588 



Lester Packer 



the shrinkage. A more rapid and reversible type of structural 

 change in chloroplasts has also been reported by measurement of 

 changes in "rectilinear attenuance" (l3), which are thoiight to 

 result from changes in the axial ratio of chloroplasts as shown 

 by a correlated morphological study (l2). The addition of ATP to 

 chloroplasts incubated either in the light or in the dark, brought 

 about a shrinkage which was quite rapid; however the circumstances 

 giving rise to this effect appear still somewhat uncertain. In 

 view of this, it seemed worthwhile to examine in more detail the 

 role of ATP in the control of chloroplast structure. 



The results of a typical experiment demonstrating structural 

 changes in chloroplasts isolated from spinach leaves is shown in 

 figure 1. Initially, the chloroplasts were incubated under condi- 

 tions necessary for phosphorylation except for phosphate and acti- 

 nic light. The reaction system contained phenazine methosulfate 

 (H4S), ADP, and Mg"*^. A low concentration of chlorophyll was 

 employed to prevent pigments from interfering with light- scatter- 

 ing measurements. The light- scattering responses were measured 

 with a low intensity green light located near the minimum of the 

 photochemical action spectrum since this process minimizes the 

 possibility of activating electron flow by the incident beam. The 

 combination of low chlorophyll concentration and low intensity 

 green light is the trick for demonstrating scattering responses. 

 The experiment shows that in the absence of phosphate a small 

 light scattering increase of approximately 5^ was brought about by 

 Illumination of the reaction mixture with actinic red light (6 x 

 lO-'-^ quanta/sec in the range 600-700 my-i). The 5/0 increase in 

 scattering was reversible upon removal of actinic light. Phos- 

 phate (5 mM) was then added in the dark; this led to no change in 

 the scattering level, but upon illumination with actinic light, an 

 enormously enhanced scattering was obtained and it now seen that 

 the scattering increase was about 70fo above the initial level. It 

 may be further noted that these responses are repeatable; alterna- 

 ting periods of light and darkness continued to give the scatter- 

 ing response. 



Similar studies established that light- scattering increases in 



1. Control experiments showed that these optical changes were de- 

 pendent on the angle at which the emitted green light irzas measured 

 in a manner characteristic of scattering and that they are there- 

 fore not fluorescence changes. Absorbance changes were ruled out 

 by showing that the /» change was independent of the chloroplast 

 concentration in the range used in the experiments, and also be- 

 cause the absorbance of the pigments was too low to effect an 

 apparent scattering measurement. 



