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. 2001 Sep;183(17):5122–5127. doi: 10.1128/JB.183.17.5122-5127.2001

FIG. 3.

FIG. 3

HPLC chromatograms showing production of [14C]UDP-MurNAc-hexapeptide by W. viridescens fraction III (A) and purified E. faecalis ORF2 (B). The standard assay (Materials and Methods) contained purified AlaSEfa (1.8 μg) and W. viridescens fraction III (0.17 μg of protein) or ORF2 (1 μg). The standard assay was scaled up for purification of UDP-MurNAc-hexapeptides produced by the W. viridescens (C) or E. faecalis (D) ligases. The reaction mixture (60 μl) contained Tris-HCl (50 mM, pH 7.2), MgCl2 (12.5 mM), ATP (2.5 mM), l-alanine (0.75 mM), UDP-MurNAc-pentapeptide (0.6 mM), tRNA (8 μg, W. viridescens fraction II), purified AlaSEfa (7.2 μg), and W. viridescens fraction III (30 μg of protein) or ORF2 (12 μg). Ala, l-alanine; Hexa, UDP-MurNAc-hexapeptide; Penta, UDP-MurNAc-pentapeptide.