Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.
Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.
D000596:Amino Acids; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D004386:Duodenum / Q000378:metabolism*; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D013552:Swine; D014357:Trypsin
D000596:Amino Acids; D064030:S100 Calcium Binding Protein G; D008345:Manganese; D014357:Trypsin; D008274:Magnesium; D002118:Calcium