Metabolism and mode of action 



disappearance is of a considerably smaller degree. For example, experiments 

 with mitochondrial preparations from castor bean are shown in Figure 2. 

 Paired aliquots of CoA were incubated in the presence and in the absence of 

 enzyme with different acid substrates. In the first place a non-enzymatic 

 disappearance of CoA sulphydryl in the order of 10~* M took place in 

 all the preparations measured. When acetic acid was provided as a substrate, 



SxW''*V[ 



I 



//o w"^v\ io'^\^ lO'^n 



subsfrafe acetate 2:'t--D TIBA 



Figure 2. Sulphydryl disappearance with acetate activating enzyme. Final concentrations of constituents : 



5 X 10-* M CoA reduced with KBH^ 

 10-2 M ATP 

 10-1 M TRIIS pH 8 

 10-2 M MgCla 

 10-2 M K^c 

 ca. 0-4 mg proteinjml where added. 



Incubation time: 60 minutes except for portions of 'no substrate' as indicated. 

 Temperature : 30"". 

 Volume: 0-5 ml diluted immediately prior to assay with 2-5 ml sat. NaCl and 0-3 ml each of 



nitroprusside and sodium carbonate-cyanide. 

 Concentration of free sulphydryl determined by £520- 



there followed an enzymatic disappearance of roughly 4 X 10~* M sulphydryl 

 concentration. The figure shows similar experiments with 2:4-D and TIBA, 

 and it can be seen that the disappearance of sulphydryls in the presence of 

 these growth substances was scarcely greater than the disappearance in the 

 non-enzymatic circumstances. 



This experiment warns us of the possible complication of the auto- 

 oxidation of CoA. Not only is the extent of sulphydryl change due to auto- 

 oxidation in the same order of magnitude as the greatest change reported by 

 Leopold and Guernsey (1953), but it is somewhat larger than the change 

 reported with 2:4-D by Millerd and Bonner (1954). Their experiments are 

 roughly comparable in that, like ours, a relatively large disappearance of 

 sulphydryl was obtained with acetate. 



Turning to the measurement of thio-esters formed, a wide range of auxin 

 concentrations was tested with a castor bean mitochondrial preparation 

 which was active in the esterificadon of CoA. Results of such an experiment 



272 



