The FASEB Journal, Vol 1, 388-393, Copyright © 1987 by The Federation of American Societies for Experimental Biology

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Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

SH Ryu, SY Lee, KY Lee and SG Rhee
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second- messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3- kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5- P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins- 1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3- 32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5- P3.

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