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
✍ Scribed by Valerie L. Leathers; Professor Anthony W. Norman
- Publisher
- John Wiley and Sons
- Year
- 1993
- Tongue
- English
- Weight
- 763 KB
- Volume
- 52
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
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 CaL+ there was a greatly diminished crosslinking process. There were also at least two membrane components of chick intestinal brush border membranes, with MR = 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-I 50 chromatography of Triton X-I00 solubilized incubations between calbindin-D Z ~K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin-DL8K undergoes calcium-dependent conformational chavges which alter its subsequent interactions with cellular proteins in a way consistent with other calcium-binding proteins such as calmodulin or troponin C.