Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance

A general scheme is proposed for the determination of spatial protein structures by proton nuclear magnetic resonance. The scheme relies on experimental observation by two-dimensional nuclear magnetic resonance techniques of complete throughbond and through-space proton-proton connectivity maps. These are used to obtain sequential resonance assignments for the individual residues in the amino acid sequence and to characterize the spatial polypeptide structure by a tight network of semi-quantitative, intramolecular distance constraints.     

Detection by electron spin resonance of an exchange-coupled Cob(II)alamin...free radical pair species generated by anaerobic photolysis of polycrystalline adenosylcobalamin

Exposure to nitrous oxide (N₂O) $\text{in vivo}$ is accompanied by oxidation of cob[I]alamin to the inactive cob[III]alamin [1] and to loss of methionine synthetase activity [2]. There is a steady increase in thymidylate synthetase activity in marrow collected from rats exposed to N₂O and this returns to normal on restoring the animals to an air environment.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Synthesis and relative stereochemistry of the four mercapturic acids derived from styrene oxide and N-acetylcysteine

The chemical reaction between (±)-styrene oxide and N-acetylcysteine produces both positional isomers ($\text{1}$ and $\text{2}$) as a mixture of diastereoisomers with a preference for the benzylic thioether isomer $\text{1}$ (2 : 1). Synthesis of the mercapturic acid conjugates from either (+)- or (?)-styrene oxide produces only two of the four possible stereoisomers. The single diastereoisomers of $\text{1}$ and $\text{2}$ were separated by high pressure liquid chromatography (HPLC) and identified by ¹H- and ¹³C-nuclear magnetic resonance (NMR). The relative stereochemistry at the benzylic carbon center of the mercapturic acid conjugates was assigned on the basis of the established chemical correlation between optically pure styrene oxide and its precursor mandelic acid, and considerations on the mechanism of ring opening of epoxides by sulfur nucleophiles. The stereochemical definition of the isomers $\text{3}$–$\text{6}$ should prove useful in investigations of the biotransformation of the glutathione (GSH) conjugates of styrene oxide.     

Induction of both cytochromes P-450 and P-448 by 2,3',4,4',5-pentabromobiphenyl, a component of fireMaster

The synthesis and purification of a component of fireMaster BP-6 and fireMaster FF-1, 2,3′,4,4′,5-pentabromobiphenyl, is described. The compound was found to be a potent inducer of liver microsomal drug-metabolizing enzymes in the rat, enhancing those enzymic activities induced by both phenobarbitone and 3-methylcholanthrene (i.e. cytochromes P-450 and P-448). The pentabromobiphenyl enhanced the activities of benzo[a]pyrene hydroxylase, dimethylamino-antipyrine N-demethylase and NADPH-cytochrome c reductase. The hepatic cytochromes b₅ and P-450 were increased and the Soret peak maximum of the latter was shifted to 448.5 nm. The relative peak intensities and spectral shifts for the ethylisocyanide-binding difference spectra confirmed the mixed induction characteristics of 2,3′,4,4′,5-pentabromobiphenyl.     

Polychlorinated biphenyls as inducers of hepatic microsomal enzymes: Structure-activity rules

A number of highly purified polychlorinated biphenyl (PCB) isomers and congeners were synthesized and administered to male Wistar rats at dosage levels of 30 and 150 μmol · kg⁻¹. The effects of this in vivo treatment on the drug-metabolizing enzymes were determined by measuring the microsomal benzo[a]pyrene (B[a]P) hydroxylase, dimethylaminoantipyrine (DMAP) N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b₅ content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450: CO and ethylisocyanide (EIC) binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC) and PB plus MC (coadministered) to the test animals. The synthetic PCB congeners used in this study included 3,4,4′,5-tetrachlorobiphenyl (TCBP-1), 2,3′,4,4′-tetrachlorobiphenyl (TCBP-2), 2,3′,4,4′,5′-pentachlorobiphenyl (PCBP-1), 2,3,4,4′,5-pentachlorobiphenyl (PCBP-2), 2,3,3′,4,4′,5-hexachlorobiphenyl (HCBP-1), 2,3,3′,4′,5,6-hexachlorobiphenyl (HCBP-2), 2,3,3′,5,5′,6-hexachlorobiphenyl (HCBP-3), 2,2′,3,5,5′,6-hexachlorobiphenyl (HCBP-4) and 2,3,3′,4,5,5′-hexachlorobiphenyl (HCBP-5) and were used to reappraise the structure-activity rules for PCBs as hepatic microsomal enzyme inducers. The results suggested that (a) PCBs which induce MC or mixed-type activity must be substituted at both para positions, at least two meta positions but not necessarily on the same phenyl ring and can also contain one ortho chloro substituent; (b) due to the considerable structural diversity of the PB-type inducers the rules for induction of this activity by PCB congeners are not readily defined.     

Polychlorinated biphenyl isomers and congeners as inducers of both 3-methylcholanthrene- and phenobarbitone-type microsomal enzyme activity

Highly purified synthetic polychlorinated biphenyls substituted in the meta and para positions of both phenyl rings and at one ortho position were administered to male Wistar rats and the effects of these compounds on the microsomal drug-metabolising enzymes were evaluated. The in vivo effects of these compounds were determined by measuring the microsomal benzo[a]pyrene hydroxylase, dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase enzyme activities, the cytochrome b5 content and the relative peak intensities and spectral shifts of the reduced microsomal cytochrome P-450 : CO and ethylisocyanide binding difference spectra. The results were compared to the effects of administering phenobarbitone (PB), 3-methylcholanthrene (MC), 2,2',4,4'-tetrachlorobiphenyl (TCBP-II) (a PB-type inducer), 3,3',4,4'-tetrachlorobiphenyl (TCBP-I) (an MC-type inducer), PB plus MC (coadministered) and TCBP-II + TCBP-I (coadministered) to the test animals. At dosage levels of 30 and 150 mumol . kg-1, pretreatment with 2,3,3',4,4'-pentachlorobiphenyl (PCBP-II), 2,3',4,4',5-pentachlorobiphenyl (PCBP-I), 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-II) and 2,3,3',4,4',5-hexachlorobiphenyl (HCBP-III) gave hepatic microsomes with enzymic and spectral properties consistent with a mixed pattern of induction. These polychlorinated biphenyl (PCB) isomers and congeners have been identified as either major or minor components of the commercial PCB mixtures and must contribute to their activity as MC-type inducers. The only PCB isomer in this series which was not a mixed type inducer was 2,3',4,4',5,5'-hexachlorobiphenyl (HCBP-I) which appeared to be a PB-type inducer. This contrasted to the mixed-type activity observed for 2,3',4,4',5,5'-hexabromobiphenyl which was isolated from a commercial polybrominated biphenyl (PBB) mixture.     

Evidence for a role of specific isoacceptor species of tRNA in amino acid transport.

Soybean leghemoglobins ā and b?were compared by microscale peptide mapping after heme removal with acid-acetone. Maps generated by trypsin or the combined action of trypsin and thermolysin indicated a large amount of homology between the proteins with the only variations detected being the N-terminal peptides. The N-terminal tryptic peptide of leghemoglobin b? was found to be both blocked and to lack the first amino acid of the corresponding leghemoglobin ā peptide. Nuclear magnetic resonance and gas chromatography/mass spectroscopy studies showed that the N-terminal of leghemoglobin b? was N-acetyl-alanine. It is possible that leghemoglobin b? arises from leghemoglobin ā by a two-stage modification involving cleavage of the N-terminal valyl residue and subsequent acetylation of the exposed alanyl residue.