Radiolabel associated with WGA-SPA beads when reaction products were captured in the presence or the absence of Sarkosyl

InhibitorRadiolabel (cpm) by capture with:
WGA-SPA beadsWGA-SPA beads with Sarkosyl
Activity by SPAaRadiolabel on washed beadsbActivity by SPARadiolabel on washed beadsb
Penicillin G (100 μM)3,965 (51)2,0981,989 (94)307
Moenomycin (1 μM)7,964 (2)8,361347 (99)72
  • a The counts on the plates were determined 17 h after bead addition. The numbers in parenthesis are percent inhibition compared to that in the reaction with no inhibitor. The values reported as activity are those after subtraction of the values for the blank (i.e., the reaction with no UDP-MurNAcpp); the blank values were as follows: for WGA-SPA bead capture set, 3,043 cpm by SPA and 861 cpm for the radiolabel remaining on the washed bead; for the WGA-SPA-Sarkosyl capture set, 1,997 cpm by SPA and 82 cpm for the radiolabel remaining on the washed bead.

  • b Following reading of the results obtained by SPA, the beads were centrifuged, washed twice with 100 μl of 50 mM HEPES-ammonia buffer (pH 7.5) without detergent or with 0.2% Sarkosyl, and resuspended in 100 μl of the same buffer; and the counts in 10 μl were determined after addition of 200 μl scintillation fluid. In this experiment the following conditions were different: the enzyme reaction was performed with 1.5 μCi of UDP-[3H]GlcNAc, 15 μM UDP-MurNAcpp, the buffer used was 50 mM HEPES-ammonia (pH7.5), and the final volume after bead addition was 100 μl.