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Large scale purification of myo ‐inositol monophosphate phosphatase from porcine brains
Author(s) -
Kwok Francis,
Wang Xing Hua,
Cheng Christopher H. K.,
Lo Samuel C. L.
Publication year - 1995
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260480517
Subject(s) - chemistry , chromatography , enzyme , size exclusion chromatography , phosphatase , inositol , homogenization (climate) , phosphate , tris , ammonium sulfate precipitation , biochemistry , uncompetitive inhibitor , gel electrophoresis , specific activity , non competitive inhibition , biology , biodiversity , ecology , receptor
myo ‐Inositol monophosphate phosphatase (IMPase) has been purified 888‐fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion‐exchange and gel‐filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol · min −1 · mg −1 . The molecular mass of the enzyme was estimated to be 29kDa by SDS poly‐acrylamide gel electrophoresis and 58 ± 5 kDa by HPLC gel filtration in 10m M Tris‐HCI, pH 7.4. Kinetic measurements have shown that the apparent K m value of the phosphatase for the utilization of inositol‐1‐phosphate and β‐glycerol phosphate are 3.20 × 10 −4 and 8 × 10 −3 M , respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K 1 was determined to be 6.38 × 10 −4 M and the inhibition is uncompetitive. © 1995 John Wiley & Sons, Inc.