RGD Reference Report - 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. - Rat Genome Database

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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.

Authors: Khadapkar, SV  Vakil, UK 
Citation: Khadapkar SV and Vakil UK, Biochim Biophys Acta. 1982 Dec 17;719(3):619-32.
RGD ID: 1624372
Pubmed: (View Article at PubMed) PMID:7150665

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.

Annotation

Gene Ontology Annotations    

Molecular Function

Objects Annotated

Genes (Rattus norvegicus)
Cblif  (cobalamin binding intrinsic factor)


Additional Information