Penicillins radioactive

A comparison of the structures of penicillin and Dalanyl-Dalanine (cf. structures 41 and 42) shows that there is a great deal of similarity between the two molecules. Penicillin is essentially an acylated cyclic dipeptide of Dcysteine and Dvaline (84). As such, it contains a peptide bond, that of the /3-lactam ring, that can acylate the enzyme. Labeling studies of the peptidoglycan transpeptidase of Bacillus subtilis indicate that radioactive penicillin reacts with a sulfhydryl group of a cysteine residue of the enzyme (86). 

DA Preston, CYE Wu, LC Blaszczak, DE Seitz, NG Halligan. Biological characterization of a new radioactive labeling reagent for bacterial penicillin-binding proteins. Antimicrob Agents Chemother 34 718-721, 1990. 

The mixed lymphocyte reaction (MLR) test was performed in 96-well flat-bottomed microtiter plates. Selected experimental agents were prepared as 10 mM stock solution in DMSO and a 50-fold dilution of this was prepared in RPMI. Serial dilutions were prepared from this subsequent solution and 10 xl of the diluted stock was added to the well to give concentrations in the assay starting at 9.5 xm. In each well was placed 1.5 x 105 donor cells to give a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% human serum, 2mM L-glutamine, and penicillin/streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide for 120 hours. 3H-Thymidine (0.5 xCi) was added in the final 6 hours of incubation and cell radioactivity levels determined, which were indicative of T-cell proliferation. 

Inactivation of certain enzyme systems involved in the oxidative metabolism of sensitive organisms by polymyxin and colistin has also been reported. This, however, might be a secondary effect . Bacitracin has been reported to interfere with cell wall synthesis. It causes Staphylococcus aureus to lyse , to form protoplasts and to accumulate cell wall precursors " . The incorporation of radioactive amino acids into cell wall mucopeptides is inhibited. Bacitracin has further been found to prevent Staphylococcus aureus from synthesizing /3-galactosidase , yet it does not interfere with the incorporation of radioactive lysine into cells . In experiments with Staphylococcus aureus, bacitracin and penicillin were shown to share a common binding site on the membrane , a result which could not be confirmed in similar experiments with B. megateriumP . Recently a direct effect of bacitracin on the cytoplasmic membrane has been demonstrated, and it was suggested that the inhibition of cell wall synthesis could be a secondary effect . ... 

Experiments with penicillin show that resistant strains of staphylococci, even those that produce no penicillinase, take up no penicillin from solution, but susceptible strains of various species combine with from 200 to 750 molecules per cell. This amount is held tightly, cannot be washed away, and does not exchange with non-radioactive penicillin (Rowley et al., 1950 Maass and Johnson, 1949). The first molecules of penicillin are bound by inert material but, when this is saturated, the penicillin combines covalently with a receptor playing a key role in the biosynthesis of cell wall. Mammalian cells do not take up penicillin, but the bacterial cell binds it in less than 2 minutes. The penicillinbinding component is on the exterior, in the cytoplasmic membrane where the cell wall is synthesized. This component occurs in a phospholipid-containing fraction of staphylococci (Cooper, 1956). 

The peptidic nature of penicillin was not recognized immediately after its isolation. In its jS-lactam bicyclic ring structure the tripeptide -(L-a-aminoadipoyl)-L-cysteinyl-D-valine is hidden but in 1960 it was discovered in extracts of Penicillium by Arnstein et al. [11]. Experiments on biosynthesis of penicillins in the intact mycelium of the producing fungi did not lead to unequivocal results, due to the poor permeability of the cell wall. Protoplasts, naked cells obtained after enzymatic removal of the outer wall, however, were able to serve in studies with radioactively labeled potential precursors, and in cell-free systems from Cephalosporium acremonium, the route to isopenicillin N was finally revealed [12]. The tripeptide L-a-aminoadipoyl-L-cysteinyl-D-valine first forms the j -lactam moiety and a second, oxidative step closes the thiazolidine ring between the -carbon of valine and the thiol of cysteine (Fig. 9). 

