Binding sites for alpha-trinositol (inositol 1,2,6-trisphosphate) in porcine tissues; comparison with Ins(1,4,5)P3 and Ins(1,3,4,5)P4-binding sites

Abstract

1. The molecular mechanism of action of the inositol trisphosphate isomer, alpha-trinositol (Ins(1,2,6)P3) which has potential therapeutic use in treatment of inflammation and burn oedema, is still unclear. Therefore we have studied binding sites for alpha-trinositol in different tissues. 2. In membranes from pig cerebellum, liver, kidney, heart, and spleen, the density of specific [3]-alpha-trinositol binding sites was maximal at pH 5.0. Cerebellum and spleen showed only one binding site (cerebellum KD = 9.1 microM, spleen KD = 7.3 microM). In the other tissues, there were a high-affinity site (heart KD = 70 nM, liver KD = 790 nM and kidney KD = 1800 nM), besides a low-affinity site with a KD ranging between 32 and 120 microM. In cerebellar membranes, the affinity and density (107 pmol mg-1 protein) of alpha-trinositol binding sites were not affected by phosphate (0 to 25 mM). 3. Binding of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to membranes from different porcine tissues was also determined. Ins(1,3,4,5)P4, the isomer stereochemically related to alpha-trinositol, binds with an affinity of 1.2 nM in cerebellum, but in the other tissues the binding site density was too low to determine the affinity. With cerebellar membranes heterologous displacement of [3H]-Ins(1,3,4,5)P4 by alpha-trinositol yielded a K1 of 11 microM. The Ins(1,4,5)P3 receptor displayed an affinity of 15 nM in cerebellum and of 5 to 7 nM in the other tissues investigated. 4. The solubilized Ins(1,3,4,5)P4 receptor preparation from cerebellum did not show Ins(1,2,6)P3 binding. Ins(1,2,6)P3 binding was found in the pellet obtained after solubilization of the membranes with the detergent Brij 58. 5. Thus, in different tissues alpha-trinositol binds to proteins with different affinity. They are obviously not related to binding sites for Ins (1,4,5)P3 or for Ins(1,3,4,5)P4. Future experiments have to unravel the identity of the binding protein(s) for alpha-trinositol.