INITIAL STAGE IN PEPTIDOGLYCAN SYNTHESIS .4. SOLUBILIZATION OF PHOSPHO-N-ACETYLMURAMYL-PENTAPEPTIDE TRANSLOCASE

The initial reaction in the biosynthesis of peptidoglycan is catalyzed by phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala:C55-isoprenoid alcohol phosphate phospho-MurNAc-pentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of uridine diphosphate-N-acetylmuramyl-pentapeptide. The solubilization of the enzyme from membrane fragments of Staphylococcus aureus Copenhagen has been achieved with the following diverse reagents: (1) sodium lauroyl sarcosinate (3 μmoles/mg of protein) at 4°; (2) 10 M urea (pH 7.8) at 20°; (3) 0.08 m KOH at 4°. In the case of sodium lauroyl sarcosinate, two distinct activity peaks for the exchange reaction were detected by sucrose density gradient centrifugation and gel filtration on Bio-Gel A-50m. The major peak of activity has an exclusion volume corresponding to a weight-average molecular weight of 2 × 106, whereas the second peak of exchange activity corresponds to a molecular weight in the range of 100,000-200,000. The activity of the translocase from urea-solubilized membrane fragments was recovered after dialysis against buffers containing KCl. The major peak of activity corresponds to a molecular weight of 2 × 106. Additional peaks of transfer and exchange activity were observed. In the case of the KOH-solubilized preparations, two major peaks of exchange activity were detected. The exclusion volumes of these peaks on Bio-Gel A-50m were identical with those observed with membrane fragments solubilized with sodium lauroyl sarcosinate. From these results, we suggest that the translocase exists in more than one form. The low molecular weight form provides a source for the purification and study of the enzyme. © 1969, American Chemical Society. All rights reserved.