Membrane-bound D-alanine carboxypeptidase activity from Pseudomonas aeruginosa is very sensitive to inhibition by piperacillin.
I N T R O D U C T I O NPiperacillin, a new semi-synthetic penicillin, is reported to have a broad spectrum of antibacterial activity against Gram-positive and Gram-negative bacteria, and to have stronger in vitro and in vivo activities against Pseudomonas aeruginosa than both carbenicillin and sulbenicillin (Ueo et al., 1977). Suginaka et al. (1974) demonstrated that the membrane fraction from P. aeruginosa contained both D-alanine carboxypeptidase and transpeptidase activities, whereas the soluble fraction contained only the D-alanine carboxypeptidase. These enzymes were inhi bited by penicillins (Suginaka et al., 1974) at concentrations comparable to those inhibiting similar activities from Escherichia coli , although most strains of P. aeruginosa are highly resistant to penicillins.We have investigated the effect of piperacillin on the membrane-bound D-alanine carboxypeptidase activity of the cytoplasmic membrane and compared it with the effect on the soluble D-alanine carboxypeptidase in the cytoplasm and/or periplasm of P. aeruginosa.
METHODSAntibiotic. Piperacillin is 6-[D( -)-~-(4-ethyl-2,3-dioxo-l-piperzinylcarbonyIamino)-a-phenyIace tamidolpenicillanate (sodium salt) and was supplied by Toyama Chemical Co. (Tokyo, Japan).Organism. The organism used was Pseudomonas aeruginosa KM338, derived from ATCC 17653 ; this was the same strain as used in our previous study (Suginaka et al., 1975) of penicillin-resistant mechanisms. As estimated by a serial tube dilution method in Trypticase Soy Broth (BBL) with an inoculum of about lo4 bacteria ml-l, the minimal inhibitory concentrations of piperacillin, carbenicillin and benzylpenicillin were 12.5, 125 and 30000 pg ml-l, respectively.Preparation ofenzyme sources. Organisms were grown in Trypticase Soy Broth at 37 "C with shaking and harvested at the late-exponential phase. Membrane (particulate) and soluble fractions were prepared from the washed organisms after alumina grinding and differential centrifugation as reported previously . These fractions were used as the sources of membrane-bound and soluble D-alanine carboxypeptidase.Assay for D-alanine carboxypeptidase activity. The method used was that of Suginaka et cil. (1974) with the following modifications. The assay mixture contained (in a total volume of 25 pl): 1 M-Tris/HCl buffer pH 7.5, 5 pl ; 1 M-MgCl,, 1 pl ; 1.84 pM-uridine 5'-diphosphate-N-acetylmuramyl-~-alanyl-~-glutamyl-meso-