-2000 



-1000 



120 ISO 240 300 360 

 ml effluent 



Fig. 10. 



The fractionation of calf lens epithelial cell proteins on 

 DEAE-cellulose after incubation in '''C-algal hydroly- 

 sate (amino acids) for 2 hours at 37°C. The elution sys- 

 tem is the same as that described for Fig. 3. The solid 

 lines denote total proteins (mg) per 3 ml fraction. The 

 dotted lines denote total counts per minute per 3 ml 

 fraction. (Fig. 8, J. Papaconstantinou, Science, in press; 

 copyright 1966 by the American Association for the 

 Advancement of Science.) 



activity data (Table H), however, show that 

 incorporation of amino acids into a-crystallins 

 was inhibited by 71%, ^-crystallins by 83% and 

 y -crystallins by 80%. 



The same experiments were performed 

 with the lens fiber cells. Elution patterns of 

 a-, ;S- and y-crystallins of cortex fiber cells 

 incubated in the absence and in the presence 

 of actinomycin D are seen in Figs. 12 and 13, 

 respectively. The incorporation of amino acids 

 into these proteins is also shown and, again, 

 both patterns are essentially identical with re- 

 spect to the distribution of the crystallins. The 

 incorporation of amino acids into the crystallins, 

 however, is significantly greater in the actino- 

 mycin treated cells. A comparison of the specific 

 activity of the a-, fi- and y-crystallins from 

 control and actinomycin treated lenses shows 

 that there is a significant stimulation of protein 

 synthesis by the antibiotic which ranges from 

 66% for the /8 -crystallins to 103% for the 

 a-crystallins (Table II). 



4000 



3000 



2000 5 



1000 



Fig. 11. 



The fractionation of calf lens epithelial cell proteins on 

 DEAE-cellulose after incubation in ^''C-algal hydroly- 

 sate (amino acids) with lO^g/ml actinomycin D. The 

 experimental conditions are the same as those described 

 in Fig. 10. (Fig. 9, J. Papaconstantinou, Science, in press; 

 copyright 1966 by the American Association for the 

 Advancement of Science.) 



A comparison of the specific activity of 

 actinomycin treated cells shows an 85% inhibi- 

 tion of r-crystallin synthesis in the elongating 

 epithelial cells and a 68% stimulation of this 

 same group of proteins in the fiber cells. Thus, 

 at the time of y-crystallin appearance the syn- 

 thesis of this protein, as well as of the a- and 

 )8-crystallins, is still sensitive to inhibition by 

 actinomycin D, whereas in the completed fiber 

 cell the synthesis of these same proteins is 

 stimulated. The mechanism by which actinomy- 

 cin D stimulates protein synthesis is unknown. 

 The mechanisms which have been proposed for 

 this effect are as follows: first, the stimulation 

 might be attributed to the availability of more 

 ATP for protein synthesis as a result of the 

 inhibition of RNA synthesis by actinomycin (40). 

 Thus, in the fiber cell the ATP normally used 

 for RNA synthesis could be channeled into the 

 synthesis of the proteins being formed on stable 

 RNA templates. 



POLLARD: Quite apart from that, however, 

 if you're just going to use one protein, aren't 

 you only using the t-RNA more efficiently on 

 that one protein when you shut off the others? 

 If all you're doing is just using one protein and 

 if you've got a long-lived message, isn't there 

 every reason why it would go up? 



PAPACONSTANTINOU: Yes, that's right; 

 if that's the explanation for it. 



58 



