Lysosomal localisation of cobalamin during absorption by the ileum of the dog

The subcellular distribution of cobalamin during absorption in the dog ileum has been studied using analytical subcellular fractionation. Animals dosed orally with cyano[57Co]cobalamin were killed 2 h later, and postnuclear supernatant fractions prepared from homogenates of the ileal mucosa were subjected to isopycnic centrifugation on reorientating sucrose density gradients. Marker enzymes for the principal subcellular organelles and cyano[57Co]cobalamin were assayed in the gradient fractions. At 2 h, the distribution of cyano[57Co]cobalamin exhibited a major lysosomal localisation with only 30% of the counts being recovered in the soluble fractions. This observation was confirmed by preparing postnuclear supernatant fractions in digitonin, which selectively disrupted lysosomes and released their contents into the soluble fractions. Lysosomal localisation during passage through the ileal enterocyte strongly supports absorption of cobalamin by a process of receptor-mediated endocytosis in the dog.