CHARACTERISTICS OF [H-3] INOSITOL(1,3,4,5)TETRAKISPHOSPHATE RECOGNITION SITES IN HUMAN CEREBELLAR MEMBRANES

The characteristics of specific [H-3]Ins(1,3,4,5)P-4 binding sites in human cerebellar membranes were determined in this study. Binding rapi dly reached steady stale, possessed a pH optimum of 4.5-5.1 and was gr eater in the absence of BSA than in its presence. Heparin inhibited bo th specific and pseudospecific binding of the ligand, whereas only the specific binding was inhibited by non-radioactive Ins(1,3,4,5)P-4. Ca lcium at a concentration of 1 mM, reduced binding by 27%. Competition studies with other inositol phosphates showed specificity for Ins(1,3, 4,5)P-4 with a pI(50) value of 6.87 and a Hill coefficient of 0.27, in dicating two sites. Ins(1,2,5,6)P-4, Ins(1,3,4,5,6)P-5, Ins(3,4,5,6)P- 4 displaced binding with IC50 values ranging from 0.1-1 mu M, Ins(1,2, 5,6)P-4 and Ins(1,3,4,5,6)P-5 being the most potent. Ins(1,4)P-2 and I ns(1,5,6)P-3 had lesser effects on binding. Rosenthal analysis of [H-3 ]Ins(1,3,4,5)P-4 saturation binding data at low ligand concentrations gave a K-D of 27 nM and a B-max of 33 pmol/mg protein. It is concluded that [H-3]Ins(1,3,4,5)P-4 binding sites in human cerebellar membranes have similar characteristics to these sites reported in the literatur e in animal cerebellar tissue, but are in greater abundance.