Purification and characterization of inositol-1,3,4-trisphosphate 5/6-kinase from rat liver using an inositol hexakisphosphate affinity column.
The metabolism of inositol 1,3,4-trisphosphate is a pivotal branch point of inositol phosphate turnover; its dephosphorylation replenishes cellular inositol pools, its phosphorylation at the 6-position supports the synthesis of inositol pentakisphosphate, and its phosphorylation at the 5-position produces inositol 1,3,4,5-tetrakisphosphate (Shears, S.B. (1989) J. Biol. Chem. 264, 19879-19886). In order to increase understanding of the control of inositol-1,3,4-trisphosphate kinase activity, the enzyme was highly purified from rat liver by precipitation with polyethylene glycol, MonoQ ion-exchange chromatography, heparin-agarose affinity chromatography, and a novel affinity chromatography procedure that utilized Affi-Gel resin to which InsP6 was coupled (Marecek, J.F., and Prestwich, G.D. (1991) Tetrahedron Lett. 32, 1863-1866). The final purification was about 26,000-fold, with a 6% yield. This final preparation performed both 5- and 6-kinase activities in the ratio of approximately 1:5. The affinity of the enzyme for inositol 1,3,4-trisphosphate was 0.04 microM, the highest yet determined for an inositol phosphate kinase. Both inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4,6-tetrakisphosphate were competitive inhibitors of the kinase (Ki values of 2-4 microM). The enzyme was determined to have a molecular mass of 36 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Kinase activity was unaffected by Ca2+/calmodulin, protein kinase A, or protein kinase C.
Abdullah
M
M
Inositol Lipid Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Hughes
P J
PJ
Craxton
A
A
Gigg
R
R
Desai
T
T
Marecek
J F
JF
Prestwich
G D
GD
Shears
S B
SB
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013379:Substrate Specificity
D007295:Inositol Phosphates; C058097:inositol 1,3,4-trisphosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C056875:myo-inositol-trisphosphate 6-kinase