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JYMS : Journal of Yeungnam Medical Science

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Hung Hye Kim 1 Article
Purification of Inositol Triphosphate Kinase from Bovine Brain
Hung Hye Kim, Jae Tae Lee
Yeungnam Univ J Med. 1996;13(1):46-58.   Published online June 30, 1996
DOI: https://doi.org/10.12701/yujm.1996.13.1.46
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Inositol 1,4,5-triphosphate (InsP3) is a second messenger for obilizing intracellular Ca2+. It can be dephosphorylated by soluble and particulate forms on InsP3 5-phosphatase, or phosphorylated to produce inositol 1,3,4,5-tetrakisphosphate (InsP3) by InsP3 3-kinase. These enzymes represent possible targets for the regulation of the InsP3/AnsP4 signal. InsP3 3-kinase which catalyses the ATP-dependent phosphorylation of InsP3 was purified from bovine brain tissue. All operation were carried out at 41°C. Fresh tissue was homogenized and centrifuged. The supernatant was pooled. Proteins were precipitated from 10% polyethylene glycol, and suspended solution was applied to DEAE cellulose column for chromatography. As the result of above procedure, two isozymes of InsP3 3-kinase, I and U were obtained. Each isozyme was applied to Matriz green gel, Calmodulin-Affigel 15 column and subsequent phenyl-TSK HPLC column. Specific activites (SA) and fold of puriety were observed at each purification step of chromatography. At DEAE cellulose chromatography, SA were I, 0.6 and II, 4.8 nM/min/mg, and folds were I, 17.2 and II, 16.6. At Matrix green gel chromatography, SA were I, 18 and II, 11 nM/min/mg, folds were I, 62.1 and II, 38.0. At calmodulin-Affigel 15 column chromatography, SA were I, 19 and II, 13 nM/min/mg, folds were I, 65.5 and II, 44.8. Finally InsP3 kinase I and II were purified 3,103-fold and 2,310-fold, and SA were I, 900 and II, 670 nM/min/mg, respectively. SDS-polyacrylamide gel electrophoresis elucidated 3 distinct fractions of Mr of 145,000, 85,000 and 69,500 from isozyme I, and 2 distinct fractions of Mr of 79,000 and 57,000 from isozyme II.

JYMS : Journal of Yeungnam Medical Science