Purification and characterization of two types of soluble inositol phosphate 5-phosphomonoesterases from rat brain.
Two soluble forms of inositol phosphate 5-phosphomonoesterase have been partially purified and characterized from rat brain and are referred to as type 1 and type 2 according to their order of elution from DEAE-Sepharose. Together, these enzymes represent 26 +/- 3% (mean +/- S.E., n = 4) of the total inositol 1,4,5-triphosphate (Ins(1,4,5)P3) phosphatase activity assayed in crude brain homogenate and are present in approximately equal total activities in a 100,000 x g supernatant, with the remainder being membrane-bound. Both soluble enzymes require Mg2+ for activity, are moderately inhibited by Ca2+ in the micromolar range, and can be inhibited by millimolar concentrations of a variety of phosphorylated compounds. The type 1 enzyme has been purified to a specific activity of 1.06 mumol/min/mg protein. It elutes as a 60-kDa protein on Sephacryl S-200. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the type 1 enzyme correlates with a pair of protein bands of 66 and 60 kDa. It has apparent Km values of 3 and 0.8 microM for Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), respectively, and hydrolyses Ins(1,4,5)P3 approximately 12 times faster than Ins(1,3,4,5)P4. The type 2 enzyme has been purified to a specific activity of 15.2 mumol/min/mg protein, elutes as a protein of 160 kDa on Sephacryl S-300, and migrates as a similarly sized subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has an apparent Km for Ins(1,4,5)P3 of 18 microM. Its apparent Km for Ins(1,3,4,5)P4, however, is greater than 150 microM, suggesting that this enzyme is primarily an Ins(1,4,5)P3 5-phosphomonoesterase. The relationship of these two enzymes to the inositol tris/tetrakisphosphate pathway is discussed.
Hansen
C A
CA
Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
Johanson
R A
RA
Williamson
M T
MT
Williamson
J R
JR
D000818:Animals; D001921:Brain / Q000201:enzymology*; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000378:metabolism; D000072181:Inositol Polyphosphate 5-Phosphatases; D007527:Isoenzymes / Q000302:isolation & purification*; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012995:Solubility
D007295:Inositol Phosphates; D007527:Isoenzymes; C081655:inositol-1,3,4,5-tetrakisphosphate; D015544:Inositol 1,4,5-Trisphosphate; C035215:inositol triphosphate phosphomonoesterase; D010744:Phosphoric Monoester Hydrolases; D000072181:Inositol Polyphosphate 5-Phosphatases