Smooth muscle aldolase C-bound inositol 1,4,5-trisphosphate studied in vitro under physiological conditions.

C B Baron, P Greeley, R F Coburn

Index: Biochim. Biophys. Acta 1401(1) , 81-92, (1998)

Full Text: HTML

Abstract

Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding to aldolase C tetramer (aldolase4) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) under conditions which approximated the in vivo state. Anions were found to have major effects. Decreasing [KCl] from 100 to 10mM, at 0 degrees C and pH 7.0, increased maximal Ins(1,4,5)P3 binding to 1.0 to 2.4mol per mol aldolase4. At 10 and 30mEq/l [Cl-], an additional high affinity site was detected (Kds = 0.43 and 0.86 microM, respectively). Increasing concentrations of other anions (SO42-, propanoate-, HCO3-, acetate-) also inhibited binding, but effects would be minimal at concentrations of these anions present in the cytoplasm of living cells. Ins(1,3,4)P3 displacement of aldolase C-bound Ins(1,4,5)P3 was sensitive to [Cl-]; at 30mEq/l [Cl-] and 37 degrees C, Ins(1,3,4)P3 released 20% of bound Ins(1,4,5)P3 at concentrations of 100nM. Changing temperature from 0 to 37 degrees C increased Kds for Ins(1,4,5)P3 binding. Changes in free [Ca2+], [Mg2+], [Na+] and [K+] and changes in osmolality had no effect on Ins(1,4,5)P3 binding to aldolase C. In vivo Ins(1,4,5)P3-aldolase4 binding at 30mEq/l [Cl-] and 37 degrees C were calculated for different [Ins(1,4,5)P3]free over the range 0.2 to 1.0 microM. For different cytoplasmic [Ins(1,4,5)P3]free. Ins(1,4,5)P3 binding to aldolase4 was sufficient, if acutely released, to nearly double cytoplasmic [Ins(1,4,5)P3]free. We proposed a schema whereby release of aldolase C-bound Ins(1,4,5)P3 evoked by Ins(1,3,4)P3 amplifies effects of phospholipase C-formed Ins(1,4,5)P3.