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 Km and Vmax values obtained are 128.2 X 10(-12) M/I and 117.6 pg X h-1 100 micrograms 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.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D005750:Gastric Juice / Q000378:metabolism*; D006023:Glycoproteins / Q000302:isolation & purification / Q000378:metabolism*; D007082:Ileum / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D007437:Intrinsic Factor / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008871:Microvilli / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014805:Vitamin B 12 / Q000378:metabolism*
D006023:Glycoproteins; C036746:intrinsic factor-vitamin B 12 complex; C036747:intrinsic factor-vitamin B12 complex releasing factor, rat; D000255:Adenosine Triphosphate; D007437:Intrinsic Factor; D014805:Vitamin B 12; D002118:Calcium