Penicillins and cephalosporins contain a P-lactam ring, which is fused with a thiazolidine ring. These types of compounds are synthesized by both prokaryotic and eukaryotic microorganisms, including Streptomyces, Penicillium, Aspergillus, and Cephalosporium species (Luckner, 1990). Important precursors are L-2-aminoadipic acid, L-cysteine, and L-valine. An important intermediate is 6-(L-aminoadipyl)-L-cysteinyl-D-valine (Luckner, 1990). The valine unit with a d-configuration found in one portion of the molecule is labeled when radioactively labeled L-valine is added to the culture. The most important antibiotic of this group, penicillin G (3) from Penicillium spp., is derived from similar precursors. 

Pollock and Perret [13] detected radioactive penicillin irreversibly bound to cells of B. cereus after treatment at 0° C, and showed a remarkable parallel between the amount of binding and the extent of penicillinase induction during subsequent growth of the culture (Fig. 2). 

The balance of evidence is against the penetration of penicillin inside the cell [Cooper, 5], and the bound penicillin forms part of a lipoprotein complex loosely associated with the membrane, which is 95 % released in a high-molecular weight form on protoplast formation [Duersken, 14], Radioactive penicillin is not associated with ribosomes, messenger RNA, purified cell wall, or purified cytoplasmic membrane. Imsande [2] finds that more radioactive penicillin (up to 75 %) remains associated with the protoplast, however. The amount of bound penicillin was estimated by Imsande to be 1000 molecules per cell the figure of 80 to 200 molecules per cell [Pollock and Perret, 13] may be low for technical reasons. 

Exposure of bacterial plasma membrane preparations to radioactively labelled penicillin gives protein complexes covalently linked to the antibiotic. All bacterial membranes give rise to these complexes which can be detected by sodium dodecyl sulphate (SDS)-gel electrophoretic separation and... 

B. Alternatively, again using the radio-autographs as a guide, a strip is cut into sections each containing the whole of one penicillin species. Each section is then extracted by boiling for a few minutes with very dilute phosphate buffer. An aliquot of each extract is evaporated down on a planchette and the radioactivity measured... 

In order to shed further light on the nature of the peptidyl intermediates in penicillin biosynthesis Arnstein et al. (235) incubated P. chrysogenum mycelium with L-[U- C]-valine and then extracted the intracellular peptide and amino acid material into boiling aqueous ethanol. After applying various purification procedures the authors obtained a radioactive peptide with properties similar to a synthetic sample of y-glutamylcysteinylvaline. Arnstein and Morris (236) went on to sequence this peptide and concluded that it was 5-(a-aminoadipyl)cysteinylvaline (265). Due to lack of material the authors were unable to determine the stereochemistry of the tripeptide. 

Cephalosporin C (179), like penicillin N, is derived from the amino acids a-aminodipic acid, cysteine and valine as shown below. The acetoxy side chain is derived from acetate. The incorporation of the three amino acids was first demonstrated by Trown et al. (285) who fed DL-a-amino-[2- C]-adipic acid, DL-[ 1 - C]-valine or a mixture of DL- and we5 o-[3- C]-cystine to fermentations of a Cephalosporium sp. In each case the resulting cephalosporin C was found to be radioactive. The cephalosporin C samples... 

Treatment of labelled a-aminoadipic acid sjmthesized by the Cephalosporium sp. in the presence of acetate-l- C with L-amino acid oxidase from Crotalus adamanteus indicated that more than 98% of this amino acid was the L-isomer (Warren et al., 1967). Hence, the apparent concentration of endogenous D-a-aminoadipic acid in the intracellular pool appeared to be too low to cause substantial dilution of the radioactivity of D-a-aminoadipic acid- C which entered the pool from the extracellular fluid and which remained largely unchanged in the cell. The relatively high dilution of radioactivity which accompanied the incorporation of from the labelled D-a-aminoadipic acid into penicillin N and cephalosporin C suggested that either free D-a-aminoadipic acid was not involved... 

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