APPBP1-UBA3-catalyzed formation of the thioester-linked Ubc12−NEDD8 covalent intermediate was assayed in 20 μL volumes containing 100 nM of APPBP1-UBA3 or mutants, 3 μM Ubc12, 4 μM wild-type NEDD8 or mutants, and 50 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 5 mM ATP, 1 mM DTT, pH 7.6.
Following preliminary fractionation of cross-linked amino acids by partition chromatography, the intermediate compounds (DHLNL and HLNL) were assayed by ion-exchange chromatography with post-column derivatization and the mature bonds (PYD and DPD) were quantified using RP-HPLC using their natural fluorescence, as described previously [25].
Intracellular intermediates of glycolysis, tricarboxylic acid (TCA) cycle and nucleotides were assayed as indicators for metabolic changes of central metabolism.
Cleavage of a half-site by Tyr-324 of Cre, with formation of the Cre-linked DNA intermediate (Figure 2B), was assayed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
Potential hydrolysis in a half-site, with either the DNA or the tyrosyl intermediate as target for water attack, was assayed by denaturing urea-polyacrylamide gel electrophoresis (denaturing PAGE).
As expected [ 29], when the purine-pyrymidine mapping rule was assayed, only structures at intermediate, but not at large, scale were detected (not shown).
Creatinine was assayed using a spectrophotometric assay.
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EMCP intermediates were extracted during exponential growth.
Nonsterol intermediates are not detected in our assay, so we cannot exclude the hypothesis that the differences observed are due to concentrations of nonsterol these intermediates in the pathway.
