TABLE I 



The Electrophoretic Mobility x 10^ (cm^ volts"! sec.'^ ) of the lens a-,p- and y-CrystalUnsfrom Adult 

 Bovine Lens Cortex and nucleus fibers. The crystallinswere fractionated on DEAE-cellulose columns. The 

 peaks were precipitated and analyzed by free boundary electrophoresis. 



method is quite good for separating the proteins 

 into the a-, ^- and y-crystallin groups. The 

 r-crystallins, which are the proteins we are 

 interested in for this discussion, are eluted 

 cleanly from the column as peaks a 1,02 and b 

 in the cortex fiber pattern (Fig. 3B) and as 

 peak a in the nucleus fiber pattern (Fig. 3C). 



POLLARD: What' s the separation process? 

 Is it on a column? 



PAPACONSTANTINOU: This is a DEAE- 

 cellulose column, using a stepwise elution 

 system starting with 0.005 M phosphate buffer 

 pH 7.0 and going to 0.02 M phosphate buffer 

 pH 5.7. After this, further elution is achieved 

 by increasing the ionic strength with NaCl. 

 We have done linear gradients on this more 

 recently and they are essentially the same. 

 We've used two linear gradients: the first is a 

 sodium phosphate gradient ranging from 0.005 

 M phosphate pH 7.0 to 0.02 M phosphate pH 5.7. 

 With this, the y- and ^-crystallins are eluted 

 from the column. Then the phosphate concen- 

 tration is kept constant at 0.02 M pH 5.7 and a 

 NaCl gradient is initiated. This results in the 

 elution of the a-crystallins. 



A comparison of these elution diagrams 

 shows that the epithelial cells (Fig. 3 A) contain 

 only traces of y-crystallins in comparison to the 

 amounts found in adult cortex (Fig. 3B) and 

 adult nucleus (Fig. 3C) fiber cells. Furthermore, 

 it can also be seen that the y-crystallins of the 

 adult cortex and adult nucleus fiber cells are 

 both qualitatively and quantitatively different 

 with respect to their chromatographic properties 



on DEAE-cellulose columns. These observations 

 indicate, firstly, that the y-crystallins are pro- 

 teins which are characteristic of the fiber cell 

 and secondly, that y-crystallins formed in fiber 

 cells of young animals (cells found in the nucleus 

 region of the adult lens) are chromatographically , 

 and possibly chemically, distinct from 

 y-crystallins synthesized in fiber cells of older 

 animals (cells found in the cortex region of the 

 adult lens). 



If the first proposal is correct, i.e., that 

 y-crystallins are proteins specific to the fiber 

 cells, then epithelial cells from animals of all 

 ages should lack these proteins. The elution 

 pattern of proteins from epithelial cells of 3 

 month calf lenses (Fig. 4A) indicate that this 

 is indeed the case. Although traces of y-crystal- 

 lins are detected by this procedure, the amount 

 detected is significantly less than that detected 

 in the fiber cells (Figs. 4B, 4C). In addition, the 

 traces of y-crystallins that are detected in the 

 epithelial cells are due to the adherence of the 

 elongating cells to the lens capsule. It is, we 

 believe, in these elongating fiber cells, where 

 the activation of y-crystallin synthesis occurs. 

 Thus, when we compare the elution patterns of 

 proteins extracted from epithelial cells, cortex 

 fiber cells and nucleus fiber cells of adult and 

 calf lenses, we see that (a) at both ages the 

 epithelial cells do not contain y-crystallins and 

 conclude that y-crystallin synthesis is initiated 

 during fiber cell formation in young and adult 

 lenses. Similarly, it has been reported that 

 y-crystallin synthesis is associated with fiber 



50 



