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A wall-plus-membrane preparation from a Bacillus licheniformis mutant incorporated radioactivity from a peptidoglycan precursor in which the free amino group of diaminopimelic acid was blocked by 14C-labelled acetyl group. This incorporation was penicillin-sensitive. The enzymically degraded product contained cross-linked dimers, showing that newly synthesized peptidoglycan chains had been cross-linked to the pre-existing cell wall.

The synthesis in vitro of peptidoglycan by Neisseria gonorrhoeaewas studied in organisms made permeable to nucleotide precursors by treatment with ether. Optimum synthesis occurred at 30°C in tris(hydroxymethyl)aminomethane-maleate buffer (0.05 M; pH 6) in the presence of 20 mM Mg2+. The incorporation from uridine 5′-diphosphate-N-acetyl-[14C]glucosamine into peptidoglycan, measured after precipitation of the cells with trichloroacetic acid, was sensitive to the β-lactam antibiotics, bacitracin, diumycin, and tunicamycin and relatively resistant to spectinomycin and tetracycline. Differences in sensitivity between preparations from a β-lactamase producer and a laboratory segregant derived from it were not great. Synthesized peptidoglycan was also fractionated into sodium dodecyl sulfate-soluble and -insoluble portions. β-Lactam antibiotics at concentrations equivalent to the minimal inhibitory concentrations for growth of the organisms did not inhibit peptidoglycan synthesis, but rather caused a small enhancement. At higher concentrations, above about 0.5 μg/ml, incorporation into sodium dodecyl sulfate-insoluble material was progressively inhibited, whereas the amount of sodium dodecyl sulfate-soluble product increased greatly, more than compensating for the loss of the precipitable fraction. Similar observations were made with three strains, and also with the β-lactam clavulanic acid, normally considered as a β-lactamase inhibitor rather than as itself an effective antibiotic.

1. The membrane from Bacillus megaterium KM contained a dd-carboxypeptidase with optimum activity under the following conditions: pH5.2, bivalent cation, 3mm; ionic strength, 40mm; temperature, 35°C. It was inactivated by treatment with p-chloromercuribenzoate but was fairly insensitive to 2-mercaptoethanol. 2. The enzyme was inhibited by penicillins and cephalosporins. The inhibition of this enzyme was partially reversed on dialysis but 0.2m-2-mercaptoethanol could neither prevent nor reverse the inhibition. 3. The enzyme was extremely sensitive to changes in the configuration and size of the side chain of the C-terminal dipeptide of the substrate. An aliphatic side chain of a well-defined length and polarity was required in the residue that precedes the C-terminal dipeptide. 4. The enzyme was inhibited by a wide range of analogues of the peptidic portion of the natural substrate.

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl2 buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10−6, 1.5×10−6and 0.63×10−6s−1(half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [14C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [14C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10−5s−1and 0.8×10−4s−1respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.

The exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 was purified to protein homogeneity and in milligram amounts. The isolated enzyme consisted of one polypeptide chain of molecular weight about 53300. Its amino acid composition and several physicochemical properties were determined and compared with those of the exo-cellular dd-carboxypeptidase–transpeptidase from Streptomyces R61.

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an125I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate–1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5′-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate–Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.

A cell-free membrane preparation from a poorly lytic mutant of Bacillus licheniformis was used to synthesize radioactive peptidoglycan. The product was apparently un-cross-linked. When UDP-N-acetyl[14C]glucosamine was used and the final peptidoglycan subjected to Smith degradation, no radioactive glycerol was found. On the other hand, when peptidoglycan labelled with meso-diamino[14C]pimelic acid was first hydrolysed in 0.1m-HCl at 60°C for 2h and then subjected to alkaline conditions, radioactive lactyl-peptides were eliminated. The proportion of radioactive lactyl-peptide decreased with increasing time of incorporation. It is concluded that the glycan chains grow by extension at their reducing ends while remaining attached by some linkage labile to mild acid, such as a glycosyl link to undecaprenol pyrophosphate.

