The inositol trisphosphate phosphomonoesterase of the human erythrocyte membrane

Abstract

Human erythrocyte ghosts exhibit an inositol trisphosphate phosphomonoesterase activity that rapidly converts inositol 1,4,5-trisphosphate into inositol 1,4-bisphosphate and Pi. Degradation of the released inositol 1,4-bisphosphate is not observed. This activity is dependent on Mg2+ (or Mn2+) and it is not activated by Ca2+. Optimum activity is around pH 7 and activity is abolished by heat denaturation. The Km for inositol trisphosphate is approx. 25 microM. 2,3-bisphosphoglycerate is a competitive inhibitor, with a Ki of approx. 0.35 mM. Glycerophosphoinositol 4,5-bisphosphate is attacked at about one-eighth of the rate for inositol trisphosphate, but glycerophosphoinositol 4-phosphate is not a substrate. Incubation of 32P-labelled erythrocyte membranes with Mg2+ causes little breakdown of phosphatidylinositol 4,5-bisphosphate, the parent compound from which both glycerophosphoinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate are derived. On the basis of its substrate specificity and the inhibition by 2,3-bisphosphoglycerate, we suggest that this enzyme is selective for the 5-phosphate in those water-soluble phosphate esters of inositol that possess the vicinal pair of 4,5-phosphates but that it may also interact less strongly with other water-soluble compounds that have pairs of vicinal phosphates.

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain

“…Cullen-et al [12] recently showed that Ins(1,3,4,5)P4 causes release of Ca2+ by its rapid conversion to Ins(1,4,5)P3 in electroporated mouse lymphoma L1210 cells via a 3-phosphatase. Oberdisse et al [13] found that Ins(l ,4,5)P3 formation from Ins (1,3,4,5)P4 is stimulated by thrombin and phorbol esters in saponin-permeabilized human platelets, indicating that in this system the 3-phosphatase may be regulated by a protein kinase C. Here we present some characteristics of the soluble Ins(1,3,4,5)P4 3-phosphatase from porcine brain.…”

“…4a) [15]. The intracellular level of Ins (1,3,4)P3 is assumed to range from 1.1 uM (basal) to 3.6 /M (stimulated) [24]. Therefore [25] and other avian cells, also occurs in murine macrophages [26], bovine adrenal glomerulosa cells [27] and differentiated HL-60 cells [16].…”

“…All nucleotides and Ins (3,4,5,6) [5-32P]Ins(1,3,4,5)P4 was prepared as described previously [10] and was checked for isomeric purity using a Partisphere WAX column as described [8], which enables the separation of InsP4 isomers. Analysis of radiolabelled InsP4 showed that the prepared [5-32P] Estimation of molecular mass The molecular mass of the 3-phosphatase was determined by gel filtration at 4 'C on a Superose 6 column (1 cm x 30 cm) equilibrated with buffer consisting of 100 mM-NaCl, 1 mM-EDTA, 1 mM-DTT and 20 mM-Hepes/NaOH, pH 7.0.…”

“…Ins (1,4,5)P3 is generated from PtdIns(4,5)P2 by phospholipase C [2]. For signal termination, Ins (1,4,5)P3 is either dephosphorylated to Ins( 1,4)P2 by a 5-phosphatase [3] or phosphorylated to Ins(l,3,4,5)P4 by a 3-kinase [4]. The physiological significance of Ins(1,3,4,5)P4 is not yet clear.…”

“…Ins (1,3,4,5)P4 is dephosphorylated to Ins(1,3,4)P3 [4], which is a precursor of more highly phosphorylated inositol polyphosphates such as Ins(l,3,4,5,6)P5, formed from Ins(1,3,4,6)P4 [7,8]. Up until now, Ins (1,3,4)P3 has been assumed to be the only degradation product of Ins(l,3,4,5)P4, but there is growing evidence that Ins(l,3,4,5)P4 is also degraded to Ins(l,4,5)P3 by an Ins(1,3,4,5)P4 3-phosphatase.…”