Purification of rat intestinal receptor for intrinsic factor-vitamin B12 complex

The intrinsic factor (IF) in a rat gastric mucosal extract was bound efficiently to vitamin B12-sepharose without significant change in its nature to produce IF-vitamin B12-sepharose. The purification of the intestinal receptor for the IF-vitamin B12 complex was performed by the affinity chromatography using the IF-vitamin B12-sepharose as the affinity adsorbent. As a result of admixing the gastric mucosal extract sample with B12-sepharose while stirring for 4 hours, the adsorption was performed without any break through. Further, it was recognized that the B12-bound protein purified by the affinity chromatography using B12-sepharose was not much changed as compared with that before purification. Furthermore, it was recognized that IF-B12-sepharose was able to be made by binding IF with B12-sepharose which was made by coupling B12 with the market-available AH-sepharose. The IF-B12-sepharose was washed with buffer solution, and then was loaded with the small intestine mucosal extract. Thereafter, the receptor was eluted by making di-valent cation inert with the buffer solution. After the removal of EDTA in the eluted solution by dialysis, the activity of the receptor was measured. 48.5% of the receptor activity loaded was recovered by the elution with EDTA. The specific activity of the receptor represented by the final amount of B12 (pg)/the amount of protein (mg) in the purified substance was 335 folds of the original activity. (Iwakiri, K.)