Preparation and characterization of proteins in the alimentary tract of the dog which bind cobalamin and intrinsic factor.

The pyloric mucosa and the gastric and intestinal juices of the dog were found to contain two cobalaminbinding proteins which have been identified as intrinsic factor and R binder. T h e ileal mucosa also contains unsaturated R binder and, in addition, a specific receptor protein which couples the intrinsic factormcobalamin complex. This receptor protein was solubilized using mechanical sharing forces alone. Another cobalamin-binding protein, identified as transcobalamin 11, was present in the dog plasma. Intrinsic factor constitutes 8 to 10% of the total unsaturated cobalamin-binding capacity in pylorus and gastric juice, while it comprises 50% and 1 to 2% of the unsaturated binding capacity of the intestinal juice and ileal mucosa, respectively. This intrinsic factor also binds to antiserum to human intrinsic factor. The remaining unsaturated cobalamin-binding capacity in all these preparations was due to R binder which, unlike intrinsic factor, could be specifically saturated by the cobalamin analogue cyanocobinamide. The molecular radius and mass of each binder was determined by gel filtration after appropriate methods were used to analyze each protein selectively. The respective values for these parameters are: for intrinsic factor, 3.54 nm and 57,000 daltons; for R binder, 4.35 nm and 71,000 daltons; for transcobalamin 11, 2.58 nm and 39,000 daltons; and for the receptor protein, 12.9 nm and 1.5 X 10‘ daltons. This receptor protein also binds cobalamin-saturated human intrinsic factor with an association constant in the order of 10” liters/mol.