Molecular dissection of cholinesterase domains responsible for carbamate toxicity.

Carbamate compounds marked for their cholinesterase (ChE) inhibition are widely used as therapeutics and as insecticides. Groups of closely related carbamate molecules provide an important tool in the understanding of the domains responsible for binding these ligands to ChEs. Comparative inhibition profiles were derived for five N-methyl carbamates, mostly carbofuran derivatives, differing in length and branching of their hydrocarbonic chain towards human erythrocyte acetylcholinesterase (H.AChE), human serum butyrylcholinesterase (H.BChE) in its normal form or in a mutant form containing the point mutation Asp70-->Gly, and Drosophila nervous system ChE. Carbofuran was more toxic to all three ChEs than any of the other derivatives, with IC50 values which differed by more than 1000-fold. Drosophila ChE appeared to be most sensitive to all of the examined carbamates, and H.AChE was consistently more sensitive than H.BChE. Moreover, inhibition efficiency for H.BChE decreased more effectively than it did for H.AChE with increased length and complexity of the side chain, indicating less flexible carbamate binding site in BChE as compared with AChE. The Asp70-->Gly mutation had no apparent effect on H.BChE inhibition by N-methyl carbamates, suggesting that the Asp70 domain localized near the rim of the active site groove is not important in carbamate binding. Comparison of the carbamate IC50 values with published LD50 values demonstrated correlation between the in vivo toxicity and inhibition of BChE by carbamates, suggesting a biological in addition to scavenging importance for BChE in mammals. Pinpointing different domains characteristic of carbamate binding in each member of the ChE family can thus shed light on the variable toxicity of these inhibitors to insects and mammals, predict the toxicity of yet untested inhibitor molecules and help in designing novel and improved ChE inhibitors.

Structural, dynamic, and metal-ion binding studies of the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr by 13C-N.m.r. spectroscopy.

13C-N.m.r. spectral data as well as spin-lattice relaxation times (T1 values) are presented for the core glycopeptides beta-D-Gal-(1----3)-alpha-D-GalNAc----Ser, Thr. The binding of Gd3+ to these model compounds containing N-terminal blocking groups and esterified carboxyl groups indicates that the disaccharide contains a rather weak, but unique, binding-site in the vicinity of C-2 of alpha-D-GalNAc (possibly involving N-2', the acetamido carbonyl group, O-3' and/or possibly the glycosidic oxygen atom (O-3)).

13C n.m.r. study of the structure and the metal ion binding sites of neuropeptides composed of L-Asp and L-Glu.

13C NMR spectral data are presented for a variety of possible neuropeptides composed of L-Asp, Ac-L-Asp, and L-Glu which contain alpha and beta peptide linkages. The data for the various compounds are compared to the data presented for Ac-Asp-Glu, a known neuropeptide, in order to gain structural information about the related compounds. Indications are that for compounds 1 and 5, the cis peptide bond configuration exists due to the interaction of zwitterionic species. This interaction appears to be eliminated when the beta peptide bonds are formed, as in the case of compounds 3 and 7. Spin-lattice relaxation times were used to confirm the structures. Electron-nuclear relaxation rates are also used to elucidate the metal ion binding sites of these species.