The two membrane precursors (pentapeptide lipids I and II) of peptidoglycan are present in Escherichia coli at cell copy numbers no higher than 700 and 2,000 respectively. Conditions were determined for an optimal accumulation of pentapeptide lipid II from UDP-MurNAc-pentapeptide in a cell-free system and for its isolation and purification. When UDP-MurNAc-tripeptide was used in the accumulation reaction, tripeptide lipid II was formed, and it was isolated and purified. Both lipids II were compared as substrates in the in vitro polymerization by transglycosylation assayed with PBP lb or PBP 3. With PBP lb, tripeptide lipid II was used as efficiently as pentapeptide lipid II. It should be stressed that the in vitro PBP lb activity accounts for at best to 2 to 3% of the in vivo synthesis. With PBP 3, no polymerization was observed with either substrate. Furthermore, tripeptide lipid II was detected in D-cycloserine-treated cells, and its possible in vivo use in peptidoglycan formation is discussed. In particular, it is speculated that the transglycosylase activity of PBP lb could be coupled with the transpeptidase activity of PBP 3, using mainly tripeptide lipid II as precursor.It is now established that in Escherichia coli there are at least two distinct modes for the insertion of newly polymerized peptidoglycan material, one for elongation and the other for septation (30). Several proteins have been described as directly involved in the polymerization reactions (34, 47). In particular, certain penicillin-binding proteins (PBPs) were found to catalyze in vitro polymerization reactions with Nacetylglucosaminyl -N-acetylmuramyl(pentapeptide) -pyrophosphate-undecaprenol as a substrate (17,18,22,29,39,41). In vivo, this membrane intermediate (lipid II) is formed from UDP-N-acetylmuramoyl-pentapeptide by a two step process (34). First, a translocase catalyzes the transfer of the phospho-MurNAc-pentapeptide moiety of UDP-MurNAcpentapeptide to the membrane acceptor undecaprenol phosphate, yielding MurNAc(pentapeptide)-pyrophosphate-undecaprenol-(lipid I). Thereafter, a transferase catalyzes the addition of N-acetylglucosamine, yielding lipid II. The translocase also catalyzes the reverse reaction, and the equilibrium is in favor of the formation of UDP-MurNAc-pentapeptide (10,26 cell-free system in which it was allowed to accumulate to a certain extent by incubation of membranes with UDPGlcNAc and radiolabelled UDP-MurNAc-pentapeptide (44).In this study, a set of optimal conditions for a maximal in vitro formation of lipid II with particulate fraction from E. coli was sought, as well as substantial improvements in its purification. A lipid II containing tripeptide Ala-y-D-Glumeso-DAP instead of the pentapeptide was prepared in a similar way. Its possible in vivo presence was investigated. Both types of lipid II were compared as substrates in the in vitro polymerization reactions assayed with PBP lb or PBP 3. The possible physiological significance of such reactions are discussed. MATERUILS AND METHODSBacterial strains, ...
The serum thymic factor (FTS) utilized in its synthetic or natural form loses its biological activity in a rosette assay after treatment with a metal ion-chelating agent, Chelex 100. This activity is restored by the addition of Zn salts and, to a lesser extent, certain other metal salts. FTS activation is secondary to the binding of the metal to the peptide. The metal-to-peptide molar ratio of 1:1 provides the best activation. These data indicate the existence of two forms of FTS. The first one lacks Zn and is biologically inactive; the second one contains Zn and is biologically active, for which we propose the name of "thymulin" (FTS-Zn). The presence of Zn in synthetic FTS was confirmed by atomic absorption spectrometry. The interaction between Zn and FTS was fiurther suggested by microanalysis demonstrating the presence of this metal in thymic reticuloepithelial cells.
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