356 A. W. Galston and R. Kaur 



indicator of some fundamental change in the configuration of the 

 protein molecules, the exact nature of which remains to be delineated. 



MATERIALS AND METHODS 



'Alaska' peas were used throughout these investigations, being 

 grown under conditions already described in another article in this 

 volume (2). Etiolated and light-grown sub-apical stem sections were 

 grown overnight in the presence or absence of an auxin. After meas- 

 urement of growth, the sections were homogenized in ice-cold 0.25M 

 sucrose -\- .00 IM ethylenediaminetetraacetic acid (EDTA) in a pre- 

 chilled mortar and pestle to yield a suspension of 12.5 mg. fresh wt/ 

 ml. This suspension was repeatedly centrifuged at various speeds in 

 an International Refrigerated Centrifuge (Model PR-2) to yield, suc- 

 cessively, cell wall fragments and unbroken cells, chloroplasts (when 

 present), mitochondria, microsomes, and a final centrifugal superna- 

 tant fraction containing the soluble proteins of the cell. The fate of the 

 nuclei in such a fractionation is unknown, but they or their fragments 

 are presumed to sediment along with the plastids or possibly mi- 

 tochondria. 



In certain of the experiments, C^^-carboxyl-labeled 2,4-D ^vas 

 used as the auxin. This was purchased from Nuclear Chicago Com- 

 pany, and had an activity of 950 microcuries per millimole. Aliquots 

 from the fractionation were pipetted on to l^^" X %2" stainless steel 

 planchets, and were counted in a stream of Q-gas with a Nuclear Chi- 

 cago model D-47 gas flow counter equipped with a micromil window, 

 mounted in a model M-5 sample changer, and attached to a model 

 186 decade Scaler. 



RESULTS 



The fractionation scheme employed for tracing the intracellular 

 localization of exogenously applied labeled 2,4-D is shown in Figure 

 1. W'ith green sections, the 2,4-D concentration applied was the 

 3 X 10-^M, which is approximately optimal for growth in the light 

 and in the presence of 1 per cent sucrose. Etiolated sections were 

 given this same concentration of 2,4-D, although it is approximately 

 100 times too high for their optimal growth in length, but about 

 optimal for increase in fresh weight. 



The distribution of radioactivity in the various fractions of green 

 and etiolated cells is seen in Table 1. It should be noted that the final 

 supernatant contains a major part of the activity, as does the first 

 precipitate, P,, consisting of unbroken cells and cell walls. ^Vhen 

 the ?! fractions were reground and recentrifuged, considerable ac- 

 ti\ity was lost from the precipitate to the wash medium, two such 



