Binding sites for α‐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, a-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 a-trinositol in different tissues. 2 In membranes from pig cerebellum, liver, kidney, heart, and spleen, the density of specific [3H]-atrinositol binding sites was maximal at pH 5.0. Cerebellum and spleen showed only one binding site (cerebellum KD= 9.1 JiM, spleen KD= 7.3 gM). 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 ,uM. In cerebellar membranes, the affinity and density (107 pmol mg-' protein) of actrinositol 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(l,3,4,5)P4, the isomer stereochemically related to ax-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 ax-trinositol yielded a K1 of 11 jIM. 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.

Translocation between membranes and cytosol of p42IP4, a specific inositol 1,3,4,5‐tetrakisphosphate/phosphatidylinositol 3,4,5‐trisphosphate‐receptor protein from brain, is induced by inositol 1,3,4,5‐tetrakisphosphate and regulated by a membrane‐associated 5‐phosphatase

“…Another explanation could be that under the conditions used for membrane preparation, some vesicles and multilayer structures are formed which prevents the fraction of p42 IP4 not present at the outer membrane surface from being accessible for ligand interaction. This is also consistent with our previously described finding that the number of [ 3 H]Ins(1,3,4,5) P 4 ‐binding sites detectable on pig cerebellar membranes increased upon detergent solubilization [29]. Moreover the data shown in Fig.…”

“…Under the conditions used, only a fraction of the total protein was translocated from the membranes. We have previously reported that part of the p42 IP4 protein pool found associated with membranes was resistant to solubilization with detergent [29], indicating that some p42 IP4 might have strong interactions with components of the cytoskeleton. Another explanation could be that under the conditions used for membrane preparation, some vesicles and multilayer structures are formed which prevents the fraction of p42 IP4 not present at the outer membrane surface from being accessible for ligand interaction.…”

“…The samples were incubated for 1 h on ice followed by centrifugation (50 000 g , 10 min, 4 °C, Beckmann Avanti 30 centrifuge) and the resulting supernatants were analyzed by SDS/PAGE (12.5% separation gel), transfer to poly(vinyl difluoride) membranes and Western blotting with an anti‐peptide antiserum directed against a 19 amino acid peptide comprising amino acids 353–371 of p42 IP4 [19]. SDS/PAGE, immunoblotting, other standard procedures and materials used were described previously [27–29]. For determining the time course of the release of p42 IP4 and the stability of Ins(1,3,4,5) P 4 in the presence of Mg 2+ , membranes were resuspended in buffer pH 7.5, as described above, with varying concentrations of Mg 2+ , and incubated either in the absence or presence of 10 µ m Ins(1,3,4,5) P 4 on ice.…”

Determination of Specificity of a High-Affinity Inositol 1,3,4,5-Tetrakisphosphate Binding Site at a 42 kDa Receptor Protein, p42IP4: Comparison of Affinities of All Inositoltris-, -Tetrakis-, and -Pentakisphosphate Regioisomers

“…Data analysis. K d values and the number of binding sites were estimated by using the RADLIG data-analysis computer program (Version 4; BIOSOFT, Cambridge, UK) as described previously [24]. In competition assays the data obtained with the displacing inositolphosphate were fitted to the data obtained for the displacement of…”

Recombinant p42IP4, a brain‐specific 42‐kDa high‐affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd‐Ins(3,4,5)P3

“…In brief, purified recombinant protein (70±500 ng) was incubated for 20 min on ice in a total volume of 280 mL of 25 For the analysis of the binding data, K d values and the number of binding sites were estimated by using the radlig dataanalysis computer program (Biosoft) as described previously [28]. In competition assays the data obtained with the displacing inositol phosphate were fitted to the data obtained for the displacement of …”