Inositol trisphosphate metabolism in Saccharomyces cerevisiae - identification, purification and properties of Inositol 1,4,5-trisphosphae 6-kinase

TY - JOUR

T1 - Inositol trisphosphate metabolism in Saccharomyces cerevisiae - identification, purification and properties of Inositol 1,4,5-trisphosphae 6-kinase

AU - ESTEVEZ, Francisco

AU - PULFORD, David

AU - STARK, Michael J. R.

AU - CARTER, A. Nigel

AU - Downes, C. Peter

PY - 1994/9/15

Y1 - 1994/9/15

N2 - Ins(1,4,5)P-3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P-3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P-3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity similar to 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of similar to 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P-3. The K-m for ATP was 2. 1 mM, and that for Ins(1,4,5)P-3 was 7.1 mu M. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P-3 into an InsP(5), and the partially purified preparation contained another activity that converted the InsP(4) product into an InsP(5). The InsP(4) product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P-4 [L-Ins(1,4,5,6)P-4] standard, identifying it as D-Ins(1,4,5,6)P-4. The yeast enzyme is thus an Ins(1,4,5)P-3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP(5) and InsP(6) in S. cerevisiae.

AB - Ins(1,4,5)P-3 metabolism was examined in Saccharomyces cerevisiae extracts. S. cerevisiae contains readily detectable Ins(1,4,5)P-3 kinase activity that is predominantly soluble, but phosphomonoesterase activity acting on Ins(1,4,5)P-3 was not detected in either soluble or particulate preparations from this organism. We have purified the kinase activity similar to 685-fold in a rapid four-step process, and obtained a stable preparation. The enzyme has an apparent native molecular mass of similar to 40 kDa, and displays Michaelis-Menten kinetics with respect to its two substrates, ATP and Ins(1,4,5)P-3. The K-m for ATP was 2. 1 mM, and that for Ins(1,4,5)P-3 was 7.1 mu M. The enzyme appeared to be the first step in the conversion of Ins(1,4,5)P-3 into an InsP(5), and the partially purified preparation contained another activity that converted the InsP(4) product into an InsP(5). The InsP(4) product of the partially purified kinase was not metabolized by human erythrocyte ghosts and co-chromatographed with an Ins(3,4,5,6)P-4 [L-Ins(1,4,5,6)P-4] standard, identifying it as D-Ins(1,4,5,6)P-4. The yeast enzyme is thus an Ins(1,4,5)P-3 6-kinase. This activity may be an important step in the production of inositol polyphosphates such as InsP(5) and InsP(6) in S. cerevisiae.

M3 - Article

SN - 0264-6021

VL - 302

SP - 709

EP - 716

JO - Biochemical Journal

JF - Biochemical Journal

ER -