METHODS OF LOCALIZATION 519 



state of the components when these donors or acceptors are added. The 

 development of many acceptor dyes, such as phenazine methosulfate, di- 

 ethylsafranin, 2,6-dichlorophenol-indophenol, and others, acting at different 

 loci in the electron-transport sequence, has increased the versatility of this 

 method. 



Distribution of Labeled Substrate Atoms 



A rather laborious but often informative technique for the localization 

 of inhibitor action involves the addition of a substrate labeled at certain 

 positions with radioactive atoms and the determination of the metabolic 

 pathways followed by these atoms in the absence and presence of an in- 

 hibitor. The refinement in various types of chromatographic analysis has 

 allowed this procedure to be applied to a great variety of metabolic block- 

 ades. The localization achieved is usually restricted to metabolic pathways 

 rather than to individual enzymes but nevertheless may be valuable in 

 the initial mapping out of the action of a new inhibitor. This method is 

 also useful in establishing whether an inhibitor, which has been shown by 

 other techniques to block some enzyme or pathway in vitro, actually does 

 this in the living cell. 



Many examples of this procedure will be discussed in connection with 

 the individual inhibitors but one illustration of the method may be taken 

 from studies on the inhibition of photosynthesis by iodoacetate and io- 

 doacetamide. These inhibitors were shown by Kohn (1935) to depress po- 

 tently the formation of sucrose and indirect evidence indicated that the 

 block was not primarily on the initial photochemical reaction (light reac- 

 tion) but on the subsequent pathway whereby CO2 is utilized in the forma- 

 tion of carbohydrate (dark reaction). Recently it has been possible to de- 

 termine the distribution of carbon- 14 when plant tissue is illuminated for 

 very brief periods in the presence of C^^Og and it is apparent that the first 

 stable intermediate is 3-phosphoglycerate, which showed the highest spe- 

 cific activity initially. The formation of hexoses could thus occur through 

 the reversal of the glycolytic pathway, the first step being the reduction 

 of the 3-phosphoglycerate to glyceraldehyde-3-phosphate. If this occurs, 

 iodoacetate and iodoacetamide, which inhibit the phosphoglyceraldehyde 

 dehydrogenase, should block the photosynthesis of carbohydrate in the 

 same manner as they inhibit glycolysis, and perhaps would cause an ac- 

 cumulation of 3-phosphoglycerate. Calvin and his co-workers (Calvin et 

 al, 1951) treated Chlorella with 0.15 mM iodoacetamide so that C^^Oa 

 fixation was inhibited 90% and yet it was found that the rate of sucrose 

 formation was not decreased. Also no phosphoglycerate accumulation oc- 

 curred. It was suggested that enzymes other than phosphoglyceraldehyde 

 dehydrogenase could have been inhibited, blocking other pathways of 

 phosphoglycerate utilization and allowing a more channeled sucrose syn- 



