A spectrophotometric assay of D-glucuronate based on Escherichia coli uronate isomerase and mannonate dehydrogenase.
Escherichia coli uronate isomerase and mannonate dehydrogenase were overexpressed in E. coli BL21(DE3)pLysS cells and purified to near-homogeneity. The kinetic properties of the two enzymes were investigated. The isomerase was found to be inhibited by EDTA and to be stimulated by Zn(2+), Co(2+), and Mn(2+), but not by Mg(2+) or Ca(2+). Both enzymes were used to develop a sensitive spectrophotometric assay, in which D-glucuronate is converted to D-mannonate with concomitant oxidation of NADH to NAD(+). The sensitivity of this assay permits the detection of less than 1 nmol D-glucuronate. This assay can also be used to determine the concentration of beta-glucuronides and glucuronate 1-phosphate after enzymatic hydrolysis of these compounds with beta-glucuronidase or alkaline phosphatase.
Linster
Carole L
CL
Laboratory of Physiological Chemistry, Université de Louvain and the Christian de Duve Institute of Cellular Pathology, B-1200 Brussels, Belgium.
Van Schaftingen
Emile
E
D019747:Aldose-Ketose Isomerases / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D002118:Calcium / Q000378:metabolism; D002412:Cations; D002845:Chromatography; D004926:Escherichia coli / Q000201:enzymology*; D005637:Fructuronate Reductase / Q000737:chemistry*; D005965:Glucuronates / Q000032:analysis / Q000737:chemistry*; D005966:Glucuronidase / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*; D013997:Time Factors
D002412:Cations; D005965:Glucuronates; D009243:NAD; D005637:Fructuronate Reductase; D005966:Glucuronidase; D019747:Aldose-Ketose Isomerases; C020589:glucuronate isomerase; D002118:Calcium