ABSTRACT
PnTx3-1 is a peptide isolated from the venom of the spider Phoneutria nigriventer that specifically inhibits A-type K(+) currents (I(A)) in GH(3) cells. Here we used a bacterial expression system to produce an NH(2)-extended mutant of PnTx3-1 (ISEF-PnTx3-1) and tested whether the toxin is functional. The recombinant toxin was purified from bacterial extracts by a combination of affinity and ion-exchange chromatography. The recombinant toxin blocked A-type K(+) currents in GH(3) cells in a fashion similar to that observed with the wild-type toxin purified from the spider venom. These results suggest that recombinant cDNA methods provide a novel source for the production of functional Phoneutria toxins. The recombinant ISEF-PnTx3-1 should be useful for further understanding of the role of A-type K(+) currents in biological processes.
Subject(s)
Neuropeptides/biosynthesis , Potassium Channel Blockers , Recombinant Fusion Proteins/biosynthesis , Spider Venoms/genetics , Amino Acid Sequence , Animals , Chemical Fractionation , Chromatography, Affinity , Chromatography, Ion Exchange , Cloning, Molecular , Molecular Sequence Data , Neuropeptides/genetics , Neuropeptides/pharmacology , Patch-Clamp Techniques , Potassium Channels/drug effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology , Spider Venoms/pharmacology , Tumor Cells, CulturedABSTRACT
The irreversible binding of the radical cation of promethazine (PMZ+.) to DNA and protein in vitro and bacterial macromolecules in situ has been studied. Binding experiments were performed with synthesized [35S] promethazine. The results are compared to those with the chlorpromazine radical cation (CPZ+.). Secondary reaction products which result from fission of the alkylamino side chain are involved in the macromolecular binding of PMZ+. Compared to CPZ+. the covalent DNA binding of PMZ+. is significantly less. A larger amount of PMZ+. binds to single-stranded DNA than to double-stranded DNA. The extent of binding to proteins and RNA is of the same order as that of CPZ+. Bacterial mutagenicity tests show that the low genotoxicity of PMZ+. is related to the low DNA binding. The bacterial cytotoxicity is possibly related to the covalent protein binding. Similar results have been obtained with photoactivated promethazine (PMZ) and chlorpromazine (CPZ). The role of radical cations in the photosensitization and metabolic activation of phenothiazine drugs is discussed.
Subject(s)
Chlorpromazine/toxicity , Mutagens , Promethazine/toxicity , Animals , Binding Sites , Biotransformation/radiation effects , Cations , Cattle , Chlorpromazine/metabolism , Chlorpromazine/radiation effects , DNA/metabolism , DNA Repair/drug effects , Escherichia coli/drug effects , Escherichia coli/genetics , Mutagenicity Tests , Photochemistry , Promethazine/metabolism , Promethazine/radiation effects , Protein Binding , RNA/metabolism , Salmonella typhimurium/drug effects , Salmonella typhimurium/geneticsABSTRACT
The degradation products of the promethazine radical cation, generated from promethazine with horseradish peroxidase/H2O2, have been investigated. Several products have been identified which resulted from fission of the bond between the two ethanamine carbon atoms of the N10 side chain. The main product (approx. 90%) was identified as 10-formyl-5-oxophenothiazine. The likely structure of three minor products was also elucidated. The degradation of the promethazine radical cation is different from that of radical cations derived from the propanamine side chain containing phenothiazine drugs.
