We utilized high specific activity, [32P]-labelled ligands to measure the binding of Ins(1,3,4,5)P4 and Ins(1,4,5)P3 to membanes prepared from bovine parathyroid glands. [32P]Ins(1,3,4,5)P4 bound rapidly and reversibly to parathyroid membranes, and the binding data could be fitted by the interaction of the ligand with two sites, one with Kd=6.8×10-9 M and Bmax=26 fmol/mg protein and a second, lower affinity site, with Kd=4.1×10-7 M and Bmax=400 fmol/mg protein. InsP5 was 10-20 fold less potent than InsP4, and Ins(1,3,4)P3 and Ins(1,4,5)P3 were nearly 1000-fold less potent in displacing [32P]Ins(1,3,4,5)P4. [32P]Ins(1,4,5)P3, on the other hand, bound to a single class of sites with Kd=7.6×10-9 M and Bmax=34 fmol/mg. While the binding of [32P]Ins(1,3,4,5)P3 increased markedly on raising pH from 5 to 8, the binding of [32P]Ins(1,3,4,5)P4 decreased by 75% over this range of pH. Thus, [32P]-labelled Ins(1,3,4,5)P4 and Ins(1,4,5)P3 may be used to identify distinct binding sites which may represent physiologically relevant intracellular receptors for InsP3 and InsP4 in parathyroid cells.
|Number of pages||9|
|Journal||Biochemical and biophysical research communications|
|Publication status||Published - Feb 28 1989|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology