Effect of glycosidases and proteinases on cobalamin binding and physicochemical properties of purified saturated haptocorrin and intrinsic factor.
The effect of exoglycosidase, N-glycanase, trypsin and chymotrypsin was studied on the binding capacity and physicochemical properties of intrinsic factor and of haptocorrin using Superose 6 gel filtration. Intrinsic factor was purified as recently described by us. Haptocorrin was purified 6000-fold from human saliva using thermolabile affinity chromatography and high-performance cationic exchange chromatography with a specific activity of 20.6 nmol of cobalamin (Cbl) per mg protein and a yield of 44.7%. Exoglycosidases provoked a decrease of 54.3 and 78.2% of the Cbl binding capacity of haptocorrin and intrinsic factor, respectively. The sequential incubation of haptocorrin and intrinsic factor wit exoglycosidases and proteinases provoked a decrease of, respectively, 100 and 92.7% of their Cbl binding capacity, whereas the incubation with proteinase decreased the Cbl binding capacity of, respectively, 67.9 and 7.9%. The result of the incubation of [3H]intrinsic factor or [3H]haptocorrin with chymotrypsin and trypsin gave, respectively, no change in the elution position and a shift corresponding to a decrease of 50% of the estimated molecular mass. The estimated molecular mass of Cbl-intrinsic factor and of Cbl-haptocorrin decreased, respectively, to 57.1 kDa and to 88.1 kDa after incubation with exoglycosidases. It was concluded that (1) the carbohydrate core of intrinsic factor protects the whole protein whereas the carbohydrate core of haptocorrin protects only half part of the protein and (2) the carbohydrates are implicated in the formation of the cobalamin binding site of haptocorrin and intrinsic factor.
Guéant
J L
JL
Equipe de Biochimie-Immunologie, Unité INSERM U 308, Vandoeuvre-lès-Nancy, France.
Monin
B
B
Djalali
M
M
Wahlstedt
V
V
Bois
F
F
Nicolas
J P
JP
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D002918:Chymotrypsin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000494:pharmacology*; D005750:Gastric Juice / Q000032:analysis; D006026:Glycoside Hydrolases / Q000494:pharmacology*; D006801:Humans; D007437:Intrinsic Factor / Q000302:isolation & purification / Q000378:metabolism*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D012463:Saliva / Q000032:analysis; D014155:Transcobalamins / Q000302:isolation & purification / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D014805:Vitamin B 12 / Q000378:metabolism*
D014155:Transcobalamins; D007437:Intrinsic Factor; D006026:Glycoside Hydrolases; C020668:glycanase; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D014805:Vitamin B 12