A HIGH-AFFINITY INOSITOL 1,3,4,5-TETRAKISPHOSPHATE RECEPTOR PROTEIN FROM BRAIN IS SPECIFICALLY LABELED BY A NEWLY SYNTHESIZED PHOTOAFFINITY ANALOG, N-(4-AZIDOSALICYL)AMINOETHANOL(1)-1-PHOSPHO-D-MYO-INOSITOL 3,4,5-TRISPHOSPHATE

A photolabile arylazido analogue of Ins(1,3,4,5)P4 selectively substituted at the 1-phosphate group was synthesized by coupling 2-aminoethanol(1)-1-phospho-D-Myo-inositol 4,5-bisphosphate with N-hydroxysuccinimidyl-4-azidosalicylic acid [Schafer, Nehls-Sahabandu, Grabowsky, Dehlinger-Kremer, Schulz & Mayr (1990) Biochem. J. 272, 817-8251 and subsequently phosphorylating the product by bovine brain Ins(1,4,5)P3 3-kinase. The product, N-(4-azidosalicyl)-aminoethanol(1)-1-phospho-D-myo-inositol 3,4,5-trisphosphate [AsaIns(1,3,4,5)P4] was radioiodinated and purified by anion-exchange chromatography. AsaIns(1,3,4,5)P4 bound to a high-affinity Ins(1,3,4,5)P4 receptor from pig cerebellum with an affinity only 3-fold lower than that of Ins(1,3,4,5)P4. Photoirradiation of I-125-AsaIns(1,3,4,5)P4 in the presence of the receptor preparation revealed that the radioactive label was specifically associated with a protein band of apparent molecular mass 42 kDa, which Donie & Reiser [(1991) Biochem. J. 275, 453-457] had previously tentatively assigned to the Ins(1,3,4,5)P4 receptor protein. The radioactive label was displaced from the receptor when the binding reaction with I-125-AsaIns(1,3,4,5)P4 was carried out in the presence of 5-mu-M-Ins(1,3,4,5)P4.