Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney

B. Seetharam, J. S. Levine, M. Ramasamy, D. H. Alpers

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Abstract

High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an M(r) of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of M(r) to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer M(r) to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of M(r) 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an M(r) of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.

Original languageEnglish
Pages (from-to)4443-4449
Number of pages7
JournalJournal of Biological Chemistry
Volume263
Issue number9
StatePublished - 1988

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