Development of a continuous coupled enzymatic assay for myo-inositol monophosphatase.
Myo-inositol monophosphatase, an enzyme purified from brain tissues, catalyses the dephosphorylation of myo-inositol 1-phosphate. This enzyme has become the subject of intense research interest since myo-inositol is needed for the resynthesis of phosphatidylinositol in cell membranes. Since phosphate contamination has always been a problem for the assay of this enzyme activity, we have developed a coupled enzymatic assay for detecting the activity of the phosphatase with no interference by the presence of phosphate. The assay is based on the measurement of inositol release after dephosphorylation and subsequent conversion of inositol into scyllo-inosose by a second enzyme, inositol dehydrogenase from Enterobacter aerogenes. Since the second reaction requires the presence of beta-NAD+, the activity of the dephosphorylation reaction can be monitored continuously by the increase of absorbance at 340 nm spectrophotometrically.
Kwok
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F
Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic, Hung Hom.
Lo
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SC
D000818:Animals; D001921:Brain / Q000201:enzymology*; D007202:Indicators and Reagents; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013401:Sugar Alcohol Dehydrogenases; D013552:Swine
D007202:Indicators and Reagents; D013401:Sugar Alcohol Dehydrogenases; C021448:myo-inositol 2-dehydrogenase; D010744:Phosphoric Monoester Hydrolases; C021822:myo-inositol-1 (or 4)-monophosphatase