Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes
Palmer, S and Hughes, K T and Lee, D Y and Wakelam, M J (1989) Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes. Cellular Signalling, 1 (2). pp. 147-56. ISSN 1873-3913
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The binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (KD = 6.82+/-2.3 nM, Bmax = 370+/-38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P3 was able to compete with [3H]Ins(1,4,5)P3 whereas Ins(1,3,4)P3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P3 were calculated to be 0.22+/-0.15 microM basal and 2.53+/-1.8 microM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P3 concentrations in the picomolar range.
Creators(s): |
Palmer, S ![]() | Item type: | Article |
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ID code: | 49653 |
Keywords: | adrenal Cortex, animals, binding sites, binding, competitive, calcium, cattle, inositol 1,4,5-trisphosphate, kinetics, liver, male, microsomes, radioligand assay, rats, rats, inbred strains, vasopressins, Biology, Cell Biology |
Subjects: | Science > Natural history > Biology |
Department: | Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences |
Depositing user: | Pure Administrator |
Date deposited: | 07 Oct 2014 10:46 |
Last modified: | 20 Jan 2021 21:27 |
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URI: | https://strathprints.strath.ac.uk/id/eprint/49653 |
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