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Biomedical subjects

H R Perkins

Publications and source records attributed to H R Perkins.

At least 55 records · Page 3Linked to original sources
PubMed1974

Peptidoglycan biosynthesis by preparations from Bacillus licheniformis: cross-linking of newly synthesized chains to preformed cell wall.

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 (14)C-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.

Amino Acids, Diamino↗
PubMed1974

Membrane-bound DD-carboxypeptidases from Bacillus megaterium KM general properties, substrate specificity and sensitivity to penicillins, cephalosporins and peptide inhibitors of the activity at pH5.

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 degrees 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.

Bacillus megaterium↗
PubMed1973

Peptide inhibitors of Streptomyces DD-carboxypeptidases.

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.

Carbon Isotopes↗
PubMed1973

Streptomyces DD-carboxypeptidases as transpeptidases. The specificity for amino compounds acting as carboxyl acceptors.

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, omega-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 alpha-amino acids of the d-configuration did function thus. These were d-cycloserine and the lactams of meso- or racemic-diaminoadipic acid.

Carbon Isotopes↗
PubMed1973

Kinetics of concomitant transfer and hydrolysis reactions catalysed by the exocellular DD-carboxypeptidase-transpeptidase of streptomyces R61.

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac(2)-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac(2)-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac(2)-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.

Acyltransferases↗
PubMed1973

Fluorescence and circular dichroism studies on the Streptomyces R61 DD-carboxypeptidase-transpeptidase. Penicillin binding by the enzyme.

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 beta-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.

Acyltransferases↗
PubMed1973

The direction of glycan synthesis in a bacterial peptidoglycan.

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[(14)C]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[(14)C]pimelic acid was first hydrolysed in 0.1m-HCl at 60 degrees 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.

Amino Acids, Diamino↗
PubMed1973

Immunochemical characterization of proteins from mouse liver plasma membranes.

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an (125)I-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.

Animals↗
PubMed1973

DD-carboxypeptidase-transpeptidase and killing site of beta-lactam antibiotics in Streptomyces strains R39, R61, and K11.

Additional evidence is given that in Streptomyces strains R39, R61, and K11 the same enzyme performs dd-carboxypeptidase and transpeptidase activities and that this enzyme is the killing site of beta-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, beta-lactamase activity was not involved in susceptibility to beta-lactam antibiotics.

Acyltransferases↗
PubMed1972

Reversal by a specific peptide (diacetyl-alpha gamma-L-diaminobutyryl-D-alanyl-D-alanine) of vancomycin inhibition in intact bacteria and cell-free preparations.

Vancomycin inhibited the growth of Bacillus megaterium, Staphylococcus aureus and Micrococcus lysodeikticus, and in cell-free preparations from B. megaterium it inhibited the formation of mucopeptide and enhanced the accumulation of the lipid intermediate in the biosynthetic pathway. All these inhibitory processes were reversed by the presence of a synthetic peptide analogous to un-cross-linked mucopeptide side chains, namely diacetyl-l-diaminobutyryl-d-alanyl-d-alanine. A considerable amount of vancomycin was found in recovering cells, whether recovery was caused by peptide or took place naturally because a low initial concentration of antibiotic was used. In cell-free preparations pretreated with vancomycin, continued inhibition of mucopeptide synthesis depended on the presence of cell-wall material. This inhibition was also reversible by added peptide.

Alanine↗