Purification of an inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity from rat liver and the evaluation of its substrate specificity.

摘要: Hepatic inositol (1,3,4,5)-tetrakisphosphate 3-phosphatase activity was detected in a 100,000 x g soluble fraction and detergent-solubilized particulate fraction. Activity both fractions increased up to 40-fold after anion-exchange chromatography due removal of endogenous inhibitors (Hodgson, M.E., Shears, S.B. (1990) Biochem. J. 267, 831-834); at this stage the comprised over 90% total activity. The phosphatase further purified by affinity using heparin-agarose red-agarose. latter column resolved two peaks enzyme (designated 1 2 their order elution from column). Their proportions varied between experiments, but peak generally predominated so hydroxylapatite chromatography. final preparation typically 38,000-fold with 7% yield. apparent molecular mass 66 kDa, as determined sodium dodecyl sulfate-polyacrylamide gel electrophoresis filtration. had little or no for following: (1,3,4,6)-tetrakisphosphate, (1,3,4)-trisphosphate, (1,3)-bisphosphate, (3,4)-bisphosphate, para-nitrophenylphosphate. At pH 7.4 Km 130 nM Vmax 4250 nmol/mg protein/min. also dephosphorylated (1,3,4,5,6)-pentakisphosphate (1,4,5,6)-tetrakisphosphate (Km = 40 nM, 211 protein/min), hexakisphosphate least five isomers pentakisphosphate 0.3 12 protein/min). is highest yet defined an involved phosphate metabolism. Determinations IC50 values, Dixon plots, revealed that substrate, pentakis- hexakisphosphates were potent competitive inhibitors; Ki values (25 0.5 respectively) similar substrate values. kinetic properties enzyme, well estimates cellular levels its potential substrates, indicate are likely be preferred substrates vivo.