RT Journal Article
SR Electronic
T1 ENZYMATIC CONVERSION OF THE DIMETHOXY ESTER OF BENZOTRIAZINE DITHIOPHOSPHORIC ACID TO AN ANTICHOLINESTERASE AGENT
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 572
OP 583
VO 119
IS 4
A1 Sheldon D. Murphy
A1 Kenneth P. Dubois
YR 1957
UL http://jpet.aspetjournals.org/content/119/4/572.abstract
AB A quantitative method was developed for measuring the metabolic conversion of the dimethoxy ester of benzotriazine dithiophosphoric acid (DBD; Guthion) to an anticholinesterase agent by liver homogenates. The method involves aerobic incubation of DBD with liver homogenates fortified with diphosphopyridine nucleotide for 10 minutes at 38°C. The anticholinesterase action of the metabolite was used as a bioassay to measure the amount of metabolite produced by incubation with liver. The activity of the enzyme which converts DBD to an anticholinesterase agent was expressed in terms of arbitrary units of metabolite produced/5 mgm. of liver (wet weight)/hour. Studies on the stability of the metabolite of DBD in the presence or liver homogenates demonstrated that loss of activity through enzymatic hydrolysis occurs at a rapid rate. Thus, when the metabolite was incubated with 10 mgm. of homogenized liver from rats, mice and guinea pigs in a final reaction volume of 3 ml. the loss of activity amounted to 44, 40 and 60 per cent respectively. Complete loss of activity occurred after incubation of the metabolite with 50 mgm. of liver from the three species. The more rapid destruction of the metabolite by guinea pig liver may be partly responsible for the greater resistance of this species than rats and mice to the acute toxic effects of DBD. Measurements of the concentration of the enzyme which converts DBD to an anticholinesterase agent gave the following average values in terms of units of metabolite produced/5 mgm. of liver/hour: female rats 4.7; male rats 11.2; female mice 3.8; male mice 3.0; male guinea pigs 2.5; and female guinea pigs 2.1. No enzyme activity was observed in several other tissues indicating that this metabolic reaction is confined to the liver. By differential centrifugation of rat liver homogenates and enzyme assays on the resultant cell fractions it was found that the enzyme system which oxidizes DBD to an anticholinesterase agent is located in the microsomes. The enzyme system which converts DBD to an active metabolite loses activity rapidly at 38°C. It is inhibited by β-diethylaminoethyl diphenylpropylacetate (SKF 525A), cytochrome c and pyridoxal phosphate when the latter compound is incubated with the enzyme in the absence of DBD. Riboflavin, pyridoxine and sodium arsenite had no appreciable effect on the enzyme activity.