Activation of cell-surface receptors releases inside cells Ins(1,4,5)P(3) which mobilizes cellular Ca(2+) stores as part of a ubiquitous signalling cascade. This project studies the enzymes metabolizing Ins(1,4,5)P3, since this deactivates signalling, while introducing down-stream metabolites such as Ins(1,3,4,5)P4 that may themselves have key functions. We have purified and characterized an active Ins(1,3,4,5)P(4) 3-phosphatase and show its true substrates in vivo to be InsP(5), and InsP(6). We have shown from our kinetic data that InsP(6) phosphatase activity in situ is usually tightly constrained. However, we have also found novel examples of apparent overexpression of this enzyme upon cell transformation which deserve further attention. In order to improve the yield of the enzyme during purification, with a view to a molecular biological approach to further understanding its significance, a novel InsP(6) affinity column is being used. With the aim of understanding the physiological significance and mechanisms behind receptorregulated InsP(5) turnover (itself a novel effect recently uncovered in this laboratory), two major enzymes of InsP(5) synthesis have also been highly purified: Ins(3,4,5,6)P(4) 1-kinase and Ins(1,3,4)P3 6-kinase (the InsP6 column is used for the 1-kinase; an Ins(1,3,4)P3 column is being prepared for the 6-kinase). With an underlying goal of Studying the impact of environmental toxins on signalling processes, we have made progress in elucidating the mechanisms by which aluminum toxicity may manifest itself; although our work shows no direct effect upon Ca(2+) signalling, we have discovered this ion may redirect the synthesis of the cell's complement of InsP(5) and InsP(6).
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