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 S-kinase was purified from immatu re soybean seeds harvested approximately 5 weeks post-anthesis. A crud e extract was clarified using polyethyleneimine and purified by chroma tography on DEAE-cellulose, Cibacron Blue 3GA-agarose, Toyopearl DEAE 650M, and Toyopearl phenyl 650M columns. The enzyme had a relative mol ecular mass, M(r), of 52,000 as determined by sodium dodecyl sulfate-p olyacrylamide gel electrophoresis and retained 50% of its activity aft er 6 weeks at 0 degrees C. The K-m values for inositol 1,3,4,5,6-penta kisphosphate and MgATP, respectively, were 2.3 mu M and 8.4 mu M, and the V-max was 243 nmol/min/mg. The pH and temperature optima, respecti vely, were 6.8 and 42 degrees C. Maximum activity was obtained when th e magnesium ion concentration was 4 mM. The kinase specifically phosph orylated 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 react ion was 14, indicating that the enzyme may be involved in both inosito l hexakisphosphate formation in maturing seeds and ATP resynthesis in germinating seeds. Substrate concentrations in mature seeds were favor able for ATP formation, whereas additional factors appeared to drive t he accumulation of inositol hexakisphosphate in maturing seeds.