Schäfer R, Nehls-Sahabandu M, Grabowsky B, Dehlinger-Kremer M, Schulz I, Mayr G W
Max-Planck Institut for Biophysics, Frankfurt, Germany.
Biochem J. 1990 Dec 15;272(3):817-25. doi: 10.1042/bj2720817.
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 (Kd = 66 and 70 nM) than Ins(1,4,5)P3 (Kd = 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, Kd = 81 nM) or much lower affinities for the binding sites [Ins(1,3,4,5)P4, Kd = 4 microM; Ins(1,4)P2, Kd = 80 microM]. Binding of AbaIP3 was also tested on a microsomal preparation of rat cerebellum [Kd = 300 nM as compared with Ins(1,4,5)P3, Kd = 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.
我们合成了两种光不稳定的1-磷酸基团选择性取代的肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）芳基叠氮类似物，用于测定Ins(1,4,5)P3结合蛋白。这两种光亲和衍生物，即N-(4-叠氮苯甲酰基)氨基乙醇-1-磷酸-D-肌醇4,5-二磷酸（AbaIP3）和N-(4-叠氮水杨基)氨基乙醇-1-磷酸-D-肌醇4,5-二磷酸（AsaIP3），在富含内质网（ER）的大鼠胰腺腺泡细胞组分中，与高亲和力的Ins(1,4,5)P3特异性结合位点结合，其亲和力（Kd = 66和70 nM）比Ins(1,4,5)P3（Kd = 7.15 nM）低9倍。测试的其他肌醇磷酸对结合位点的亲和力相当（DL-肌醇1,4,5-三磷酸硫代物，Kd = 81 nM）或低得多[Ins(1,3,4,5)P4，Kd = 4 microM；Ins(1,4)P2，Kd =
