02 

 0.1 h 



- A 



_L 



02r B 



200 400 600 800 

 83 84 



E 



200 400 600 800 



200 400 600 800 

 ml effluent 



Fig. 6. 



The fractionation of y-crystallins from adult cortex, adult 

 nucleus and embryonic lenses. The proteins, which had 

 been stored as an ammonium sulfate precipitate were 

 spun down and dissolved in 0.01 A/ Tris, pH 10. Ammon- 

 ium sulfate was eliminated by dialysis against 0.01 M 

 Tris. The protein solution was placed on a DEAE- 

 cellulose column and was eluted from the column by the 

 stepwise addition of Tris-HCl buffers in the following 

 order: I. 150 ml 0.01 M Tris, pH 10; II. 150 ml 0.02 /W 

 Tris, pH 9; III. 150 ml 0.04 M Tris, pH 8.6; IV. 150 ml 

 0.06 M Trls-HCl, pH 8.2; V. 150 ml 0.08 M Trls-HCl, 

 pH 7.6; VI. 150 ml 0.1 M Tris-HCl, pH 7.2. In each 

 fractionation the column size was 17 mm diameter x 20 

 cm height; 5 ml aUquots were collected. (Fig. 4, J. 

 Papaconstantinou, Biochim. Biophys. Ada 107, 81, 1965; re- 

 produced by permission of Elsevier Publishing Company.) 



presently involved in experiments designed 

 to determine whether epithelial cells in vivo and 

 in tissue culture can be induced to synthesize 

 r-crystallins and at the same time retain both 

 their epithelial cell structure and their ability 

 to replicate. 



B. The loss of LDH-5 isozyme synthesis 

 in lens cell differentiation: gene 

 repression. 



I would like to present you with another 

 example of differential gene action associated 

 with fiber cell differentiation as well as with 

 the aging of the epithelial cells, i.e., the specific 

 repression of one of the lactate dehydrogenase 

 isozymes. These experiments were carried out 

 in collaboration with Mr. James A. Stewart (12, 

 13). 



Lactate dehydrogenase isozymes have been 

 shown to occur in many vertebrate tissues in 

 5 electrophoretically distinct forms, and to vary 

 in activity during embryonic and post-embryonic 

 development (14-19). In addition, it is now well 

 established that all 5 isozymes are composed 

 of 4 protein subunits and that only the extreme 

 cathodal (LDH-5) and anodal (LDH-1) forms of 

 these enzymes are homogeneous with respect to 

 their subunits. Furthermore the subunits of 

 LDH-1 do not have the same amino acid com- 

 position as the subunits of LDH-5. Thus, by 

 dissociation and reassociation of the subunits in 

 a mixture of LDHs-1 and 5, all 5 isozymes can 

 be formed (20). These experiments show that 

 LDH's-2, 3 and 4 are composed of combinations 

 of LDH-1 and 5 subunits. Since there is now 

 good evidence that the synthesis of LDH-1 and 

 LDH-5 subunits is genetically regulated (21), 

 we felt that any alterations in the isozymic 

 patterns during fiber cell differentiation would 

 be another indication of the differential regula- 

 tion of protein synthesis which in the lens could 

 be localized to a very specific stage of cellular 

 differentiation, namely, the differentiation of an 

 epithelial cell to a fiber cell. 



Our electrophoretic analyses of the lactate 

 dehydrogenase isozymes show that the epithelial 

 cells of the adult lens and calf lens have five 

 forms of the enzyme. Typical isozymic patterns 

 of calf and adult epithelial and fiber cells are 

 diagrammatically presented in Fig. 9. Concen- 

 trating on the epithelial cell diagrams alone, 

 a comparison of the patterns from calf and adult 

 cells shows that a change occurs from pre- 

 dominantly cathodal forms to predominantly 

 anodal forms. These data show that there is 

 a transition of epithelial cell LDH isozyme ac- 

 tivity during the post-natal aging of these cells. 

 This is a very interesting change since the 

 epithelial cells of the lens, during embryonic, 

 early post-natal and adult life, carry out the 

 same functions, i.e., to either replicate for the 

 formation of more epithelial cells or to differen- 



53 



