Fractionation of the DD‐Carboxypeptidase‐Transpeptidase Activities Solubilized from Membranes of Escherichia coli K12, Strain 44

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Abstract
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.