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Catalytic properties of inositol trisphosphate kinase: activation by Ca 2+ and calmodulin
Author(s) -
Ryu Sung Ho,
Lee Sang Yeol,
Lee KeeYoung,
Rhee Sue Goo
Publication year - 1987
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.1.5.2824270
Subject(s) - calmodulin , extracellular , chemistry , second messenger system , inositol , endoplasmic reticulum , intracellular , kinase , inositol phosphate , biochemistry , cytosol , enzyme , receptor
Inositol 1,4,5‐trisphosphate (Ins‐1,4,5‐P 3 ) is an important second‐messenger molecule that mobilizes Ca 2+ from intracellular stores in response to the occupancy of receptor by various Ca 2+ ‐mobilizing agonists. The fate of Ins‐1,4,5‐P 3 is determined by two enzymes, a 3‐kinase and a 5‐phosphomonoesterase. The first enzyme converts Ins‐1,4,5‐P 3 to Ins‐1,3,4,5‐P 4 , whereas the latter forms Ins‐1,4‐P 2 . Recent studies suggest that Ins‐1,3,4,5‐P 4 might modulate the entry of Ca 2+ from an extracellular source. In the current report, we describe the partial purification of the 3‐kinase [ ~ 400‐fold purified, specific activity = 0.12 μmol/(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 Ca 2+ /calmodulin complex. Therefore, we propose that Ca 2+ mobilized from endoplasmic reticulum by the action of Ins‐1,4,5‐P 3 forms a complex with calmodulin, and that the Ca 2+ /calmodulin complex stimulates the conversion of Ins‐1,4,5‐P 3 , an intracellular Ca 2+ mobilizer, to Ins‐1,3,4,5‐P 4 , an extracellular Ca 2+ mobilizer. A rapid assay method for the 3‐kinase was developed that is based on the separation of [3‐ 32 P]Ins‐1,3,4,5‐P 4 and [γ‐ 32 P]ATP by thin‐layer chromatography. Using this new assay method, we evaluated kinetic parameters ( K m for ATP = 40 μ m , K m for Ins‐1,4,5‐P 3 = 0.7 μ m , K i for ADP ‐ 12 μ m ) and divalent cation specificity (Mg 2+ > > Mn 2+ > Ca 2+ ) for the 3‐kinase. Studies with various inositol polyphosphates indicate that the substrate‐binding site is quite specific to Ins‐1,4,5‐P 3 . Nevertheless, Ins‐2,4,5‐P 3 could be phosphorylated at a velocity approximately 1/20‐1/30 that of Ins‐1,4,5‐P 3 .—R yu , S. H.; L ee , S. Y.; L ee , K.‐Y.; R hee , S. G. Catalytic properties of inositol trisphosphate kinase: activation by Ca 2+ and calmodulin. FASEB J. 1: 388‐393; 1987.