The circular dichroism of the dd-carboxypeptidase–transpeptidase from Streptomyces R61 shows in the near u.v. a set of weak extrema at 289nm (positive) and at 282, 275 and 268nm (all negative). In the far u.v. it shows negative extrema at 217–218 and 208nm, crossover at 202nm and a positive maximum at about 194nm. The u.v. absorption of the enzyme shows it to contain tyrosine and tryptophan in approx. 3.4:1 ratio. The enzyme is fluorescent with a maximum emission at 318–320nm. The near-u.v. circular dichroism of the protein is extensively affected by binding of penicillin G, but the far u.v. is unaffected. Binding of the antibiotic also causes quenching of the fluorescence of the enzyme. The latter effect has been used to study the binding of penicillin G to the enzyme and the influence exerted upon it by salts, denaturants and peptide substrates and inhibitors. High-affinity binding of penicillin appears to be comparatively slow and reversible, and can occur under conditions in which the protein is enzymically inactive. The thermal denaturation of the enzyme in guanidinium chloride at pH7 is affected by binding of the antibiotic. The presence of even large concentrations of β-mercaptoethanol neither impaired the activity of the enzyme nor prevented its inhibition by penicillin G or cephalosporin C. A new hypothesis for the molecular mechanism of the interaction of the enzyme with penicillin is proposed.

The ability of the water-soluble dd-carboxypeptidases of Streptomyces strains albus G, R61, K11 and R39 to perform transpeptidation was studied. The donor was diacetyl-l-lysyl-d-alanyl-d-alanine, and a whole range of amino acids, peptides and structurally related amino compounds were tested for acceptor function. No compound tested was an acceptor for the enzyme from strain albus G whereas the enzymes from strains R61 and K11 could utilize with varying efficiency a wide range of substances including peptides with N-terminal glycine or d-alanine, ω-amino acids, aminohexuronic acids, 6-aminopenicillanic acid and d-cycloserine. Certain peptides, when present in higher concentration, inhibited the transpeptidation observed at lower concentration. The enzyme from strain R39 would not use any dipeptide as an acceptor, but a few compounds that were not glycine or α-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid.

1. Peptides that inhibit the dd-carboxypeptidases from Streptomyces strains albus G and R61 were synthesized. They are close analogues of the substrates of these enzymes. The enzymes from albus G and R61 strains are in general inhibited by the same peptides, but the enzyme from strain R39 differs considerably. 2. The two C-terminal residues of the peptide substrates and inhibitors appear to be mainly responsible for the initial binding of the substrate to the enzymes from albus G and R61 strains. The side chain in the third residue from the C-terminus seems critical in inducing catalytic activity. 3. Experimental evidence is presented suggesting that the amide bond linking the two C-terminal residues has a cis configuration when bound to the enzymes from strains albus G and R61. 4. The peptide inhibitors are not antibiotics against the same micro-organisms.

1. Antiserum to purified mouse liver plasma membranes was prepared and the partially purified γ-globulin antibody fraction was iodinated with 125I. The reaction of the 125I-labelled γ-globulin antibody with isolated mouse liver plasma membranes was studied. 2. The γglobulin antibody bound specifically to mouse liver plasma membranes and there was little reaction with mouse liver intracellular membranes or with surface-membrane fractions from either rat liver or pig lymphocytes. 3. ‘Light’ and ‘heavy’ mouse liver plasma-membrane subfractions bound similar amounts of γ-globulin antibody, and this is consistent with a surface origin for the light fraction. 5. Plasma membranes were fractionated by sequential extraction with 50mm-NaHCO3–Na2CO3 buffer, pH10.2, containing 10mm-EDTA and aq. 33% (v/v) pyridine. The alkali-soluble and -insoluble fractions and the pyridine-soluble and -insoluble fractions all reacted with the antiserum, and the cross-reactivity among the various fractions and with the total plasma membranes was investigated. 5. The results are discussed in terms of the arrangement of the antigenic determinants within the membrane.

Additional evidence is given that in Streptomycesstrains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of ß-lactam antibiotics. With strain R61, it was found that the exocellular enzyme has a sensitivity towards some antibiotics different from that of the membrane-bound enzyme. Under the growth conditions used in the present investigations, ß-lactamase activity was not involved in susceptibility to ß-lactam antibiotics.