Solubilization of the ileal receptor for intrinsic factor--vitamin B-12 complex by digestion with papain.
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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D006224:Cricetinae; D007082:Ileum / Q000187:drug effects / Q000378:metabolism*; D007437:Intrinsic Factor / Q000378:metabolism*; D046911:Macromolecular Substances; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D010206:Papain / Q000494:pharmacology*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018000:Receptors, Peptide; D012995:Solubility; D014805:Vitamin B 12 / Q000378:metabolism*
D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; C028199:intrinsic factor receptor; D007437:Intrinsic Factor; D010206:Papain; D014805:Vitamin B 12; D002118:Calcium