SMOOTH-MUSCLE ALDOLASE C-BOUND INOSITOL 1,4,5-TRISPHOSPHATE STUDIED IN-VITRO UNDER PHYSIOLOGICAL CONDITIONS

Our goal was to quantitate inositol 1,4,5-trisphosphate (Ins(1,4,5)P-3 ) binding to aldolase C tetramer (aldolase(4)) and its displacement by inositol 1,3,4-trisphosphate (Ins(1,3,4)P-3) under conditions which a pproximated the in vivo state. Anions were found to have major effects . Decreasing [KCl] from 100 to 10 mM, at O degrees C and pH 7.0, incre ased maximal Ins(1,4,5)P-3 binding to 1.0 to 2.4 mol per mol aldolase( 4). At 10 and 30 mEq/l [Cl-], an additional high affinity site was det ected (K(d)s = 0.43 and 0.86 mu M, respectively). Increasing concentra tions of other anions (SO42-, propanoate(-), HCO3-, acetate(-)) also i nhibited binding, but effects would be minimal at concentrations of th ese anions present in the cytoplasm of living cells. Ins(1,3,4)P-3 dis placement of aldolase C-bound Ins(1,4,5)P-3 was sensitive to [Cl-]; at 30 mEq/l [Cl-] and 37 degrees C, Ins(1,3,4)P-3 released 20% of bound Ins(1,4,5)P-3 at concentrations of 100nM. Changing temperature from 0 degrees to 37 degrees C increased K(d)s for Ins(1,4,5)P(3)binding. Cha nges in free [Ca2+], [Mg2+], [Na+] and [K+] and changes in osmolality had no effect on Ins(1,4,5)P-3 binding to aldolase C. In vivo Ins(1,4, 5)P-3-aldolase(4) binding at 30 mEq/l [Cl-] and 37 degrees C were calc ulated for different [Ins(1,4,5)P-3](free) over the range 0.2 to 1.0 m u M. For different cytoplasmic [Ins(1,4,5)P-3] (free) Ins(1,4,5)P-3 bi nding to aldolase(4) was sufficient, if acutely released, to nearly do uble cytoplasmic [Ins(1,4,5)P-3](free). We proposed a schema whereby r elease of aldolase C-bound Ins(1,4,5)P-3 evoked by Ins(1,3,4)P-3 ampli fies effects of phospholipase C-formed Ins(1,4,5)P-3. (C) 1998 Elsevie r Science B.V.