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KMID : 0578319940040030335
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Molecules and Cells
1994 Volume.4 No. 3 p.335 ~ p.341
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Immunoaffinity Purification of Inositol 1,4,5-trisphosphate Receptor (Subtype-I) from Rat Cerebellum and Its Functional Reconstitution into Liposomes
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Rhee, Seung keun
Lee, chang Hee/Mikoshiba, Katsuhiki
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Abstract
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Most of cell membrane receptors stimulate the phosphoinositide (PI) cycle, which produces complex intracellular Cal+ signals that regulate diverse cellular processes such as growth, differentiation and development_ A major messenger of this cycle, inositol 1,4,5-trisphosphate (InsP3), stimulates its receptor on the endoplasmic reticulum to release Cal+ into cytosol. So far, three subtypes of Insl`, receptors have been identified, and they are shown to express tissue-specificity. The subtype-I is dominantly expressed in neuronal tissue, especially in cerebellum. To elucidate the Cat+-releasing activity of each subtype of InsP3 receptor, in this study, we have isolated the subtype-I receptor from rat cerebellum by using a polycolnal antibody raised against a synthetic peptide corresponding to the C-terminal region of a subtype-I receptor. This immunoaffinity method allowed us to purify the InsP3 receptor to homogeneity by a single chromatographic step. The structural analyses with chemical cross-linking and electron microscopic studies showed that the purified InsP3 receptor is a tetrameric structure. In addition, we observed InsP3-dependent Cal+ channel activity of the receptor after incorporating it into phospholipid liposomes.
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