Microassay for UDP-galactose 4-epimerase activity

✍ Scribed by Carl R. Merril; Asis K. Das; Robert J. LaPolla; Igor Prissovsky

• Publisher: Elsevier Science
• Year: 1976
• Tongue: English
• Weight: 524 KB
• Volume: 72
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(76)90573-x

✦ Synopsis

A sensitive microassay technique for determining IJDP-galactose 4-epimerase activity in cell-free lysates has been devised. This method measures directly the conversion of UDP-galactose to UDP-glucose by thin-layer chromatographic separation of the two compounds. Radioactive labeling of the substrate serves as the quantitative basis for the assay. This method has been found to be approximately 100 times more sensitive than previously described spectrophotometric assay methods. Using the chromatographically based assay method a polar epimerase mutant and a promoter mutant of the galactose operon, which could not be distinguished enzymatically by the spectrophotometric assay method, could now be distinguished on the basis of their epimerase activity levels.