Fractionation of the DD-carboxypeptidase-transpeptidase activities solubilized from membranes of Escherichia coli K12, strain 44

A transpeptidase activity in Escherichia coli was measured independently of other enzymes involved in peptidoglycan synthesis by quantitating the formation of UDP-N-acetylmuramyl-l-alanyl-y-d-glutamyl-(l)-meso-diaminopimelyl-(l)-d-alanyl-[14C]glycine when UDP-N-acetylmuramyl-l-alanyl-y-d-glutamyl-(l)-meso-diaminopimelyl-(l-d-alanyl-d-alanine was used as donor substrate and [14C]glycine as acceptor in a transfer reaction. After extraction of membrane envelopes with Brij-36T and subsequent ammonium sulfate precipitation, DEAE-cellulose chromatography revealed two major fractions; one not adsorbed to the ion-exchange resin and the other adsorbed. The fraction which was bound to DEAE-cellulose was bound to and could be eluted from an ampicillin affinity chromatography system while the fraction not bound to DEAE-cellulose was also not bound to the ampicillin column. Both unbound and bound ampicillin fractions exhibited dd-carboxypeptidase and transpeptidase activities although for equivalent dd-carboxypeptidase activity, the bound ampicillin fraction required about five times more glycine acceptor to achieve the same amount of transpeptidation as the unbound ampicillin fraction.