Isolation and characterization of proteolytically derived ileal receptor for intrinsic factor-cobalamin.

B Seetharam,S.S Bagur,D.H Alpers

doi:10.1016/s0021-9258(19)68344-4

Abstract

Isolation and characterization of proteolytically derived ileal receptor for intrinsic factor-cobalamin.

Highlights

Receptor for intrinsic factor-cobalamin prepared using a nonionic detergent such as Triton X-100 and has been purified from canineileal mucosa byan initial those prepared using proteolytic enzymes such aspapain, solubilization with papain
The purified receptor was trypsin, and cathepsin, Such a comparison has revealed that obtained first b y papain digestion from ileal mucosa in in some integral membrane enzymes, polypeptides of molecwhich endogenous protease activity was not inhibited. ular weight 8,OOO-lO,OOO are inserted in the lipid bilayer [3,4,5,6,7,8,9], This receptor revealed heterogeneity on disc gel elec- and these pieces have ahigh proportion of hydrophobic amino trophoresis for protein staining and activity
In theintestinal brush-bordermembrane, alkaline phosphatase and sucrase-isomaltase complexeshave been shown to be inserted into themembrane with hydrophobic anchor pieces of molecular weight 6,000 and 20,000, respectively, and that both of these proteins can be solubilized by proteolytic enzymes [10, 11].Receptor for intrinsic factor-cobalamin complex is located in the ileal brush-border [12] and has recently been purified inourlaboratory from Triton X-100-treated canine ileal mucosa homogenates and shown to have amolecular weight of about 222,000 [13]

Summary

Purification of the Receptor

Procedure I-Ileal mucosa, 2.5 kg, was thawed and homogenized for 2 min at top speed in a Waring blender with 10 liters of 0.25 M mannitol in 5 mM K-phosphate buffer, pH 7.4. Digested receptor was applied on the column, homogenate was centrifuged again as mentioned above and the re- but eluted with phosphate-buffered saline buffer without Triton X-. SDS-polyacrylamide gel enate was cooled to 5 "C and centrifuged for 60 min a t 105,000 electrophoresis with the receptor and treatment with antiprotease. For SDS gel electrophoresis studies dialyzed for 18 h against 18 liters of the same solution with three with the receptor obtainedby procedure 2, Triton X-100 was washed changes of the dialysate. Five-hundred Experiments with antireceptor antibody were carried out using the g of ileal mucosa was homogenized with 2 liters of0.25 M mannitol antiserumdirectedagainstthepurifiedTriton X-100 form of the containing 0.5 m~ phenylmethanesulfonyl fluoride and centrifugeadt receptor [13]. The particulate material was homogenized in0.25 M mannitol and centri-

RESULTS

Crude homogenate

Total aminoacids

Antireceptor antihndv

Findings

DISCUSSION