Figure 1.

Formation of modified ricin A chains and their reconstitution with the B chain to form holotoxin. (a) The C-terminal end of wild-type ricin A chain and A chain modified to contain sulfation and glycosylation sites. The C-terminal nonapeptide of rat cholecystokinin precursor (indicated with boldface type) was added to the C-terminal end of ricin A chain to form ricin A-sulf-1. To form ricin A-sulf-2, a nonapeptide containing three partially overlapping N-glycosylation sites was added to the C terminus of ricin A-sulf-1. The constructs were produced as fusion proteins with maltose-binding protein (MBP) and purified on an amylose column. (b) Lanes: 1, ricin B chain; 2, uncleaved fusion protein containing ricin A-sulf-1; 3, the same as lane 2, but after cleavage with factor Xa; 4, reconstituted ricin obtained by dialyzing together material as in lanes 1 and 3. (c) Material as in b, lane 4, labeled with ¹²⁵I (lane 1) was added to Vero cells at 4°C for 30 min in the absence (lane 2) and presence (lane 3) of 10 mM lactose. The cells were then washed and analyzed by SDS/PAGE under nonreducing conditions. (d) Increasing amounts of wild-type and reconstituted ricin or the free A and B chains were added to Vero cells and incubated for 4 h. Then the ability of the cells to incorporate [³H]leucine was measured.