39 



addition of a centrif ugation step after each wash to pellet 

 the suspended cells. 



Cell Binding 



125 

 Synthesis of I-Con A . Evaluation of the number of 



membrane binding sites present on cultured cells for Con A 



was acheived by using Con A labeled to a high specific acti- 



125 

 vity with I. The chloramine T method developed by 



Cuatrecasas (1973) was used with minor modification. To 



400ug of Con A in 0.05ml of 0.15M PBS , pH 7.4 was added 



125 



lmCi of I (New England Nuclear, sodium salt, carrier 



free) contained in 0.1ml of . 1M sodium phosphate buffer, 

 pH 7.4. Freshly prepared chloramine T (lOOpg in 0.025ml 

 water) was quickly added and mixed for 50 seconds. Sodium 

 metabisulf ate (200yg in 0.025ml water) was added to stop the 

 reaction followed by addition of 0.30ml PBS . The entire 

 mixture was applied to a 2.5ml column of Sephadex G-100 pre- 

 equilibrated with PBS . The column was washed with 25 vol- 

 umes of PBS after which time negligible activity eluted from 



the gel. D-glucose (0.3M) was added to the elution buffer 



125 

 and the bulk of the I-labeled Con A was collected in a 



125 

 single 1.0ml fraction. The I-Con A was dialyzed versus 



two three liter changes of PBS at 5°C over a 48 hour period. 



The labeled protein was analyzed for total activity, TCA 



precipitable activity and total protein by the fluorometric 



assay of Bohlen et al. (1973). Following analysis the 



preparation was diluted with native Con A to a final specific 



5 

 activity of 2 x 10 cpm/pg and stored at -20°C in Nunclon 



