic activity following this conversion. Papain and trypsin brought about an almost complete conversion to the smaller form of enteropeptidase whereas chymotrypsin, pancreatin and an intestinal peptidase preparation were only partially effective. It is concluded that membrane bound enzymes such as enteropeptidase and aminopeptidase are bound to the intestinal brush border membrane in a similar manner and are not actively secreted into the lumen but rather are largely released or solubilised by the combined action of the bile and pancreatic secretions.
从新鲜十二指肠液丙酮粉中纯化得到的猪肠肽酶（EC 3.4.21.9），经Ultragel AcA - 34测定，分子量为190000。已使用1%（v/v）的 Triton X - 100从猪肠黏膜中溶解出肠肽酶。当在DEAE - 纤维素上进行部分分级分离后的冻干黏膜的Triton X - 100提取物在Sephadex G - 200上进行层析时，大部分活性在空体积中洗脱，而不是以预期的Ve/V0比值约1.24（对应分子量约200000）洗脱。在不存在Triton X - 100的情况下获得的水性黏膜提取物的凝胶过滤显示出两个大致相等比例的酶活性区域，一个在空体积中，另一个具有预期的Ve/V0比值1.24，而上述提取物残渣的Triton X - 100提取物仅显示存在肠肽酶的大分子形式。该物种被Sepharose 4B排除。已证实氨肽酶也以被Sepharose 4B排除的分子形式被Triton X - 100提取。结果表明，Triton X - 100提取的肠肽酶带有附着的膜成分，与此一致的是，发现蛋白水解作用迅速将大分子形式转化为稳定的较小分子物种，其大小与在十二指肠液中溶液中发现的相对应。这种转化后酶活性完全恢复。木瓜蛋白酶和胰蛋白酶几乎完全将其转化为较小形式的肠肽酶，而胰凝乳蛋白酶、胰酶和一种肠肽酶制剂仅部分有效。结论是，诸如肠肽酶和氨肽酶等膜结合酶以类似方式与肠刷状缘膜结合，并非主动分泌到肠腔中，而是在很大程度上通过胆汁和胰腺分泌物的联合作用而释放或溶解。
