Intracellular Locale of Auxin Action 



359 



operations sufficing to reduce the radioactivity in the precipitate to 

 11 to 14 per cent of the original level (Table 2). We thus consider 

 that the wall fractions themselves contain little or no firmly bound 

 2,4-D. The same appears to be true of the other particulate fractions, 

 in which a single washing in sucrose-EDTA reduces the radioactivity 

 to under 10 per cent of the original value (Table 3). It is thus clear 

 that the great bulk of the applied 2,4-D is not bound to any visible 

 particulate in the cell, and must be assumed to be in the soluble 



phase. 



Our next experiments were designed to test the possibility that 

 the 2,4-D was bound to some macromolecule in the final supernatant 

 (S4) fraction, since several previous investigations had reported the 

 presence or formation of auxin-protein complexes in plant cells (3, 

 6, 10). Dialysis of the final supernatant against the sucrose-EDTA 

 medium used in preparing the homogenate revealed a ready outward 

 passage of the label (Table 4). Two successive 16-hr. dialyses at 2° C. 

 against 100 volumes of medium reduced the residual counts to about 

 3 per cent of the original value. Since this did not differ significantly 

 from the behavior of 2,4-D added in vitro to control supernatant pro- 

 tein (from sections not treated with auxin during the overnight 

 growth test), this experiment offers no evidence for the binding of ap- 

 plied 2,4-D to any non-dialyzable component of the final supernatant. 



Attempts were next made to precipitate proteins by 0.5M tri- 

 chloroacetic acid, by saturated (NH4)oS04 (pH 6.1), and by boiling 

 (100° C. for 8 min.), and to test the precipitates for C^^ content. The 

 first two methods yielded only very weak activity in the washed pre- 

 cipitates; however, the boiling experiment, while providing no direct 

 evidence for protein-bound 2,4-D, led to an observation of great in- 

 terest. We noted that those homogenates derived from auxin-treated 

 cells yielded little or no precipitate, the solution merely turning opal- 

 escent after immersion for 8 min. in a boiling water bath. The con- 

 trols, on the other hand, invariably yielded a copious bulky-white 

 precipitate. This experiment could be rendered quantitative by cen- 



Table 4. Dialysis of final supernatant of green sections. 



