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Evidence for calcium mediated conformational changes in calbindin‐D 28K (the vitamin D‐induced calcium binding protein) interactions with chick intestinal brush border membrane alkaline phosphatase as studied via photoaffinity labeling techniques
Author(s) -
Leathers Valerie L.,
Norman Anthony W.
Publication year - 1993
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.240520216
Subject(s) - brush border , calcium , calmodulin , vitamin d dependent calcium binding protein , biochemistry , alkaline phosphatase , calcium binding protein , chemistry , photoaffinity labeling , membrane , binding site , enzyme , vesicle , organic chemistry
The role of the vitamin D‐induced calcium binding protein termed calbindin‐D (CaBP) in the biological response to 1,25‐dihydroxyvitamin D 3 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‐D 28K . Calcium‐dependent interactions were evident with purified chick intestinal CaBP‐immunoglobulins and bovine intestinal alkaline phosphatase; in the absence of Ca 2+ 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‐D 28K and chick intestinal BBMS, with 17% of the radiolabelled CaBP comigrating with alkaline phosphatase activity. These studies collectively demonstrate that calbindin‐D 28K 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.