PRINCIPAL INVESHGATORPROGRAM DIRECTOR Sandra HanHwpr^pr. M . T) , 
synthesis, and the exact site of vancomycin's inhibitory action 
has not been demonstrated in vivo. 
Early investigations suggested that the primary site of 
vancomycin binding was to the cell wall. Best and Durham 
described adsorption of vancomycin to whole cells and cell walls 
of Bacillus subtilis , in proportions of up to 750 ug per 1 mg of 
wall (3) . Perkins and Nieto found that when log phase cultures 
of Micrococcus luteus were incubated with vancomycin, the 
majority of antibiotic bound was initially in the cell wall 
fraction, but as time elapsed, the proportion of antibiotic 
found in the membrane fraction increased. There was no 
accumulation of vancomycin in the cytoplasm. Although vancomycin 
binds in vitro to UDP-N-acetylmuramyl pentapeptide (51) , the 
absence of labelled vancomycin in the cytoplasm argues against 
binding to the UDP-linked precursors as a mechanism of action in 
vivo (50) . 
Sinha and Neuhaus observed that vancomycin inhibited 
incorporation of muramyl-pentapeptide from UDP-N-acetylmuramyl 
pentapeptide into peptidoglycan (61) . This inhibition was 
reversed by addition of cell walls, suggesting that cell walls 
have a higher affinity for vancomycin than the lipid-linked 
membrane acceptor. Such affinity evidence suggested that the 
newly formed uncrosslinked peptidoglycan was the site of 
vancomycin's action, preventing addition of newly synthesized 
subunits. However, the very large amounts of vancomycin bound by 
the cell walls of Gram positive bacteria are clearly in enormous 
excess of the peptapeptide component of these walls (3) . 
Vancomycin also binds to some structures found in peptide cross 
bridges, and to the cross-linked disaccharide dimers of some 
bacterial species (45) . Complex formation of this type might 
account for a significant portion of vancomycin binding to cell 
walls, and the contribution of such binding, if any, to the 
antimicrobial activity of vancomycin remains unclear. Thus the 
results of affinity studies can not be considered predictive of 
vancomycin's mechanism of action. 
More recent evidence suggests that the inhibitory action of 
vancomycin may occur at the level of the lipid-linked precursor. 
Spin-label studies have shown that vancomycin will complex with 
undecaprenyl-diphospho-N-acetyl muramylpentapeptide (33) . In 
particulate enzyme preparations of M. luteus , vancomycin 
inhibited the utilization of phospholipid-disaccharide- 
pentapeptide for peptidoglycan synthesis at concentrations 
similar to those inhibiting cell growth (1) . Effects on the 
phospho-N-acetylmuramy 1- pentapeptide translocase reaction are 
also consistent with binding to the lipid intermediates. At 
concentrations of 50 ug/ml vancomycin inhibits the exchange 
reaction but stimulates the overall transfer reaction, probably 
by complexing with the undecaprenyl diphospho-muramyl 
pentapeptide, preventing reassociation with the translocase 
enzyme. Enhancement of transfer is not observed when 
UDP-N-acetylmuramy 1-tetrapeptide is used as the substrate (22). 
In summary, vancomycin is likely to act by inhibition of 
transfer of the lipid-linked precursors to the nascent 
peptidoglycan, by binding to D-alanyl-D-alanine residues present 
in either the lipid intermediates or nascent peptidoglycan. 
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Recombinant DNA Research, Volume 13 
