Effect of gastrointestinal proteases on purified human intrinsic factor-vitamin B12 (IF-B12) complex.
Intrinsic factor (IF) from human gastric juice was purified and complexed with vitamin B12 (IF-B12 complex) on Sepharose-vitamin B12 affinity matrix. By labeling studies, using [(57)Co] vitamin B12 and (125)I, the specific B12 binding activity of IF was found to be 23 microg B12/mg protein, and the molecular size by gel filtration 60 kDa. Proteolysis of the IF-B12 complex by sequential treatment with pepsin, trypsin, alpha-chymotrypsin and carboxypeptidase A, followed by chromatography of proteolysed complex and IF-B12 showed higher mobility of proteolysed fraction. Gel filtration, however, showed same molecular size for both proteolysed and the IF-B12 complex. On SDS-PAGE, purified IF-B12 appeared as a single band of 60 kDa. The proteolysed complex had higher mobility on SDS-PAGE and did not bind to zirconium phosphate gel. Immunodiffusion with rabbit antisera had positive reaction with IF-B12, but there was no reaction with the proteolysed sample.
Dept. of Biological Sciences, Pondicherry University, Kalapet, Pondicherry 605014, India.
K
K
Srikumar
R
R
Premalatha
D006801:Humans; D007437:Intrinsic Factor / Q000302:isolation & purification* / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D059748:Proteolysis; D013270:Stomach / Q000201:enzymology*; D014805:Vitamin B 12 / Q000302:isolation & purification* / Q000378:metabolism*
C036746:intrinsic factor-vitamin B 12 complex; D007437:Intrinsic Factor; D010447:Peptide Hydrolases; D014805:Vitamin B 12