Metabolism and mode of action 



in the coupling reaction, actually interferes with it by favouring the dissocia- 

 tion of the lAA protein into protein plus unbound lAA. This 'unbound 

 lAA' seems not to be free lAA, since much of it is not extractable into 

 diethyl ether at pH 3-0. We consider that it is either lAA linked to CoA or 

 lAA bound to a small peptide. We have, however, not had sufficient time 

 to investigate this problem properly. 



Since these investigations, the lAA-protein has been investigated further 

 by Dr. I. B. Perils. He has found the lAA-protein to be formed in all 

 portions of the etiolated pea seedling, even in those incapable of growth. 

 He has also been able to purify the protein several-fold by means of ammonium 

 sulphate and KH2PO4 fractionation. 



What is the physiological significance, if any, of this lAA-protein? 

 Unfortunately, we do not yet know. It is tempting, of course, to suppose that 

 the catalytic effect of lAA on growth is a consequence of its coupling to some 

 protein, the whole then forming an enzyme somehow critically involved in 

 the growth process. If this supposition is true, then the I AA-protein we have 

 discovered may function in this important way. However, we have not yet 

 been able to localize any enzymatic activity within this protein. This 

 situation is also true of other auxin-proteins of natural occurrence (Larsen, 

 1951). 



Another possibility is that the lAA-protein constitutes a stored, but 

 physiologically inactive form of lAA. In support of this hypothesis is the fact 

 that the lAA bound to protein can apparently not be destroyed by the lAA 

 oxidase system. The release of lAA from such a stored state may, in the cell, 

 be mediated by CoA, which could either release it from protein entirely, or 

 effect its transfer from storage to catalytically active protein. 



THE OXIDATION PRODUCT OF lAA IN PLANT BREIS 



If lAA is added to a brei prepared from roots or etiolated aerial portions of 

 certain plants, it i§ rapidly inactivated (Tang and Bonner, 1947; Wagen- 

 knecht and Burris, 1950). This inactivadon may be followed either by bio- 

 assay procedure such as the Avena coleoptile curvature test, or by chemical 

 procedures such as the Salkowski colorimetric reagent. Results with both 

 techniques are essendally the same, so that the colorimetric technique is 

 generally employed because of its greater convenience. 



It is known that in the inactivation of lAA, one mole of O2 is absorbed 

 and one mole of CO2 is released per mole of lAA inactivated. This simple 

 reladon has prompted the conjecture (Tang and Bonner, 1 947 ; Wagen- 

 knecht and Burris, 1950) that a simple oxidative decarboxylation of lAA to 

 3-indolealdehyde is involved. This conclusion is supported by the fact that 

 the oxidation product gives a colour with Ehrlich's reagent (and therefore 

 may have an indole ring), and also forms an insoluble 2:4-dinitrophenyl- 

 hydrazone (implying the presence of a carbonyl group) . Despite this evidence, 

 we are now convinced that the oxidation product is not 3-indolealdehyde, 

 but is rather of the nature of a hydroxyaminoacetophenone. 



Dr. D. T. Manning investigated this problem in our laboratories by the 

 use of paper chromatography. He added large amounts of pure lAA to 

 active lAA oxidase preparations from etiolated pea seedlings. After a major 

 portion of the lAA had been destroyed, as shown by Salkowski-reagent 



222 



