Biomedical subjects

G M Ehrenfeld

Publications and source records attributed to G M Ehrenfeld.

7 recordsLinked to original sources

PubMed1987

Copper-dependent cleavage of DNA by bleomycin.

DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.

Bleomycin↗

PubMed1985

Analysis of products formed during bleomycin-mediated DNA degradation.

By the use of DNA, copolymers of defined nucleotide composition, and a synthetic dodecanucleotide having putative bleomycin cleavage sites in proximity to the 5'- and 3'-termini, the products formed concomitant with DNA strand scission have been isolated and subjected to structural identification and quantitation via direct comparison with authentic synthetic samples. The products of DNA strand scission by Fe(II)-bleomycin include oligonucleotides having each of the four possible nucleoside 3'-(phosphoro-2''-O-glycolates) at their 3'-termini, as well as the four possible base propenals. At least for 3-(adenin-9'-yl)propenal and 3-(thymin-1'-yl)propenal, the products formed were exclusively of the trans configuration.

Base Sequence↗

PubMed1985

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission.

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.

Bleomycin↗

PubMed1983

Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly.

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.

Amino Acyl-tRNA Synthetases↗

PubMed1983

Transfer RNA pyrophosphorolysis with CTP(ATP):tRNA nucleotidyltransferase. A direct route to tRNAs modified at the 3' terminus.

The pyrophosphorolysis of tRNA by yeast CTP-(ATP):tRNA nucleotidyltransferase has been studied in an effort to define the behavior of the enzyme and the experimental parameters that lead to net loss of the 3'-terminal nucleotide or to nucleotide exchange. It was found that removal of AMP from the terminus of tRNA proceeded optimally at 1.0 mM PPi; incorporation of 2'- or 3'-dAMP was also studied and shown to proceed optimally at a 6.0 mM concentration of deoxynucleoside triphosphate. CTP was shown to inhibit the pyrophosphorolysis and nucleotide exchange observed when starting from intact tRNA, but apparently not by inhibiting removal of CMP from tRNA missing the 3'-terminal adenosine moiety. The optimized conditions for nucleotide exchange were used for the preparative conversion of tRNAs to species terminating in 2'- and 3'-deoxyadenosine.

Kinetics↗

PubMed1982

Oxygen transfer from bleomycin-metal complexes.

Both Fe(III) and Cu(II) complexes of bleomycin (BLM), but not N-acetyl BLM . Fe(III), mediated the transfer of oxygen from iodosobenzene to organic substrates. In analogy with results obtained using certain cytochrome P-450 analogs, cis-stilbene was converted cleanly to the respective oxide, while no more than traces of trans-stilbene oxide were formed from trans-stilbene under identical conditions. The possible relevance of these observations to the degradation of DNA by bleomycin was also studied. In both the presence and absence of O2, BLM . Cu(II) . C6H5IO effected DNA degradation, as judged by the release of [3H]thymine from radiolabeled Escherichia coli DNA. These findings provide a valuable new assay system for the study of bleomycin analogs and suggest the possibility that bleomycin may function as an "oxygen transferase" in its degradation of DNA in situ.

Bleomycin↗

PubMedDate not supplied

DNA strand scission by bleomycin group antibiotics.

Certain properties of the bleomycin analogs deglycobleomycin A2 and decarbamoylbleomycin A2 have been characterized. In common with bleomycin A2, both deglycobleomycin A2 and decarbamoylbleomycin A2 were found to mediate DNA degradation in the presence of Fe(II) + O2. Both analogs were found to have essentially the same sequence selectivity for DNA strand scission as bleomycin A2 when a 5'-[23P]-end-labeled linear duplex DNA derived from SV40 DNA was employed as a substrate. Product analysis for the three analogs was carried out by assay for malondialdehyde (precursors) after digestion of calf thymus DNA, and also by hplc analysis of the digestion products formed from the dodecanucleotide d(CGCTTTAAAGCG). All three Fe(II) X bleomycin A2 analogs produced the same products, albeit not in the same relative amounts.

Base Sequence↗