Release of vitamin B-12 in vitro from intrinsic factor-vitamin B-12 complex Purification and characterization of a releasing factor from rat ileal brush border

Vitamin B-12 is released from the purified gastric intrinsic factor-⁵⁷Co]vitamin B-12 (intrinsic factor- ⁵⁷Co]vitamin B-12) complex, when incubated with rat intestinal mucosa. Maximum specific activity for splitting the complex is localized in ileal brush border. Release of ⁵⁷Co]vitamin B-12 is not due to its mere exchange during incubation with endogenous non-radioactive vitamin B-12. The splitting process has specific requirement for Ca²⁺ and ATP and it is thermolabile, time- as well as temperature-dependent. It is also inactivated by the presence of p-chloromercuribenzoate. Further, the vitamin B-12-releasing factor has been isolated from solubilized brush border and is purified 70-fold by (NH₄)₂SO₄ precipitation, gel filtration and Con. A-Sepharose 4B affinity chromatography. In SDS-polyacrylamide gel electrophoresis, it is resolved into a single band of about 25 kDa, indicating its purity. The releasing factor exhibits maximum activity at pH 7.4; isoelectric focusing reveals only one major form with pI 7.52. With intrinsic factor-⁵⁷Co]vitamin B-12-complex as the substrate, apparent K_m and V_max values obtained are 128.2·10⁻¹² M/1 and 117.6 pg·h⁻¹ 100 μg protein, respectively. Amino acid and carbohydrate analyses reveal the glycoprotein nature of the factor. Intrinsic factor-⁵⁷Co]vitamin B-12 complex is not susceptible to unspecific proteolytic digestion/ Similarly, the releasing factor does not hydrolyse other proteins. Thus, the observed substrate-specificity of the releasing factor differentiates it from other known proteolytic enzymes of ileal brush borders.