Beesley R C
Biochem J. 1981 Nov 15;200(2):225-9. doi: 10.1042/bj2000225.
Brush-border-membrane vesicles isolated from hamster ileum were incubated with either papain or Pronase P and subsequently centrifuged to obtain soluble (supernatant) and insoluble (pellet) fractions. Papain (4 units/ml) solubilized 95--100% of the sucrase and leucine naphthylamide-hydrolysing activities but only 30% of the alkaline phosphatase. Digestion with papain also resulted in the solubilization of more than 75% of the ileal receptor for intrinsic factor-vitamin B-12 complex with a corresponding decrease in receptor activity in the pellet. Essentially 100% of the receptor activity was recovered. In contrast, digestion with Pronase P resulted in a decrease in total receptor activity. Papain-solubilized receptor was not sedimented by centrifugation at 105 000 g for 90 min and was eluted in the included volume of Sepharose 6B. Like the binding to more intact preparations, binding of intrinsic factor-vitamin B-12 complex to papain-solubilized receptor was rapid, reaching 50% of maximum in 8 min, and required Ca2+. Although Mg2+ could not completely substitute for Ca2+, Mg2+ did stimulate Ca2+-dependent binding at low Ca2+ concentrations. These results demonstrate that the ileal receptor for intrinsic factor-vitamin B-12 complex can be solubilized with papain, and suggest that papain solubilization may be a useful first step in the isolation and purification of this receptor.
从仓鼠回肠分离出的刷状缘膜囊泡与木瓜蛋白酶或链霉蛋白酶P一起孵育，随后进行离心以获得可溶性（上清液）和不溶性（沉淀）部分。木瓜蛋白酶（4单位/毫升）可溶解95%-100%的蔗糖酶和亮氨酸萘基酰胺水解活性，但仅能溶解30%的碱性磷酸酶。用木瓜蛋白酶消化还导致超过75%的内因子-维生素B12复合物回肠受体溶解，沉淀中的受体活性相应降低。基本上回收了100%的受体活性。相比之下，用链霉蛋白酶P消化导致总受体活性降低。木瓜蛋白酶溶解的受体在105000克离心90分钟后不会沉淀，并在琼脂糖6B的包容体积中洗脱。与与更完整制剂的结合一样，内因子-维生素B12复合物与木瓜蛋白酶溶解的受体的结合很快，8分钟内达到最大结合量的50%，并且需要Ca2+。虽然Mg2+不能完全替代Ca2+，但在低Ca2+浓度下，Mg2+确实能刺激Ca2+依赖性结合。这些结果表明，内因子-维生素B12复合物的回肠受体可用木瓜蛋白酶溶解，这表明木瓜蛋白酶溶解可能是分离和纯化该受体的有用第一步。
