Inositol polyphosphate 1-phosphatase from calf brain. Purification and inhibition by Li+, Ca2+, and Mn2+.
We recently identified an enzyme which we have designated inositol polyphosphate 1-phosphatase that hydrolyzes both inositol 1,3,4-trisphosphate (Ins-1,3,4-P3) and inositol 1,4-bisphosphate (Ins-1,4-P2), yielding inositol 3,4-bisphosphate and inositol 4-phosphate, respectively, as products (Inhorn, R. C., Bansal, V.S., and Majerus, P.W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2170-2174). We have now purified the inositol polyphosphate 1-phosphatase 3600-fold from calf brain supernatant. The purified enzyme has an apparent molecular mass of 44,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphatase activities. The enzyme hydrolyzes Ins-1,4-P2 with an apparent Km of approximately 4-5 microM, while it degrades Ins-1,3,4-P3 with an apparent Km of approximately 20 microM. The enzyme hydrolyzes these substrates at approximately the same maximal velocity. Inositol polyphosphate 1-phosphatase shows a sigmoidal dependence upon magnesium ion, with 0.3 mM Mg2+ causing half-maximal stimulation. A Hill plot of the data is linear with a value of n = 1.9, suggesting that the enzyme binds magnesium cooperatively. Calcium and manganese inhibit enzyme activity, with 50% inhibition at approximately 6 microM. Lithium inhibits Ins-1,4-P2 hydrolysis uncompetitively with a Ki of approximately 6 mM. This mechanism of lithium inhibition is similar to that observed for the inositol monophosphate phosphatase (originally designated myo-inositol-1-phosphatase; Hallcher, L.M., and Sherman, W.R. (1980) J. Biol. Chem. 255, 10896-10901), suggesting that these two enzymes are related. Lithium also inhibits Ins-1,3,4-P3 hydrolysis with an estimated Ki of 0.5-1 mM.
Inhorn
R C
RC
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Majerus
P W
PW
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007700:Kinetics; D008094:Lithium / Q000494:pharmacology*; D008345:Manganese / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D008345:Manganese; D008094:Lithium; D010744:Phosphoric Monoester Hydrolases; C053087:inositol-1,4-bisphosphate 1-phosphatase; D002118:Calcium