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Abstract

We have synthesized two photolabile arylazido-analogues of Ins(1,4,5)P₃ selectively substituted at the 1-phosphate group for determination of Ins(1,4,5)P₃-binding proteins. These two photoaffinity derivatives, namely N-(4-azidobenzoyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AbaIP₃) and N-(4-azidosalicyl)aminoethanol-1-phospho-D-myo-inositol 4,5-bisphosphate (AsaIP₃), bind to high affinity Ins(1,4,5)P₃-specific binding sites at a 9-fold lower affinity (K_d = 66 and 70 nM) than Ins(1,4,5)P₃ (K_d = 7.15 nM) in a fraction from rat pancreatic acinar cells enriched in endoplasmic reticulum (ER). Other inositol phosphates tested showed comparable (DL-myo-inositol 1,4,5-trisphosphothioate, K_d = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P₄, K_d = 4 microM; Ins(1,4)P₂, K_d = 80 microM]. Binding of AbaIP₃ was also tested on a microsomal preparation of rat cerebellum [K_d = 300 nM as compared with Ins(1,4,5)P₃, K_d = 45 nM]. Ca²⁺ release activity of the inositol derivatives was tested with AbaIP₃. It induced a rapid and concentration-dependent Ca²⁺ release from the ER fraction [EC₅₀ (dose producing half-maximal effect) = 3.1 microM] being only 10-fold less potent than Ins(1,4,5)P₃ (EC₅₀ = 0.3 microM). From the two radioactive labelled analogues ([³H]AbaIP₃ and 125I-AsIP₃) synthesized, the radioiodinated derivative was used for photoaffinity labelling. It specifically labelled three proteins with apparent molecular masses of 49, 37 and 31 kDa in the ER-enriched fraction. By subfractionation of this ER-enriched fraction on a Percoll gradient the 37 kDa Ins(1,4,5)P₃ binding protein was obtained in a membrane fraction which showed the highest effect in Ins(1,4,5)P₃-inducible Ca²⁺ release (fraction P₁). The other two Ins(1,4,5)P₃-binding proteins, of 49 and 31 kDa, were obtained in fraction P₂, in which Ins(1,4,5)P₃-induced Ca²⁺ release was half of that obtained in fraction P₁. We conclude from these data that the 37 kDa and/or the 49 and 31 kDa proteins are involved in Ins(1,4,5)P₃-induced Ca²⁺ release from the ER of rat pancreatic acinar cells.