A?ich.Recent studies have indicated that barbiturates may, in vitro, interfere with oxidative phosphorylation ( 1 ) . The significance of this interference is difficult to assess, since many compounds of apparently unrelated biological activity and structure exhibit the same effect. However, further understanding of the manner in which oxidative phosphorylation is interrupted may reveal points of departure among the various compounds involved. In a previous discussion(2), it was pointed out that certain broad-spectrum antibiotics, as well as 2 ,4-dinitrophenol(3 ) , increased the adenosinetriphosphatase ( ATPase) * activity of rat liver homogenates and mitochondria. It was also observed(2) that the ATPase activity of rat brain homogenates was not stimulated by either the antibiotics or dinitrophenol. The results obtained in the present study indicate a possible role of chelation effects in -4TPase stimulation.The purities of Sigma sodium adenosinetriphosphate ( ATP) , Sigma sodium adenosinediphosphate ( ADP) ? and Pabst sodium uridinetriphosphate (UTP) were confirmed by analyses of labile and total phosphorus. The barbituratest and other chemicals used were the purest obtainable
Materials and methods.*This term is used to identify enzyme(s) catalyzing release of inorganic phosphate from ATP.The exact mechanism involved may be more complex than that of a direct hydrolysis of substrate(4). t The au'thors are indebted to Dr. C. W. Pettinga, Eli Lilly and Co., for a sample of 1,3-dimet,hyl-.butyl-ethyl-barbiturate. from various commercial sources. Ten per cent homogenates of rat liver or brain (frontal lobes) were made in 0.25 M sucrose at 0°C by use of a glass homogenizer (5). Tubes were set up containing the following: 0.04 M KCl, 0.0001 31 MgS04, 0.05 M tris(hydroxymethyl) amino-methane buffer ( pH 7.4), 0.003 31 -4TP (or other substrate), 0.1 ml homogenate, barbiturate solution or other additions if desired, total volume 1.0 ml. All solutions were made in glass-redistilled water and adjusted to pH 7.4.Substrate was added after the other components of the reaction mixture had been equilibrated in the water bath for 5 minutes. Following 10 minute incubation at 30°C, proteins were precipitated by addition of 0.1 ml 50% trichloracetic acid. Orthophosphate was determined( 6) in the supernatant after centrifugation. Corrections were made for phosphorus liberation in the absence of homogenate and in the absence of substrate. The level of magnesium ion used in these experiments was punposely set far below the optimum in order to allow for maximum expression of stimulatory and chelation effects.Results. In these experiments, the reproducibility of results when duplicate determinations were made with the same homogenate was quite satisfactory, a variation of more than t S % being unusual. However, the activities and stimulatory responses of tissues from different animals were often subject to considerable quantitative variation. For this reason, comparisons of stimulatory activity at EMORY UNIV on August 6, 2015 ebm.sagepu...