Test for the detection of the activity of phospholipids - n - acetylmuramyl - pentapeptide - translocase

摘要

The invention provides an assay for detecting phospho-N-acetylmuramyl-pentapeptide translocase enzyme activity, which comprises the steps of: (1) incubating a reaction mixture comprising, in aqueous medium, N-succinimidyl [2,3-3H]propionate substituted UDP-MurNAc-pentapeptide, N-succinimidyl propionate substituted UDP-MurNAc-pentapeptide (non-radioactive), a source of divalent metal ions, a source of undecaprenyl phosphate, a source of translocase enzyme and a detergent, under conditions suitable for enzyme activity to occur; (2) acidification of the reaction mixture with a suitable buffer comprising a quaternary ammonium salt at pH ∩4.2 to stop the enzyme reaction of step (1); and (3) extraction of any undecaprenol-pyrophosphate-[2,3-3H]propionate-N-acetylmuramyl-pentapeptide product formed and measuring radioactivity using a scintillation counter; and also a kit for use therein.