Heterogeneity of [3H]inositol 1,4,5-trisphosphate binding sites in adrenal-cortical membranes. Characterization and validation of a radioreceptor assay

Abstract

1. The characterization of a radioreceptor assay for determining Ins(1,4,5)P2 concentration in tissue extracts is described which utilizes the binding of [32H]Ins(1,4,5)P3 to an adrenal-cortex membrane fractions. 2. Analysis of [3H]Ins(1,4,5)P3 binding by isotope dilution demonstrated an apparent single population of binding sites (KD 3.65 .+-. 0.18 nM, Bmax. 872 .+-. 70 fmol/mg of protein). Specific binding of [3H]Ins(1,4,5)P3 was enhanced at alkaline pH values (maximum at pH 8.5), with complete loss of specific binding at pH < 6. These binding sites displayed strict stereo- and positional specificity for Ins(1,4,5)P3, with L-Ins(1,4,5)P3, Ins(1,2,4)P3 and DL-Ins(1,3,4,5)P4 causing 50% displacement of specific [3H]Ins(1,4,5)P3 binding (IC50 values) at concentrations of 14 .+-. 3 .mu.M. 3.0 .+-. 0.3 .mu.M and 0.53 .+-. 0.03 .mu.M respectively. 3. Kinetic analysis of binding data, however, revealed a high-affinity [3H]Ins(1,4,5)P3 binding site (KD 0.052 nM) in addition to the lower-affinity site (KD 2.53 nM) already demonstrated in displacement studies. 4. It is shown that the presence of the high-affinity site can be exploited to increase the sensitivity of the [3H]Ins(1,4,5)P3 radioreceptor assay, allowing accurate detection of 20 fmol of Ins(1,4,5)P3 in 300 .mu.l of tissue extract. 5. Further validation of the specificity of the above assay for Ins(1,4,5)P3 was provided by incubating tissue extracts with either a 5-phosphatase or 3-kinase preparation. It was shown that identical loss occurred for both Ins(1,4,5)P3 mass and [3H]Ins(1,4,5)P3, added to parallel incubations. 6. The ability of the assay to measure basal and agonist-stimulated increases in Ins(1,4,5)P3 concentration has been demonstrated with rat cerebral cortex and bovine tracheal smooth-muscle slices and a range of cultured and isolated cell preparations.