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

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

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21 Scopus citations

Abstract

Receptor for intrinsic factor-cobalamin (vitamin B12) has been purified from canine ileal mucosa by an initial solubilization with papain. The purified receptor was obtained first by papain digestion from ileal mucosa in which endogenous protease activity was not inhibited. This receptor revealed heterogeneity on disc gel electrophoresis for protein staining and activity. Electrophoresis in the presence of 1% sodium dodecyl sulfate revealed nine protein bands with molecular weights ranging from 170-32 x 103. However, when the ileal mucosa was first washed and then homogenized with buffer containing 0.5 mM phenylmethanesulfonyl fluoride before papain digestion, the purified receptor had only four protein bands with an apparent molecular weight in the range of 170-120x103. In both cases, the receptor activity was recovered in a large peak near the void volume on Sephadex G-200 chromatography. Quantitative amino acid analysis of the purified receptor obtained in the presence of endogenous proteases gave a value of 186,000xg of amino acid per mol of intrinsic factor-cobalamin binding activity. This value is less than the 222x103g reported for the Triton X-100 form of the receptor. Pure receptor obtained with Triton X-100 could be converted to lower molecular weight (150-180x103) active fractions by treatment with papain and other pancreatic proteases (trypsin, chymotrypsin, and elastase). In light of these findings, we propose that the ileal receptor for intrinsic factor-cobalamin is an integral protein with nearly 83% of its protein mass (equivalent to an approximate molecular weight of 186,000) facing the outside of the membrane and 17% (equivalent to a molecular weight of 36,000) included in the membrane itself.

Original languageEnglish
Pages (from-to)183-189
Number of pages7
JournalJournal of Biological Chemistry
Volume257
Issue number1
StatePublished - Jan 1 1982

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