Purification and some properties of inositol 1,3,4,5,6-Pentakisphosphate 2-kinase from immature soybean seeds.
Inositol 1,3,4,5,6-pentakisphosphate 2-kinase was purified from immature soybean seeds harvested approximately 5 weeks post-anthesis. A crude extract was clarified using polyethyleneimine and purified by chromatography on DEAE-cellulose, Cibacron Blue 3GA-agarose, Toyopearl DEAE 650M, and Toyopearl phenyl 650M columns. The enzyme had a relative molecular mass, M(r), of 52,000 as determined by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and retained 50% of its activity after 6 weeks at 0 degrees C. The Km values for inositol 1,3,4,5,6-pentakisphosphate and MgATP, respectively, were 2.3 microM and 8.4 microM, and the Vmax was 243 nmol/min/mg. The pH and temperature optima, respectively, were 6.8 and 42 degrees C. Maximum activity was obtained when the magnesium ion concentration was 4 mM. The kinase specifically phosphorylated the 2-position on the inositol ring and could also utilize D-inositol 1,4,5,6-tetrakisphosphate as a substrate. The K for the reaction was 14, indicating that the enzyme may be involved in both inositol hexakisphosphate formation in maturing seeds and ATP resynthesis in germinating seeds. Substrate concentrations in mature seeds were favorable for ATP formation, whereas additional factors appeared to drive the accumulation of inositol hexakisphosphate in maturing seeds.
United States Department of Agriculture, Southern Regional Research Center, New Orleans, Louisiana 70124.
B Q
BQ
Phillippy
A H
AH
Ullah
K C
KC
Ehrlich
D002845:Chromatography; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000302:isolation & purification* / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics
D017886:Durapatite; D017853:Phosphotransferases (Alcohol Group Acceptor); C064990:inositol pentakisphosphate kinase