Evidence for calcium mediated conformational changes in calbindin-D28K (the vitamin D-induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques.
The role of the vitamin D-induced calcium binding protein termed calbindin-D (CaBP) in the biological response to 1,25-dihydroxyvitamin D3 was assessed by photoaffinity labeling techniques. The heterobifunctional cross-linking reagent methyl-4-azidobenzoimidate was employed for studies with the 28 KD chick intestinal calbindin-D28K. Calcium-dependent interactions were evident with purified chick intestinal CaBP-immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca2+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with M(R) = 60,000 and 130,000, which were photoaffinity cross-linked with CaBP in a calcium-dependent manner. Similar interactions were demonstrated following incubations of CaBP with phosphatidylinositol-specific phospholipase C (PI-PLC)-treated supernatant fractions from chick intestinal brush borders. PI-PLC was shown to release 14% of the alkaline phosphatase from chick intestinal brush borders compared to greater than 80% for rabbit and chick kidney BBM preparations. Specific interactions between CaBP and brush border membrane proteins could also be demonstrated in the absence of photoaffinity labeling by Sephadex G-150 chromatography of Triton X-100 solubilized incubations between calbindin-D28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-D28K undergoes calcium-dependent conformational changes which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.
Leathers
V L
VL
Department of Biochemistry, University of California, Riverside 92521.
Norman
A W
AW
D000345:Affinity Labels; D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002645:Chickens; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007422:Intestines / Q000201:enzymology* / Q000648:ultrastructure; D008871:Microvilli / Q000201:enzymology / Q000648:ultrastructure; D010777:Photochemistry; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism
D000345:Affinity Labels; D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D000469:Alkaline Phosphatase; D002118:Calcium