Purification and characterization of a 43-kDa membrane-associated inositol polyphosphate 5-phosphatase from human placenta.
We have identified, isolated, and characterized a membrane-associated inositol polyphosphate 5-phosphatase (5-phosphatase) from the particulate fraction of human placenta. The enzyme was purified 3700-fold from a detergent extract of human placental membranes to apparent homogeneity, by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, and Biosil SEC 250 HPLC gel filtration. The purified 5-phosphatase has a molecular mass of 43 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. The enzyme hydrolyzes inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to inositol 1,4 bisphosphate (Ins(1,4)P2) with an apparent Km of 5 microM. The 43-kDa 5-phosphatase also hydrolyzes inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) with an apparent Km of 1.2 microM. The enzyme requires Mg2+ ions for activity and is inhibited by Ca2+ concentrations greater than 100 microM. Polyclonal antibodies developed against the membrane-associated enzyme immunoprecipitate the purified membrane-associated placental 5-phosphatase and the platelet Type I cytosolic enzyme, but not the 75-kDa platelet Type II 5-phosphatase. These results demonstrate that the purified membrane 5-phosphatase bears physical and immunological similarity with the Type I cytosolic platelet enzyme.
Laxminarayan
K M
KM
Department of Medicine, Monash Medical School, Box Hill Hospital, Melbourne, Australia.
Matzaris
M
M
Speed
C J
CJ
Mitchell
C A
CA
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D000072181:Inositol Polyphosphate 5-Phosphatases; D008566:Membranes / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D010920:Placenta / Q000201:enzymology*; D011233:Precipitin Tests
D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases