Properties of the proteins that bind vitamin B 12 in subcellular fractions of rat liver

The proteins in the subcellular fractions of rat liver that bind radioactive vitamin B₁₂ (B₁₂) after an intravascular injection of [⁵⁷Co]-cyanocobalamin ([⁵⁷Co]CN-B₁₂) have been characterized by chromatography on columns of Sephadex G-100 and earboxymethyl cellulose (CM-cellulose). Each fraction was studied at that time after the injection of [⁵⁷Co]CN-B₁₂ when it contained the maximum radioactivity (J. Biol. Chem.246, 4619, 1971). The radioactive B₁₂ in microsomal and plasma membrane fractions isolated 5 min after the injection of [⁵⁷Co]CN-B₁₂ was associated with a protein which cochromatographed with rat transcobalamin-II (TC-II) on columns of Sephadex G-100 and CM-cellulose. Seventy percent of the radioactivity in a highly purified lysosomal fraction isolated 30 min after the injection of [⁵⁷Co]CN-B₁₂ was bound to this same protein. The remainder of the radioactivity in the lysosomal fraction chromatographed as free B₁₂. After incubation of the lysosomal fraction in vitro for 2 hr at 37 °C in buffered isotonic sucrose (pH = 5.5), 70% of the radioactivity in the fraction was present as free B₁₂. Ninety percent of the radioactivity in a soluble fraction and 40% of the radioactivity in a mitochondrial fraction isolated 24 and 72 hr, respectively, after the injection of [⁵⁷Co]CN-B₁₂ was associated with proteins having M_r greater than 100,000. The data suggest that a portion of the [⁵⁷Co]CN-B₁₂ was taken into the cells of the liver as the intact [⁵⁷Co]CN-B₁₂-TC-II complex which was then further metabolized.